Bull. Org. mond. Sante 1961, 25, 227-262 Bull. Wld Hlth Org.

The Biological Disposition of and its Surrogates 1 *

E. LEONG WAY, Ph.D.' & T. K. ADLER, Ph.D.'

CONTENTS Page INTRODUCTION ...... 227 MORPHINE ...... 228 Methods of estimation ...... 229 Absorption ...... 234

Distribution ...... 236

Metabolism ...... 243

Excretion ...... 252

REFERENCES ...... 259

INTRODUCTION The last decade has seen the elucidation of the structural features that endow these molecules with stereochemistry (Rapoport & Payne, 1950, 1952; their biological potential. This potential can be Rapoport & Lavigne, 1953; Ginsburg, 1953) and realized only when the molecule has reached its structural configuration (Lindsay & Barnes, 1955) target organ, for the very nature of the pharmacolo- of the morphine molecule, as well as the verification gical response indicates a highly selective action at of the structure by total synthesis (Gates & Tschudi, specific receptor sites within the central nervous 1952); this has supplemented previous chemical system. Accordingly, the onset, intensity, and dura- knowledge of the (Small, 1932; tion of action will be governed by the relative ease Bentley, 1954). Moreover, the relationship between with which the molecules can reach the effective site; chemical structure and biological activity has been this, in turn, depends upon a series of physico- explored not only for morphine but for the synthetic chemical processes that regulate the biological dis- substances that can act as surrogates for morphine position of the compound. The present study con- in vivo (Bockmuhl & Ehrhart, 1949; Eddy, 1950; siders in detail the absorption, distribution, bio- Schwartzman, 1950; Beckett & Casy, 1954; Braen- transformation and elimination of each compound, den, Eddy & Halbach, 1955; Reynolds & Randall, as well as evaluating the analytical methods used in 1957). From these studies it is apparent that much the investigation of these processes. Whenever progress has been made towards delineating those possible, the significance of the findings in relation to the over-all pharmacology of the compound is discussed. The study is divided into four parts. The * This study on the biological disposition of morphine first three parts, which deal, successively, with and its surrogates is being published in the Bulletin of the morphine, the partially synthetic World Health Organization in four instalments. This-the derivatives of first-is devoted to morphine per se; the second and third morphine and the wholly synthetic substances with instalments will deal, respectively, with derivatives of mor- morphine-like action, are presented in the form of phine and synthetic surrogates of morphine, and the final instalment will discuss general considerations. The four individual monographs. The structure of each instalments will eventually be available as a joint reprint. agent is shown in Fig. 1. The final part is devoted to 1 Department of Pharmacology, University of California a discussion of general considerations pertinent to Medical Center, San Francisco, Calif., USA. The authors were aided in their preparation of this report respectively by this group of drugs as a whole. Grant RG-1839 from the National Institutes of Health and The material for this study has been selected by Senior Research Fellowship SF 271 from the Public Health Service, US Department of Health, Education, and mainly from papers published within the past twenty Welfare. years, attention being directed primarily to those 1042 -227- 228 E. LEONG WAY & T. K. ADLER

FIG. I MORPHINE AND MORPHINE-MIMETIC COMPOUNDS a

NATURALLY N-CM5 N-CM OCCURRING CHI cm OPIUM ALKALOIDS 4"o ON c- MOAHIM

N -CM5 N-CM5 N-CM3NCH-HUH PARTIALLY ICM SYNTHETIC DERIVATIVES OF MORPHINEMORPHINE q__O%G14- -O 0 0-C-CM5-,,csH.ooozoMo 0 0 04g-0 0 0 No 0 ON MUA0OIN DIMYDROMORPHMINNONE AYDROCODEINONE NALO AHINI

SYNTHETIC N-MHNC NC-*M COMPOUNDS 0 -H MORPHINANS MO C,5-o Mo LEVORAPNOL DEXTPOMETMORPMAN LEVALLOAPHN

CM,^-C-O Q~CM5 ,cH} 0 /CM-N CM- CMCCM N-M0CC-M- /CHM METHADONES C oCCHS_CHMN CH3-CH-CH-C-CHZ-CH-N\CCQM WsMMCN~tF0 iCMC

METHADONE ArETYLMETHADOL U AONI POPOXYPHENtE

-C-0-cma-CM5 O-C-CaCN- CM5aCH L -X0C-CMI-CH5 PHENYL- M 4M CHS It H C H PIPERIDINES HaN Ha Ha MgNgHS Ma MEPERIDINE AUM%APODINL KETOSEMIDONE ANIUDIN

-°-CH-04 NI-CH-^ MISCELLANEOUS HSM CMa N3 CHI ETMOMEPTAZINE PM4NAZOCINE

a The compound " meperidine " is henceforth referred to in these studies by its proposed international non-proprietary name, . papers appearing since the publication of the mono- more recent findings. Certain aspects of this material graph on the pharmacology of the opium alkaloids have been reviewed elsewhere in a more limited by Krueger, Eddy & Sumwalt (1941, 1943) to the fashion (Krueger, 1955; Peterson, 1955; Perrine & time of writing (December 1960). Occasionally, Eddy, 1956; Reynolds & Randall, 1957; Schaumann, references to the older literature are discussed at 1957; Stolman & Stewart, 1960; Way & Adler, length when this is pertinent to the interpretation of 1960).

MORPHINE In discussing morphine and its derivatives the carbon atoms of the side chain, which, together with present authors have adhered to the Gulland & nitrogen and carbons 13 and 14 of the Robinson formula as presented in 1925. Accord- skeleton, form a piperidine ring, are numbered 15 ingly, morphine is presented as a partially saturated and 16. No number is assigned to either the ring phenanthrene derivative containing an ethane- nitrogen or the ring oxygen. It should be noted, side chain. The fourteen carbon atoms of the however, that inasmuch as morphine is a complex phenanthrene part of the formula are drawn and molecule, the graphic formula, the numbering of the numbered in the classical manner. The remaining atoms, and the nomenclature of morphine deriva- BIOLOGICAL DISPOSION OF MORPHINE AND ITS SURROGATES. 1 229

FIG. 2 NUMBERING SYSTEMS USED FOR THE GRAPHIC FORMULA OF MORPHINE

"CLASSICAL" PHENANTH REN E PHENANTHROFURAN "" PHENANTHREN E tives will depend on the relative emphasis placed on tion and separation of the morphine from the particular structures in the molecule. Schaumann extraneous biological phase. The morphine-con- (1940) considers morphine to be primarily a piperi- taining moiety finally obtained is rarely free from dine derivative, but this proposal has not gained wide interfering substances and often the yield is far from acceptance among chemists or pharmacologists. quantitative. In addition, the small size of the dose For the most part, the phenanthrene structure has of morphine required to elicit the pharmacological been emphasized during the thirty-seven years since or toxicological response has often necessitated the Gulland & Robinson (1923) pointed out the striking use of large amounts of the biological material for tendency of the morphine alkaloids to give phenan- the analysis. threne derivatives during chemical degradation. In speaking of morphine recovered from biological Even here several methods of numbering the atoms media, the terms frequently encountered are " free are in current use. The Ring Index (Patterson & morphine ", " bound, combined or conjugated Capell, 1940) regards morphine as a derivative of morphine" and " total morphine ". Free morphine imino-ethano-phenanthrene-furan, and both the is generally assumed to be the unchanged parent furan oxygen and the imino nitrogen are included in compound. The terms bound, combined and con- the numbering system. Rasmussen & Berger (1955) jugated have been used interchangeably to indicate have suggested that the resemblance between mor- the morphine released after acid hydrolysis, and the phine and the be stressed, and in their quantity of bound morphine is estimated by taking graphic formula the phenanthrene part of the the difference between the total and free morphine molecule is drawn and numbered in the manner used found in a given sample. Recent evidence suggests in steroid chemistry. Fig. 2 shows the various ways that bound morphine is chiefly, if not entirely, in which the formula for morphine may be drawn. morphine-3-monoglucuronide dihydrate, but since None of these conveys a feeling for the gnarled and this has not been established with absolute certainty twisted shape of the molecule revealed by X-ray in all cases, and since the of examination (Lindsay & Barnes, 1955). morphine has not been completely delineated, we have, in general, preferred to retain the more METHODS OF ESTIMATION familiar term, bound morphine. It is presumed that when morphine is referred to in papers published The major problem encountered in the estimation before the existence of bound morphine was estab- of morphine in body fluids and tissues is usually lished that the various authors mean the free or concerned with the processes involved in the isola- unchanged . However, it is probable that in 230 E. LEONG WAY & T. K. ADLER some of these studies, especially when direct tests for method utilizes multiple extraction techniques under alkaloid reactants were performed on the biological varying pH conditions, the major separation of the specimens without prior purification by adsorption or impurities from the morphine being effected by the extraction, some bound morphine was also measured. more or less differential distribution of the compo- Many methods have been proposed for the estima- nents between various solvent pairs as the pH is tion of morphine in biological fluids, but no satis- systematically changed. Unfortunately, morphine is factory procedure was available prior to 1928. The difficult to extract into the organic solvent phase, methods employed since then are summarized in owing to the concomitant presence of the phenolic Table 1, together with an evaluation of their use- hydroxyl and the tertiary nitrogen groups, which fulness. A critique of the earlier methods will be confer amphoteric properties on the compound. In found in the publications by Pierce & Plant (1932) general, optimal extraction of morphine has been and Wolff, Riegel & Fry (1933). Only those methods obtained at about pH 9, which is near its isoelectric which have direct bearing on data concerning the point, using a variety of solvents such as ethyl biological disposition of morphine have been in- acetate and mixtures of with chloroform. cluded. Some recent methods for estimating More recently, Fujimoto, Way & Hine (1954) morphine for forensic purposes have been cited in proposed that the extraction of morphine be effected the text, but are not discussed because the applicabi- with n-butanol from a highly concentrated alkaline lity of these methods has been studied only to the solution. Application of the procedure to morphine extent of noting recovery values after adding mor- present in biological media yielded good recoveries phine to tissue; the methods have not been actually along with only negligible quantities of naturally employed for investigating the fate of morphine. occurring interfering substances. Most of these forensic methods are usually only Ion-exchange and adsorption chromatography semi-quantitative but some of them appear to be have been developed for the assay of the crude drug sufficiently sensitive for estimating small amounts of and its galenical preparations (Brochmann-Hanssen, morphine in biological fluids. Biological methods for 1955), but their use in the separation of morphine estimating morphine have also been reported, but from animal material has been limited (Achor & such procedures lack the precision and sensitivity of Geiling, 1954; Tompsett, 1960). In a preliminary chemical methods (Forst & Deininger, 1949; report, Stewart, Chatterji & Smith (1937) gave pro- Fichtenberg, 1951; Janssen & Jagenau, 1956). Other mising indications of using adsorption on kaolin, but references with respect to biological procedures may the details have not been forthcoming. Stolman & be obtained from Schaumann's monograph (1957), Stewart (1949) subsequently developed the method which also includes methods for estimating morphine of adsorbing the morphine on to a column of in opium. A comprehensive list of micro-chemical magnesium trisilicate (Florisil), and fractionally tests for morphine and its surrogates has been com- eluting the morphine with methanol. Other studies piled by Clarke (1959). (Oberst, 1938-39; Fischer & Chalupa, 1950; Fischer Most of the methods for estimating morphine & Goll, 1950) demonstrate the purifying power of follow a general pattern in that the morphine is re- adsorbents. Paper chromatography methods for the covered from the biological fluid by processes that separation of morphine from its contaminants have selectively separate the compound from its con- been reported by many groups (Goldbaum & taminants. The purified morphine is then measured Kazyak, 1952; Breinlich, 1953; Jatzkewitz, 1954; by some general reaction for a or alkaloidal Kaiser & Jori, 1954; Mannering, Dixon, Carrol & substance. The reliability of the procedure is, Cope, 1954; Curry & Powell, 1954; Siebert, Williams therefore, chiefly dependent upon the adequacy of & Huggins, 1954; Woods, 1954; Schultz & Strauss, the purification processes, which usually involve 1955; Kariyone & Hashimoto, 1957; Vidic, 1955, extraction, adsorption or precipitation of morphine 1957; Way, Kemp, Young & Grassetti, 1960). A without actual isolation of the crystalline product. suitable solvent system for application of the The isolation and separation techniques used are of counter-current distribution technique has also been two general types: (a) the extraction procedures and developed; this system consists of phosphate buffer, (b) the adsorption methods. The extraction proce- pH 6.6, and 10% butanol in chloroform (Young & dures involve the partition of morphine between Way, unpublished data). different solvent phases and have, in general, been Once the morphine has been obtained in a form modifications of the classic Stas Otto method. The more or less free from impurities, a variety of chemi- BIOLOGICAL DISPOSMON OF MORPHINE AND ITS SURROGATES. 231

TABLE I METHODS FOR THE ESTIMATION OF MORPHINE IN BIOLOGICAL FLUIDS

ISample Apia ln Reference Purification Measurement Sample size Alica- Blank Remarks (g or ml) bility (Mg) (Mg/mI)

RADIOACTIVE METHODS Adler, Elliott & Addition of Isotopic dilution Plasma 0.4 0.05 Nil Sensitivity can be enhanced George (1957) carrier morphine, of crystalline deri- 0.002-0.01 0.1 by Increasing sample size extractions and vative Tissues 1.0-1.5 0.1 conversion to dinitrophenyl ether Mule &Woods Extractions Radioactivity of Brain 0.1-1.0 0.05 Nil Probably has high degree (1960) extracted alkaloid of specificity, but assess- ment not given Achor & Geiling None Radioactivity of Tissue ? Nil Method non-specific, direct (1953) plated tissue Urine plating sublect to greater homogenate or errors, insufficient detail urine Miller & Elliott None Radioactivity of Tissues 0.025-0.05 0.1-0.2 Nil Non-specific (1955) plated pepsin digest of tissue Elliott, Tolbert, None Radioactivity after Urine 15 0.01-0.1 Nil Method non-specific for Adler & combustion to Faeces 10-15 morphine but useful for Anderson (1954) C02 and precipi- Breath 3-6 min. tracing metabolites tation as BaCO3 sample

PHOTOMETRIC METHODS Way, Kemp, Extractions With Folin- Tissues 1 10 0.3 Rapid Young & Ciocalteu Grassetti (1960) Young & Way Extractions With Folin- Tissues 1 2 0.2 Modification of earlier (unpublished Clocalteu method data) Siminoff & Extractions and With methyl Blood 10 2 0.02 Combined method adopting Saunders (1958)a conversion to orange Tissues 10 2 0.02 extraction procedures of nitrobenzoyl ester Fujimoto, Way & Hine (1954) and Woods, Cochin, Fornefeld &Seevers (1954) Szerb, MacLeod, Precipitation, With Folin- Plasma 5 5 0.2 Other phenolic narcotics Moya & McCurdy extraction, Ciocalteu Tissues 1 10 0-1.2 can interfere (1957) a adsorption on resins Feldstein & Extractions and As nitroso- Urine 50 250 2 Bound blanks considerably Klendshoj (1956)a nitrosation morphine higher; other phenolic narcotics interfere Fujimoto, Way & Extractions With silico- Urine 15 45 0.3 Fairly rapid, adaptable for Hine (1954) a molybdic acid ultraviolet analysis Woods, Cochin, Extractions and With methyl Plasma 5 15 0.04 Concentration and optical Fornefeld & conversion to orange Urine 5 20 0.3 density non-linear. Bound Seevers (1954) a nitrobenzoyl ester Faeces 4 80 2 morphine blanks not stated Tissues 1.5 10 0.4-2 Guarino (1946) Extraction Oxidation with iodic acid and complexing with ferric chloride

a Adapted for measuring bound morphine. 232 E. LEONG WAY & T. K. ADLER

TABLE I METHODS FOR THE ESTIMATION OF MORPHINE IN BIOLOGICAL FLUIDS (concluded)

I Applica- BlankReak Reference Purification Measurement Sample ~~~~~~~~~Samplesize Remarks (g or ml) bilitl(ttg)y(g (gmIAglml)

PHOTOMETRIC METHODS (continued)

Oberst (1938-39) Extraction, With molybdo- Urine 50 150 0.8 Amino- would adsorption on phosphotungstic interfere permutit acid

Mull (1937) Precipitation With phospho- Blood 1 5 1 Reliability not sufflciently from deprotein- molybdic acid established Ized blood

Pierce & Plant Diazotization Urine 60 2 000 25 Low sensitivity but (1932) Extractions with sulfanilic Faeces 10 (dried) reasonably accurate with lglauer (1949) acid fresh specimens

Fleischmann Deproteinatlon With phospho- Blood 5 20-2 000 ? Not reliable (1929) with uranyl nitrate molybdic acid

OTHER METHODS

Paerregaard Extractions, paper Polarographic Urine 5 5 Nil Dihydromorphinone (1957)a chromatography, after nitrosation Blood 5 or 10 10 Nil Interferes Mllthers (1958) elution

Achor & Geiling Extraction, ad- Ultraviolet Reported to be applicable (1954) sorption on lon- spectrophoto- for tissue but no data exchange resins, metric presented elution

Deckert (1936a, 1936b) Endo & Kato (1937,1938) Extraction, pre- Nephelometric Urine 25-100 30-100 Semlquantitative Oberst cipitation as (1938-39) molybdate- Schirm (1940) vanadate complex lglauer (1949) Oettel (1950)

Balls & Wolff Extraction, pre- Gravimetric Urine 100-200 8 000 1 000- Low sensitivity, but reason- (1928) cipitation with 2 000 ably accurate Wolff, Riegel & silicotungstic Fry (1933) acid

Ikeshima (1935) Tissue digestion lodometric Blood 5 600 ? Low sensitivity, reliability Kabasawa with papain ex- Brain 500 not sufficiently established (1933-35) traction

To & RI (1938) Extractions, pre- Titration of Urine 1 000 10 000 ? Not reliable - reported cipitation with HI bound to values too high iodine-potassium morphine Iodide (Wagner's reagent)

a Adapted for measuring bound morphine. BIOLOGICAL DISPOSMON OF MORPHINE AND ITS SURROGATES. 1 233 cal, physical and biological methods is available 1954), but it has a relatively low order of sensitivity. for the identification, estimation and characteriza- A polarographic procedure has been developed which tion of the morphine. The chemical methods consist appears to be sufficiently sensitive for morphine in of qualitative and quantitative tests of crystalline concentrations as low as 1 ,ug/ml (Paerregaard, salt formation, precipitation reactions, and various 1957a; Milthers, 1958). colour reactions. The formation of crystalline salts Tracer techniques have been applied to 14C mor- is highly desirable but biological media generally phine labelled either in the N-methyl position yield neither the quantity nor the degree of purity of (Rapoport, Lovell & Tolbert, 1951; Andersen & the morphine residue required to accomplish this Woods, 1959) or randomly by cultivation of the task. Nephelometric methods of measuring the poppy in an atmosphere of 14CO2 (Achor & Geiling, amount of precipitation of morphine as an insoluble 1954). Earlier measurements using isotopic mor- complex have been used, but such methods have not phine were largely confined to determination of been perfected to the degree attained with colori- radioactivity (Achor & Geiling, 1953; Elliott,Tolbert, metric procedures. The latter have been most Adler & Anderson, 1954; March & Elliott, 1954; popular because many colour reactions are possible Miller & Elliott, 1955). While this is extremely involving the functional groups present in morphine. useful in providing information on the routes of The aromatic hydroxyl on the 3-position can be de- excretion and, possibly, on the metabolic pathways tected by a variety of phenolic reagents with varying of morphine, measurements of radioactivity do not degrees of sensitivity and precision. A coloured give specific information as to concentrations of derivative of morphine can be formed by coupling morphine in various tissues. More recently Adler, the phenol group with diazotized sulfanilic acid. Elliott & George (1957) were able to detect concen- The basic nitrogen group offers the possibility of trations ofmorphine as low as 0.028,g/ml in plasma, utilizing the general coupling reaction of certain using an isotope dilution technique. Morphine acidic dyes with organic bases to form an extractable N-14CH3 in biological media was determined by complex of morphine. These colour reactions are adding carrier morphine to the sample to be analysed. generally quite sensitive and easily performed, but After recovery and purification of the isotopically are not specific for morphine alone; hence, the diluted morphine N-14CH$ by conventional pro- extracted morphine must be in a highly purified state cedures, it was converted to crystalline dinitrophenyl- before the reaction is carried out. Most of the morphine-N-l"CH3 and the specific activity deter- methods summarized in Table 1 are based on the mined. adaptation of these colour reactions to the quantifi- The purity of the crystalline derivative was ascer- cation of morphine. tained by its powder X-ray diffraction pattern, thus Physical methods have the inherent advantage of making this method one of the most sensitive as well not requiring the chemical alteration of the morphine as specific for determination of morphine. molecule for its estimation, characterization or The chief deterrent to the development of a suit- identification. The subject has been recently able method for the estimation of morphine in reviewed (Farmilo & Levi, 1953; Farmilo, Oest- biological media may be attributed to the fact that reicher & Levi, 1954). A wide selection of methods is microgram quantities of morphine need to be mea- available for the purified material, utilizing polaro- sured in the presence of a large amount of naturally graphic (Paerregaard, 1957a; Milthers, 1958), occurring biological substances, which may react as electrophoretic (Spengler, 1958), fluorescence morphine. Most of the methods we have cited (Nadeau & Sobolewski, 1958), infra-red (Seagers, generally measure some blank material which gives a Neuss & Mader, 1952), ultraviolet (Elvidge, 1940; morphine test. This greatly limits the precision of Bradford & Brackett, 1958), optical rotation the method, especially at the lower limits of sen- (Soehring & Frahm, 1949) and X-ray diffraction sitivity. With isotopic procedures and apparently (Gross & Oberst, 1947) techniques. Unfortunately, with a recent polarographic method (Paerregaard, most of these methods are not suitable at the present 1957a; Milthers, 1958) blank contributions need not time for measuring morphine in biological fluids. be considered. The sensitivity of the methods which The ultraviolet absorption spectrum has been used we have listed in Table 1 does not necessarily agree with some success for estimating and identifying with what is claimed by the various authors, morphine in biological media (Biggs, 1952; Gold- inasmuch as we are reporting a concentration which baum & Kazyak, 1952; Fujimoto, Way & Hine, we consider will give a precision of approximately 234 E. LEONG WAY & T. K. ADLER i 15% after making allowances for the blank con- However, Rapoport and his colleagues (personal tribution. communication) have recently found that conditions The absolute specificity of the methods for deter- necessary for complete hydrolysis of bound mor- mining morphine listed in Table 1 has not been phine often need to be much more rigorous than established beyond doubt. However, there can be no those proposed in these earlier investigations. question that morphine is excreted in the urine since Furthermore, if more than one form of bound Stevenson & Rapoport (1955) reported the isolation morphine exists, as some authors claim (Thompson and crystallization of morphine from the 24-hour & Gross, 1941; Woods, 1954), until the postulated urine of six patients on morphine. It is quite prob- products are isolated in purified form for proper able that most of the methods listed show a high study it cannot be stated with certainty that the degree of selectivity for morphine. Those procedures absolute amount of morphine measured after hydro- which we considered non-specific are so indicated lysis represents the total amount of bound morphine under the column " Remarks " in Table 1. Although present. It should be noted, however, that recent the specificity of some of the procedures has not studies indicate that only one form of bound mor- always been fully evaluated, the uniformity of the phine-namely, morphine monoglucuronide-is pre- results on the excretion of morphine obtained by the sent in the urine in significant quantities (Fujimoto & various investigators with divergent techniques gives Way, 1957) and that hydrolysis of this compound a strong indication that these methods are essentially can be effected under conditions which do not cause valid for measuring morphine. While it is conceiv- decomposition of the liberated morphine (Gross & able that some biotransformation products of Thompson, 1940; Woods, 1954; Fujimoto & Way, morphine might retain the characteristics of a phenol 1957). as well as those of an alkaloid and thus react as morphine (e.g., as normorphine), the available evi- ABSORPTION dence indicates that any contribution of normorphine Considering the amount of work that has been or other metabolic products to the readings of carried out on morphine, there is a surprising dearth morphine by the above methods is very minor. of quantitative studies on the absorption of mor- Nevertheless, one should bear in mind that minor phine. Numerous citations from an earlier review contributions from alkaloidal phenols may exist. (Krueger, Eddy & Sumwalt, 1941) attest that It should be noted that while many of these morphine can enter the mammalian body by any methods are applicable for studying the number of routes. In addition to the usual means for of the morphine, this does not necessarily mean that obtaining morphine effects by parenteral administra- the method is applicable for forensic purposes, tion, morphine is reported to have produced sys- especially when the substance ingested is unknown. temic effects after being placed in contact with the In such instances some closely related narcotic lining or walls of the alimentary canal, respiratory congeners or aminophenol may be mea- tract, vagina, urethra, prepuce, bladder, abraded sured as morphine. Also, with the advent of the use skin, ear, conjunctival sac and brain. However, a of nalorphine for the treatment of acute morphinism, comparative study of the efficiency of morphine one must be cognizant of the fact that nalorphine absorption by diverse routes has not been exten- gives many of the reactions of morphine and is not sively investigated. From the vast amount of easily separated from the latter by general purifica- pharmacological, toxicological and clinical data tion procedures. published, it is possible to conclude that morphine is The more recent methods, as noted in Table 1, well absorbed by all species when injected paren- have been applied also to the estimation of bound terally, but is erratically absorbed when given by morphine. This is obtained by difference after mouth. estimation of the total (free plus bound) morphine In man the absorption of morphine appears to be present in a given sample after subjecting it to heat fairly prompt after parenteral administration. It is and strong acid. It has tacitly been assumed that well known from clinical usage that the effects of any morphine that is bound, combined or con- morphine can appear within a few minutes after jugated is completely released without decomposition hypodermic administration, but judging from animal under conditions used for hydrolysis of the bound studies about one hour is probably required to morphine. This assumption was based on the studies attain peak levels of the compound. There is a need of Gross & Thompson (1940) and of Oberst (1940). for reliable quantitative studies on man concerning BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 235 the rate of appearance of morphine in the blood (Fleischmann, 1929). Peak blood levels were noted after its administration. Only recently have some 1 hour after intraperitoneal administration of objective data been reported concerning the rate of 20 mg/kg; 30-minute levels were almost as high absorption of morphine. Elliott, Tolbert, Adler & (Siminoff & Saunders, 1958). Anderson (1954) administered 10-15 mg of morphine- In the rat, detectable levels of 14C in various parts N-methyl-l4C intramuscularly and found measurable of the central nervous system were found within amounts of 14CO2 in the breath within 10-15 minutes, 15 minutes after hypodermic injection of 5 mg/kg 14C- with the peak rate of excretion occurring between labelled morphine and peak levels of radioactivity, 30 and 90 minutes. equivalent to between 0.2 and 0.6 ,ug/g tissue, occur- Absorption of morphine by mouth appears to be red at 60 minutes (Miller & Elliott, 1955). Over 90% poor. Isbell & Fraser (1953) found that orally of the radioactivity disappeared from the subcuta- administered morphine produced little, if any, neous injection site within 1 hour after injecting pupillary constriction and respiratory depression in morphine-N-methyl-14C. Absorption was equally former addicts. Beecher, Keats, Mosteller & rapid following injection into the popliteal space, Lasagna (1953), in assessing analgesia, were unable and was not markedly altered by vasopressin, ACTH to distinguish between a placebo and 10 mg of or adrenalectomy (Adler, Elliott & George, 1957). morphine orally, but found that 0.6 g of Pretreatment with an antihistamine (mepyramine) produced analgesia greater than that of the placebo. or depletion of tissue histamine with compound Comroe & Dripps (1948) reported minimal side- 48/80 (a condensation product of p-methoxyphenyl- effects in normal volunteers given 15-30 mg of ethylmethylamine and formaldehyde) enhanced pro- morphine by mouth. Walton & Lacey (1935a, nouncedly the subcutaneous absorption of morphine 1935b) administered morphine sublingually in doses as measured by blood levels of morphine 10 minutes ranging between 20 and 120 mg and noted negligible after injection (Milthers & Schou, 1958). The expla- pharmacological effects. nation was offered that morphine, being a histamine In contrast to the dearth of objective evidence on releaser, causes self-depression of absorption. This the absorption of morphine in man, ample data to conclusion was based on the belief that any substance this effect exist for animals. In monkeys, a level of releasing tissue histamine would tend to inhibit its 6 ,ug/ml of the free alkaloid was attained in plasma own absorption after subcutaneous administration, within 30 minutes after subcutaneous administration since histamine given locally was found to decrease of 30 mg/kg and the maximum level of 8 ,tg/ml the absorption of (Schou, 1958). The occurred after 120 minutes. The biological half-life concept of self-depression of absorption was exten- was reported to be about 3 Y2-4 hours (Mellett & ded to include 5-hydroxytryptamine liberators Woods, 1956). (Milthers, 1959), since enhanced absorption of mor- As to dogs, 25% and 42% of the alkaloid were phine was noted also after pretreatment with the recovered after 30 minutes from the injection site in serotonin antagonist, BOL 148, and with depletion of two dogs given 100 mg/kg morphine sulfate intra- tissue 5-hydroxytryptamine. It was pointed out muscularly (Hatcher & Gold, 1929). A peak plasma further that newborn animals, being resistant to the level of 7 ,tg/ml was attained about 45 minutes after action of histamine and 5-hydroxytryptamine, should subcutaneous administration of 30 mg/kg morphine, absorb morphine more rapidly than adult animals. but the same dose given orally yielded barely de- As evidence it was noted that blood levels of mor- tectable or no plasma levels of morphine (Cochin, phine 10 minutes after subcutaneous administration Haggart, Woods & Seevers, 1954). In the same com- of 100 mg/kg morphine hydrochloride were nearly munication, it was reported that absorption of 6 times higher in 12-day-old rats than in adult morphine by the intramuscular route does not differ animals (Milthers, 1960) and these findings were greatly from that found with subcutaneous adminis- related to the increased susceptibility of younger tration, but the former route yielded an earlier and animals to morphine. With respect to the latter greater peak. point, Kupferberg & Way (unpublished data) Rabbits given doses of morphine varying from maintain, on the basis of their disposition studies on 4 to 10 mg/kg exhibited high levels in the blood morphine, that a decreased blood-brain barrier to within 35 minutes after intramuscular injection morphine rather than enhanced absorption is the (Mull, 1937); maximum blood concentrations were primary cause for the decreased resistance of new- obtained 30 minutes after subcutaneous injection born animals to morphine. 236 E. LEONG WAY & T. K. ADLER

In the guinea-pig, morphine was found in tissues Man 15 minutes after subcutaneous administration The store of knowledge on the tissue distribution of (Keeser, Oelkers & Raetz, 1933). morphine in man is meagre. Forensic data offer In the mouse, after subcutaneous administration some qualitative information but lack of precise in the hind leg, approximately 50% of the injected knowledge of dosage levels, time relationships, dose disappeared after 15 minutes and over 90% routes of administration, and complications owing after 60 minutes (Way, Young, Kemp & Johnson, to possible simultaneous ingestion of other com- unpublished data). Within 15 minutes after intra- pounds, etc., makes it difficult to view the information peritoneal injection of approximately 10 mg 14C- in proper perspective. A satisfactory method of labelled morphine per kg, mouse blood contained, sufficient sensitivity for the measurement ofmorphine per millilitre, over 5 % of the dose of 14C and concentrations in blood well below 0.5 ,mg/ml still appreciable levels of radioactivity were present in needs to be developed. Even if one neglects animal other tissues at that time (Achor & Geiling, 1954). data indicating that morphine rapidly leaves the blood and concentrates in certain tissues, the dis- DISTRIBUTION tribution of a 20 mg/70 kg dose of morphine in body Information on the distribution of morphine has water would yield a blood level of only 0.4 ,ug/ml. It been obtained in several species. The earlier data are is known that the blood levels of morphine at any concerned only with the free alkaloid; more recent time after an intravenous dose of 20 mg are less than work includes results on bound morphine. Most 1 ,Ag/ml (Milthers, 1958). Measurable levels of extensive information is available on the dog and rat, morphine are found in the urine, bile, faeces, saliva but considerable data are available as well on and sweat and these are discussed under the section monkeys, cats, rabbits, mice, and guinea-pigs. on excretion. In general, free morphine, like most basic , rapidly leaves the blood and concentrates in tissues, Monkey particularly parenchymatous tissues. Kidney, lung, In monkeys after subcutaneous administration of liver, and spleen show a predilection for the drug, 30 mg/kg of morphine peak plasma levels of free with by far the highest concentration being found in morphine (about 8 ug/ml) were obtained 1 /2-2 hours the kidney. Certain endocrine organs, such as the after injection, the biological half-life being approxi- adrenal, thyroid and pancreas, also appear to con- mately 3 Y2-4 hours; bound morphine reached a centrate the drug. While skeletal muscle may show a peak in about 2 hours (26 ,ug/ml) and had a half-life somewhat lower level of morphine, it generally of about 6 hours. The distribution of free morphine accounts for the major fraction of the drug left in the 90 minutes after injection was found to be highest in animal body, except for what may be present in the adrenal, lung, pancreas, and kidney, the con- bladder urine and gall-bladder bile. Extensive centration range being between 11 and 16 ,ug/g. cumulation of morphine in tissues does not occur; Brain, spinal cord, and cerebrospinal fluid failed to tissue levels fall to quite low levels within 24 hours show a detectable level of free morphine with a after the last administration. Relative to the dose method sensitive to 5 ,tg/g. After 4 hours only the administered the amount of the drug found in the adrenal (9 ,tg/g) and bile (35 ,tg/ml) showed ap- brain is extremely minute and the concentration in preciable concentrations of free morphine. Bound the central nervous system is but a fraction of that morphine was found in high concentrations in the attained in most other organs. It may well be that bile (4120 ,ug/ml) and the kidneys (16 pg/g) at the wide range in toxicity of morphine found among 4 hours (Mellett & Woods, 1956). the different animal species is related to the ability of A comparison of the distribution of morphine in the compound to gain access to the central nervous four non-tolerant monkeys and in five monkeys system in the various species. maintained on 15 mg/kg morphine injected sub- Bound morphine, which has relatively low pharma- cutaneously twice a day for nearly a year indicated cological activity, is found in highest concentra- that tolerant monkeys did not differ greatly from tions in organs concerned with its excretion-namely, non-tolerant animals. With both tolerant and non- the kidney and gall-bladder. The bound morphine tolerant monkeys, high concentrations were found found in tissues is very probably identical with the in the adrenals and in the bile at 90 minutes and morphine-3-monoglucuronide isolated from urine 4 hours and no morphine was detected in the brain, and bile. spinal cord, cerebrospinal fluid, skeletal muscle, or BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 237

TABLE 2 TISSUE DISTRIBUTION OF MORPHINE IN DOGS AFTER ADMINISTRATION OF VARIOUS DOSES 1111I Dose infused 375 SC a 37.5 SC 37.5 37.5 SC 30 IV b 30 IV 30 SC 30 SC 30 30 SC (mg/kg) IV for SC SC 5 hours Time after ratiois- 0.6 4 4 24 24 1.5 4 1.5 1.5 4 4 (hours) No. of animals 1 6 6d 6 61 1 1 1d 1

g of drug per g of wet tissue Brain 230 9.8 7.8 7.5 3.2 5 3 6 5 Blood 50 5.3 5.9 2.2 3.6 5 5 c 2 4 2 Kidney 810 0.67 c 0.77 c 0.27 c 0.29 c 68 9 c 25 55 12 15 Liver 160 18.5 14.8 4.7 8.2 5 1 c 4 11 3 8 Spleen 77 7 c 20 33 9 10 Lung 230 16.9 13.9 4.7 5.0 30 5 c 12 20 5 12 Heart 110 11.3 8.2 3.4 6.4 14 3 c 6 8 6 Muscle 360 11.3 6.4 2.9 4.8 27 7 c 5 10 2 Skin 90 Bone 370 Pancreas 25 32 7 6 Adrenals 10 19 6 8 7 e 14 e6 GI tract 480 4.7c 74c 2.3c 46c 8f 32f 9f GI contents 205 c 1.7 c 8.1 1.8 C 7.6 c Bile 7 1Oc 340 52 27 29 Urine 896 c 16.1 17.1 20.9 c 32.0 c Percentage recovery 39

Wolff et al. (1933) Plant & Pierce (1933) Woods (1954)

a SC = subcutaneous administration. c Total quantity recovered in mg. e Jejunum. b IV = intravenous administration. d Tolerant. f Colon. spleen. The small intestine of tolerant animals had characteristics of the distribution of morphine in the higher levels of morphine than did that of non- dog are: (a) its rapid disappearance from the blood; tolerant ones at 4 hours (Mellett & Woods, 1956). (b) the high levels attained in the kidneys; (c) the relatively Dog large amounts recovered in skeletal muscle; and (d) the low levels attained in the The tissue uptake of morphine in dogs has been brain. studied over a wide dosage range. Since a direct com- Evidence that morphine disappears rapidly from parison of the various studies is not always possible blood and can be recovered to a large extent in because of the many differences in experimental con- skeletal muscle was furnished some thirty years ago ditions, the results obtained in three different labo- by Hatcher & Gold (1929). Using a procedure for ratories have been grouped in Table 2. General estimating morphine which was rather insensitive 238 E. LEONG WAY & T. K. ADLER

but apparently reliable for the high doses used, they variation was such that it is difficult to attach any were able to demonstrate that less than 2% of an special significance to the findings (Plant & Pierce, injected intravenous dose of 50-100 mg/kg of 1933). morphine sulfate was present in the blood of animals Woods (1954), using a dose of morphine (30 mg/kg exsanguinated after 30 minutes. Analyses of muscle subcutaneously) close to that of Plant & Pierce, 30 minutes after intravenous administration of found high concentrations of morphine in the kid- 50 mg/kg and 100 mg/kg in two animals yielded neys, pancreas, and spleen at 90 minutes. Somewhat recoveries of morphine which accounted for 47% lower levels were found in the lung and adrenals and 22%, respectively, of the injected dose. Mor- and even lower levels in cardiac, skeletal, and phine leaves the muscle rapidly, only 2% of the smooth intestinal muscle. The concentrations in the dose being present in an animal 2 hours after brain and blood were below the sensitivity of the receipt of 100 mg/kg infused intravenously for one method (5 ,g/g). The kidneys also had extremely hour. In the same animal the morphine in the liver, high concentrations of bound morphine; the liver heart, kidneys, lungs, spleen, stomach, intestines and had somewhat lower but still high concentrations of pancreas accounted for less than 1.0 % of the the bound metabolite. However, the concentration injected dose. of bound morphine in both organs was still far Wolff, Riegel & Fry (1933) injected morphine below that in gall-bladder bile. After 4 hours the intravenously (1111 mg/kg base equivalent) into a total morphine values in all tissues had decreased dog under ether anaesthesia over a 5-hour period. considerably and after 12 hours the concentration of Thirty-five minutes after the injection the various total morphine was less than 5 ,ug/g. After adminis- organs were analysed for the free morphine content. tration of 30 mg/kg morphine intravenously, brain The estimated recovery of the dose administered was levels of free morphine were barely detectable or not 39%, and more than half of this quantity could be measurable although the kidney, spleen, lung, and accounted for by the morphine in skeletal muscle skeletal muscle levels (named in order of decreasing alone. As indicated in Table 2, the kidney had the concentrations) were roughly twofold to fivefold highest concentration, but appreciable levels were higher than after subcutaneous administration. At also present in the gastro-intestinal tract, skeletal 1 hour the concentration of free morphine in the muscle and bone. It is of interest to note the rela- plasma of these animals was threefold greater and tively high brain levels of morphine which were that of bound morphine 30-fold greater than the attained at this high dosage level. corresponding concentrations in erythrocytes. Plant & Pierce (1933) studied the distribution in The distribution of N-methyl-4C-labelled mor- tolerant and non-tolerant dogs 4 and 24 hours after phine in the white and grey matter of the cerebral subcutaneous administration of 50 mg/kg of mor- cortex was determined after administration of phine sulfate. Their data, recalculated in terms of 2 mg/kg subcutaneously. At 4 hours the morphine the free base, are summarized in Table 2. High levels were 0.23 ,tg/g in grey areas and 0.13 ,ug/g in concentrations were found in the kidney. Although white areas. At 16 hours the level in both areas was the data on the kidney were reported in terms of total about 0.05 ,tg/g (Mule & Woods, 1960). The gross milligrams recovered rather than on a concentration concentration ofmorphine in dog brain appears to be basis as with most other organs a reasonable appro- little affected by the simultaneous subcutaneous ximation of kidney concentrations can be obtained administration of nalorphine (3 mg/kg) with mor- by assuming the organ to weigh 30 grams. The brain phine (30 mg/kg) (Woods, 1957). levels appear high when compared with the distribu- Woods (1954) also studied the distribution of the tion characteristics of morphine in other species and drug in animals made tolerant to morphine, 30 mg/kg. with more recent data on the dog (Woods, 1954, Although not specifically stated, it is presumed that 1957). Since Plant & Pierce (1933) claimed good the degree of tolerance and its duration in these reproducibility only down to 15 ,ug/g (actually animals were comparable to that achieved in the 0.3 mg/20 g tissue), the brain values they reported do bitches used in a previous study (Cochin, Haggart, not fall within the limits of adequate precision. We Woods & Seevers, 1954). The distribution of free feel, therefore, that these values are, at best, accept- and bound morphine was compared in three non- able only as maximum values. Tolerant and non- tolerant and three tolerant animals, using one animal tolerant animals showed some difference in tissue from each group at three different time intervals after levels, but the differences were not striking and 30 mg/kg of morphine injected subcutaneously. At BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 239

12 hours tissue levels of morphine were too low for not only from biliary free morphine secreted in the detection, but at 90 minutes and 4 hours the concen- intestine and reabsorbed, but also from biliary trations of free and bound morphine were found, in bound morphine, since the latter is readily hydro- general, to be higher for most tissues in tolerant than lysed in vivo (Blaney, Bloom & Woods, 1955). Thus, in non-tolerant animals; brain levels in both groups with chronic administration, mobilization of the were either below or barely exceeded the sensitivity of biiary stores of free and bound morphine would the method (5 ,g/g). Woods concluded that these augment the effect of injected morphine and lead to data do not provide acceptable evidence that altered higher tissue levels. distribution is responsible for the development of tolerance to morphine. To this we readily agree, but Rabbit we would add that inasmuch as some possible altera- Concentrations of tions in the distribution of morphine in rabbits after ad- morphine within the ministration of 20 mg/kg intraperitoneally were central nervous could not have system been detected determined by Siminoff & Saunders (1958). Ap- by the methods used, the study also does not refute preciable amounts of both free and such an idea. bound morphine were detected within 30 minutes in brain, blood, liver, Woods (1954) minimized the differences noted and kidney, and peak levels occurred at 1 hour. The between the tolerant and the non-tolerant dogs and kidney had high concentrations of both free and offered the explanation that " such differences are bound morphine. Brain levels at 1 hour were not of such magnitude that they cannot be explained 1.8 ,tg/g. Animals rendered tolerant to the respira- on the basis of quantitative differences in the vascular tory effects of 20 mg/kg of morphine by daily responses of tolerant as compared with non-tolerant injection of increasing doses for 100 days yielded animals resulting in altered mobilization from the tissue levels comparable to those of non-tolerant site of administration and differences in the vascular animals 1/2, 1, 2, 4, and 8 hours after intraperitoneal supply to the several tissues." The meaning of the administration of 20 mg/kg of the drug. Half of the latter part of the explanation is not quite clear to us intravenous dose of morphine hydrochloride since, in an earlier experiment, after intravenous (1-2 mg/kg) disappeared from the blood within a administration of morphine, he postulated that the few minutes after injection; the levels at 20 minutes high levels of morphine found in certain tissues were were approximately 1 ,ug/ml or less (Milthers, the consequence of a marked vasodilatory effect and 1958). pooling of blood in these organs. If we assume that Guinea-pig the vasodilatory effect of morphine is greater in non- tolerant than in tolerant dogs, then this should result In the guinea-pig studies were carried out after in higher rather than lower tissue morphine levels in injecting 400 mg/kg of morphine subcutaneously. the non-tolerant animals, but such is not the case. Morphine was found in all organs examined except We would like to offer an alternative explanation the muscles 16-24 hours after injection. The highest for the higher organ levels of morphine found in levels were found in the kidney and liver. The levels tolerant than in non-tolerant dogs. Since the in the blood and skeletal muscle were lower. The tolerant animals received daily injections of mor- brain was the organ with the lowest concentration of phine, it appears more plausible to suppose that morphine. In animals which had received morphine residual morphine may be present in this group. daily for 3-6 weeks, morphine was still detectable in That such may be the case is apparent from the the kidney and blood 24 hours after the last injection biliary excretion data of Woods (1954), where (Keeser, Oelkers & Raetz, 1933). significant concentrations of morphine in the free and especially in the bound form were found in Rat the gall-bladder bile even 72 hours after injection. Depending on the route of administration blood While this finding was obtained in a non-tolerant levels of morphine in rats reach a peak value soon animal, measurements in tolerant animals (studied after administration which is followed by a more or only up to 12 hours) yielded biliary levels of total less rapid fall. With the exception of the central morphine comparable to, if not higher than, those nervous system, the partition between blood and observed in the non-tolerant animals. In the tissues is largely in favour of tissues and, as equili- tolerant animals there would be a residual free brium is approached, this is reflected by high tissue morphine pool which receives sizeable contributions and low blood levels. 240 E. LEONG WAY & T. K. ADLER

After intravenous administration of 75 mg/kg of uniform distribution of the drug in the animal, morphine sulfate, blood levels fell rapidly from a whereas other organs show a selective preference for level of 27 ,ug/ml at 10 minutes to 16 ,ug/ml at morphine. 60 minutes and to 5 ,ug/ml at 180 minutes (Szerb & Age greatly influences the uptake of morphine by McCurdy, 1956). In rats given 150 mg/kg morphine the central nervous system. Sixteen-day-old rats subcutaneously blood levels of free morphine fell show brain levels that are roughly threefold higher from 19 ,ug/ml at 90 minutes to less than 3 ,ug/ml at than those of 33-day-old animals after either intra- 240 minutes. During the same interval, levels of peritoneal or intravenous injection of comparable free morphine in most other organs were generally doses (Kupferberg & Way, unpublished data). As several-fold higher than the blood levels (Woods, might be surmised, higher levels of free morphine in 1954). Plasma levels of free morphine 60 minutes the brain are attainable by intravenous administra- after injection of 2 mg/kg of labelled morphine, sub- tion of high doses of the compound than by other cutaneously or intrapopliteally, averaged 0.08 ,ug/ml routes. With an intravenous dose of 75 mg/kg, in Sprague-Dawley rats and 0.10 u.g/ml in Long- which produced almost immediate catalepsis in Evans rats (Adler, Elliott & George, 1957). Sprague-Dawley rats, the brain level at 15 minutes Levels of bound morphine in the blood fluctuate was 13 ,ug/g, and at 40 minutes it had decreased to more widely than free morphine levels and are de- 7 ,ug/g (Young & Way, unpublished data). On the tectable in tissue after a longer period than free other hand, in male Holtzman rats receiving 75 morphine. The variations and the lag are to be ex- mg/kg morphine intravenously, Szerb & McCurdy pected since the appearance of bound morphine is (1956) found a lower and later peak, with a morphine dependent on conjugation processes, which in turn brain level of 6.2 ,ug/g at 10 minutes rising to 9.3 are subject to influences by dosage, physiological ,ug/g at 60 minutes. The latter result is rather sur- state of the animals, prolonged morphine adminis- prising, since organic bases as a general rule leave the tration, etc. No bound morphine was detected in the blood rapidly owing to rapid tissue uptake and blood 10 minutes after intravenous administration of ordinarily one would expect peak organ levels of 75 mg/kg morphine sulfate intravenously, but at morphine to occur about 10 minutes after intra- 60 minutes the level was 3.5 ,ug/ml and at 180 minutes venous administration. it was 7.8 ,ug/ml (Szerb & McCurdy, 1956). After Administration of morphine by parenteral routes subcutaneous administration of 150 mg/kg, no other than intravenous yields considerably lower bound morphine was detected at 90 minutes but at levels of the compound in the brain. With a dose as 4 hours, when no free morphine was found, the level high as 150 mg/kg injected subcutaneously, Woods of bound morphine was 23 ,ug/ml (Woods, 1954). (1954) found no morphine in the brain, using a After injection of 2.0 mg/kg of labelled morphine method with a sensitivity of 5 ,ug/g. Hosoya (1956) subcutaneously or intrapopliteally, over 95 % of the reported values of 10± 1.9 ,tg/g in brain 40 minutes radioactivity in the plasma of Sprague-Dawley rats after intraperitoneal injection of 150 mg/kg mor- (1.9 ,ug/ml) and about 85% of that of Long-Evans phine and of 5.66 ± 2.0 ,ug/g after 4.0 mg/kg. The rats (0.67 ,ag/ml) at 60 minutes represented radio- latter value is inordinately high for such a low dose activity contributed by bound morphine. Whole of morphine and is difficult to explain. The central blood levels of 14C ranged between 66 % and 78 % of nervous system was reported to contain negligible the 14C concentration of plasma, the lower values amounts of radioactivity 1 hour after subcutaneous being associated with a high haematocrit (Adler, administration of 5 mg/kg of morphine-N-methyl- Elliott & George, 1957). Szerb & McCurdy (1956) 14C (March & Elliott, 1954). However, subsequent reported that free morphine was equally distributed studies by the same laboratory using improved between plasma and erythrocytes while there was techniques resulted in measurement of morphine about 4 times more bound morphine in plasma than levels in the brain even with doses as low as 2 mg/kg in formed element. (Adler, Elliott & George, 1957) and 5 mg/kg (Miller There appears to be a considerable blood-brain & Elliott, 1955). During the interval between 30 and barrier to morphine, although the uptake of small 60 minutes (when the pharmacological effects were quantities of the drug by the organ is relatively maximal) after a 5 mg/kg subcutaneous dose of rapid. The concentration of free morphine attained morphine-N-methyl-14C in Long-Evans rats, the in the brain on a mg/kg basis is generally but a very morphine levels in various parts of the central small fraction of that to be expected assuming nervous system were between 0.2 and 0.7 ,ug/g. BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 241

These values represent maximum values for mor- that an extremely sensitive and selective response phine since only 14C levels were measured and the to morphine is exhibited by the central nervous brain was not perfused free of blood containing system. appreciable radioactivity. The spinal cord showed In contrast to the central nervous system, other the highest radioactivity, yielding levels representing tissues usually attain morphine levels higher than between 0.6 and 0.7 ,ug of morphine per g of wet those found in blood, and those organs concerned tissue; the levels in the hypothalamic area were only with the excretion of morphine, particularly the slightly lower. Concentrations in the cerebellum, kidneys, show a considerable capacity to concen- medulla, mid-brain and cerebrum were between trate the drug. With a 5 mg/kg subcutaneous dose of 0.2 and 0.4 ,ug/ g (Miller & Elliott, 1955). If calcula- labelled morphine, the renal level in terms of radio- tions of the morphine concentration are based on activity at 60 minutes was more than three times the lipid content of the tissue rather than on the higher than that to be expected assuming uniform weight of wet tissue, the cerebrum levels actually distribution of the drug and was roughly about 80 exceed those of the spinal cord (Adler, Elliott & times higher than cerebral levels. Radioautographs George, 1957). Sixty minutes after subcutaneous or taken of the kidney of the animals given labelled intrapopliteal injection of a 2 mg/kg dose of labelled morphine indicated that the majority of the radio- morphine in Sprague-Dawley or Long-Evans rats, activity present was localized in the cortex and calyx, cerebrum levels ranged between 0.04 and 0.09 suggesting concentration in the region rich in glome- ,ug/g in all but one of eight animals (Adler, Elliott ruli and tubules (Miller & Elliott, 1955). Undoubt- & George, 1957). Despite the fact that these levels edly, a considerable portion of the radioactivity is represent maximum values, they are the lowest due to bound morphine, since with a dose of 150 measurable level of morphine recorded. mg/kg injected subcutaneously the total morphine The minute amounts of morphine needed in the level in the kidney at 90 minutes represented a free central nervous system to elicit pharmacological morphine concentration of 133 ,ug/g and a bound effects can be further emphasized by the fact that the morphine concentration of 158 ,tg/g (Woods, 1954). maximal values given above may be several-fold too At 4 hours the free morphine level had decreased to high. Adler, Elliott & George (1957) have shown 65 ,tg/g and the bound morphine level had in- that specific activity studies after isotope dilution creased to 213 ,ug/g. By 12 hours the free morphine indicated that only 10-20 % of the 14C present in the level was less than 10 ,ug/g and the bound morphine tissue could be recovered as free morphine. With level was less than 15 ug/g. In contrast to in vivo higher doses of morphine the free morphine-N-14CH3 conditions, morphine is readily taken up in vitro by recovered represented 51 % of the 14C in the cereb- rat cerebral cortex slices, so that at 30 minutes the rum; the recovery was not substantially increased concentration in the slices (240 micromoles/kg) was after hydrolysis, indicating that there is practically more than twice the initial concentration of mor- no bound morphine in the brain after injection of phine in the incubating bath (10-4 M) (Bell, 1958). small doses. Moreover, since blood levels of radio- Morphine uptake by the liver is generally con- activity were more than 20 times higher than cereb- siderably less than that by the kidney, but is still rum levels in Sprague-Dawley rats and about much greater than that by the brain. With a 2 mg/kg 6.5 times higher in Long-Evans animals, any blood dose of labelled morphine in Long-Evans rats, the trapped in the central nervous system would tend to radioactivity was approximately 20-25 times higher elevate the radioactivity of the organ and a correc- in the liver than in the cerebrum (Miller & Elliott, tion for this should be applied. Analysis of the 1955; Adler, Elliott & George, 1957). Radio- residual blood content in the brain indicated that, autographs of the liver from an animal receiving on the basis of the blood concentration, the cereb- labelled morphine showed distribution of activity rum values could be reduced roughly by one-third throughout the tissue, with points of concentration in the Sprague-Dawley rats and by one-ninth in the probably at portal areas (Miller & Elliott, 1955). Long-Evans rats. Finally, if one considers that most Appreciable free morphine levels appeared in the of the "4C after injection of N-"4CH3 morphine is not liver 90 minutes after subcutaneous administration of in the neuronal elements ofthe brain but rather in the 150 mg/kg, but by 4 hours, although the levels of highly vascularized choroid plexus and ventricles free morphine in other tissues were still quite high, as judged by radioautographs of brain sections there was scarcely any free morphine left in the liver (Miller & Elliott, 1955), it becomes quite apparent (Woods, 1954). However, as might be expected from 242 E. LEONG WAY & T. K. ADLER the role of the liver in conjugating morphine, there the total morphine levels in these organs other than were substantial amounts of bound morphine the kidney were not detectable, being lower than present at this time as well as at 12 hours (Wood 5 ,ug/g (Woods, 1954). 1954). In rats made gradually tolerant to 150 mg/kg mor- The skeletal muscle appears to be an important phine in approximately a month, the blood levels of depot for morphine and its metabolites. In Sprague- free morphine were found to decrease more rapidly Dawley rats receiving 2.0 mg/kg of labelled mor- than they did in non-tolerant animals. The difference phine by subcutaneous or intrapopliteal injection, the was attributed to impaired conjugation of morphine average maximum morphine concentration in muscle in the non-tolerant animals resulting from inhibition one hour after injection was about 0.7 ,ug/g and of glycogen synthesis by the injected morphine was about tenfold higher than the cerebrum level. (Szerb & McCurdy, 1956). The same authors (op. Approximately 15% of the injected dose was re- cit.) also reported that tolerant animals were found covered from the muscle as 14C. About one-third to be hyperactive with a free morphine brain level of of this radioactivity was represented by free mor- 6.5 ,ug/g 60 minutes after injection, while non- phine, another third by the bound form and the tolerant ones were still immobile 180 minutes after remainder by a fraction which was unidentified injection with an average brain concentration of (Adler, Elliott & George, 1957). In Long-Evans 4.3 ,ug/g, and suggested that the reaction of the rats receiving 5.0 mg/kg of labelled morphine central nervous system to morphine was altered by approximately 12% of the injected 14C was present chronic morphine administration. A comparison of in muscle (Adler, Elliott & George, 1957), but with the tissue distribution of morphine in non-tolerant higher doses of morphine a higher fraction of the rats and in rats made tolerant to 150 mg/kg over an dose can be accounted for in muscle. Thus, after a unspecified period of time did not reveal striking dose of 150 mg/kg injected subcutaneously, the free differences beyond a higher concentration of mor- morphine level at 90 minutes was reported to be phine in the thyroid and spleen of the tolerant 72 ,tg/g (Woods, 1954). On the assumption that animals (Woods, 1954). skeletal muscle makes up 40% of the total body- Although vasopressin increases and ACTH de- weight, this would mean that roughly one-fifth creases sensitivity to morphine, beyond decreasing of the total dose of morphine would be accounted the bound morphine levels in plasma, treatment with for in skeletal muscle as free morphine alone. From neither substance altered the gross distribution the data at 4 hours, indicating free and bound characteristics of morphine markedly (Adler, Elliott morphine to be present in approximately the same & George, 1957), nor did pretreatment with neo- concentrations, it may be similarly calculated that stigmine (Szerb & McCurdy, 1956), SKF525A one-tenth of the total dose can still be recovered /3-ethylaminoethyldiphenylpropyl acetate hydro- as morphine in skeletal muscle at this time. chloride) (Hosoya, 1956), or nalorphine (Woods, Other tissues also show considerable ability to 1957). On the other hand, adrenalectomy resulted localize morphine. However, the ratio of bound to in increased tissue levels of morphine without im- free morphine in all tissues is generally much lower pairing the animals' ability to conjugate morphine than that in the kidneys and liver, especially during (Adler, Elliott & George, 1957). the early stages after drug administration. Organs such as the lung, spleen, thyroid, adrenals, and heart Mouse appear to take up high concentrations of morphine. The tissue distribution of 14C in the mouse after Adrenal levels of radioactivity between 30 and 60 intraperitoneal injection of morphine has been minutes after injection of 5 mg/kg of labelled mor- studied by Achor & Geiling (1956), using 14C- phine subcutaneously reached levels that were labelled morphine obtained from radioactive pop- roughly 25-fold higher than those in the cerebrum, pies. In general, all tissues studied showed maximal and considerable radioactivity was still present in the levels of radioactivity 15 minutes after injection of adrenal at 150 minutes (Miller & Elliott, 1955). 10 mg/kg. The tissue levels of 14C declined rapidly After a dose of 150 mg/kg of morphine injected sub- with time except in the case of the liver and gastro- cutaneously, the levels of free morphine in the intestinal tract where, after an initial decline at thyroid, spleen, lung, heart, and skeletal muscle at 60 minutes, the 14C levels rose to a secondary and 90 minutes were roughly one-fourth to one-half the higher peak at 120 minutes after injection. Some level of 133 ,tg/g found in the kidney. At 12 hours changes in the tissue distribution pattern of radio- BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 243

FIG. 3 KNOWN AND POSTULATED METABOLIC PATHWAYS OF MORPHINE

H CH3 N

0 ~~=O 0 0 OH /6H\ MORPHINE-5-MONOGLUCURO- MORPHINE ,CH-CH/ NIDE ,CH3 HOH H N

N- DEMETYLA4TION

Ho 0 HO 0 OH NORMORPH I N E

CH3 PSEUDOMORPHINE CODEINE

activity were noted as a result of treatment of the barrier to speculation concerning their presumed role mice with nalorphine or 5-monoamino-acridine, but in producing the pharmacological effects of mor- the meaning of this is not clear. phine. In past years the withdrawal syndrome has been attributed to the vainly sought oxidation METABOLISM product of morphine, pseudomorphine. More recently N-demethylation has been the focus of A schematic representation of known and postu- interest and the spur to much experimental work in lated pathways of morphine metabolism in vivo is attempts to correlate this metabolic pathway with presented in Fig. 3. The metabolism of morphine analgesia and with the development of tolerance. A has been of particular interest because, notwithstand- discussion of the recent hypotheses concerning ing the fact that the disposition of an appreciable N-demethylation and pharmacological effects will be fraction of the administered dose is still unknown, found in a later instalment. important roles in producing analgesia and physical dependence have been assigned at various times to Conjugation known and unknown biotransformation products. It is now known that conjugation with glucuronic However, of the several metabolic pathways in- acid is a major pathway for the detoxication of dicated above, only one-the conjugation of mor- morphine. Development of the evidence leading to phine with glucuronic acid-has been conclusively this conclusion can be traced from early experiments established in vivo by isolation of the metabolite in showing that morphine is excreted in a bound form crystalline form. No such firm foundation supports to the present knowledge of both the chemical the other pathways, although the equivocal nature of structure of bound morphine and the sequences of the evidence of their existence in vivo has been no enzyme reactions leading to its formation. '2AA E. LEONG WAY & T. K. ADLER

Before the turn of the century, earlier workers the derived morphine and the mixed melting-point (Stolnikow, 1884; Marquis, 1896) suggested the after conversion to heroin were also determined. possibility that morphine might be bound or con- Most early workers were of the opinion that jugated in the body, but the evidence was not con- bound morphine was a glucuronide. Ashdown clusive. In 1938 Endo reported that when the urine (1890) and Mayer (1899) reported that a glucuronide from morphinized rabbits was allowed to stand in was excreted in the urine after morphine administra- dilute sulfuric acid, a larger amount of morphine was tion and acknowledged that the original observation recovered than from untreated urine. The existence was reported by Mering in 1874. Mayer postulated of bound morphine as a morphine metabolite in that morphine was conjugated as glucuronide on the urine was demonstrated in a convincing fashion in basis of optical rotation measurements made on 1940 by Gross & Thompson using dogs, and shortly hydrolysed urine from patients receiving morphine. after by Oberst (1940) in human addicts. The Endo (1938) felt that morphine was conjugated with isolation of crystalline bound morphine from the glucuronic acid, but the English abstract of his urine of the dog and its identification as the glucu- original manuscript (in Japanese) cited no experi- ronide was reported by Woods in 1954, and sub- mental observations to support his conclusion. sequently from the urine of human addicts by Oberst (1941) observed a correlation between the Fujimoto & Way (1954, 1957, 1958). It has been amount of glucuronic acid excreted in the urine and suggested (Thompson & Gross, 1941; Woods, 1954) the dose of morphine. Direct proof was lacking, that more than one form of bound morphine exists, however, that morphine and glucuronic acid were but this view has not been universally accepted. It is paired with each other. Oberst & Gross (1944) of interest, therefore, to consider in some detail the prepared a bound morphine, morphine sulfuric more recent studies with respect to the nature of ether, and studied its actions and fate, but no evidence bound morphine. exists that such a derivative may be biosynthesized Gross & Thompson (1940) hydrolysed samples of from morphine. urine from morphinized dogs with one-tenth the More recently three groups of workers reported volume of concentrated hydrochloric acid in an almost simultaneously that they had obtained autoclave for 30 minutes at a pressure of 15 pounds evidence that conjugated morphine was a glucuronide per square inch (I atm.). They found an increase in (Fujimoto & Way, 1954; Seibert, Williams & phenolic substances and reported that this was due Huggins, 1954; Woods, 1954). Convincing proofwas primarily to morphine. The morphine liberated from furnished by Woods (1954), who was the first to the conjugated or bound morphine was isolated by report the isolation of bound or conjugated mor- repeated extraction and reprecipitation to obtain a phine in crystalline form from the urine and bile of yellow-white residue. This residue gave the usual dogs. Subsequently, a crystalline product was colour test for morphine and, when injected into isolated from the urine of addicts by Fujimoto & dogs, produced effects similar to equivalent doses of Way (1957) which yielded an infra-red curve identical authentic morphine. The excretion pattern of the with that obtained from the dog (Fujimoto & Way, biologically obtained morphine was similar to that 1958). of pure morphine in dogs (Gross & Thompson, The presence of morphine in bound morphine was 1940). In a follow-up study (Thompson & Gross, established by powder X-ray diffraction analysis of 1941) further evidence was obtained. Specific rota- the dinitrophenyl derivatives of the hydrolysed tion measurements of the isolated morphine matched morphine conjugate (Fujimoto & Way, 1958). those of an authentic sample. The diacetyl derivative Liberated morphine after hydrolysis was also was also made and the mixed melting-point with identified by paper chromatography (Fujimoto & pure heroin showed no change. Way, 1954; Seibert, Williams & Huggins, 1954; Oberst (1940) demonstrated the presence of bound Woods, 1954) and by mixed melting-point de- morphine in the urine of human addicts. He acidified terminations of the free base, the picrate (Seibert, the urine with one-fifth its volume of acid and after Williams & Huggins, 1954) and the diacetyl (Woods, refluxing for three hours he found an increased 1954) derivatives. amount of morphine. The identity of the latter The product conjugated with morphine was compound was established by mixed melting-point identified as glucuronic acid by various chemical and determinations and by colorimetric and nephelo- physical tests. The morphine conjugate after metric tests for morphine. The specific rotation of counter-current distribution was hydrolysed and BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 245 analysed for morphine and glucuronic content. The lysable. Woods (1954) also suggests that two forms morphine and glucuronic curves were found to be of bound morphine may be excreted in dog urine almost identical within the limits of experimental the monoglucuronide, which is crystalline and error (Fujimoto & Way, 1957). The infra-red curve possesses low water solubility, and another com- of the conjugate was found to give a very strong pound, possibly a di-conjugated morphine, which is band in the 3 ,u region, which is in agreement with the amorphous and possesses high water solubility. OH stretch frequency broadened by association to be Fujimoto & Way (1957), however, concluded that expected from a polyhydroxy glucuronide com- 3-morphine-monoglucuronide is the only bound pound. A strong absorption in the 6-7 ,u region was morphine present in appreciable quantity in the interpreted as being in agreement with the presence urine of addicts. While they did not completely of a carboxylate ion (Fujimoto & Way, 1958). The exclude the possibility that other bound bound morphine, separated by paper chromato- could be formed, they felt that any quantities graphy, on hydrolysis with acid or /-glucuronidase formed would be of a low order of magnitude. yielded a positive test for glucuronide as well as for Thompson & Gross (1941) concluded that there morphine (Seibert, Williams & Huggins, 1954). were two forms of bound morphine from following Elemental analyses were found to be consistent with the rate of hydrolysis of the bound morphine in dog the assay procedures for a conjugate of morphine urine. Upon adjusting the pH of the urine to with glucuronic acid being combined in the ratio of between 1 and 2 and heating at 100°C, morphine one to one, and associated with two molecules of appeared to be set free at a fairly uniform rate for the water (Woods, 1954). first 60 minutes, but thereafter the reaction proceed- The molecular site of conjugation of bound ed at a very slow rate. The morphine fraction liber- morphine with glucuronic acid was established to be ated by two hours of hydrolysis under these condi- at the 3-phenolic position on the basis that the tions was designated the " easily" hydrolysable phenol reagent used in the morphine determination fraction. The remainder of the bound morphine, procedure did not give the *olour with the con- which yielded morphine only after 30 minutes in the jugated material until after acid pressure hydrolysis autoclave in the presence of 5 % hydrochloric acid, (Fujimoto & Way, 1957). The ultraviolet absorption was called the " difficultly" hydrolysable fraction. characteristics of the conjugate in acid and base gave Of the two forms, the latter was present in much no evidence as to the presence of a free phenol. larger quantities in both tolerant and non-tolerant Morphine, like other phenols, shows a bathochromic dogs, but significant amounts of the " easily " hydro- shift with an increase in pH, whereas the ultraviolet lysable fraction were also found. Although it curve for morphine conjugate was more similar to appears plausible to accept the conclusions of that for codeine, in which the phenol group is Thompson & Gross, kinetic studies on a system as masked (Fujimoto & Way, 1958). complicated as urine place restrictions on the inter- The experimental evidence indicated that the pretations. Any substance in urine which might morphine conjugate is a zwitterion (Fujimoto & catalyse the hydrolysis of bound morphine and is Way, 1958). The infra-red curve of bound morphine slowly destroyed by heat would show a drop in the showed a maximum at 6.2 , with no band between absolute rate of hydrolysis of bound morphine 5.6 and 6.2 ,t. This was interpreted as meaning that over a given time. Thus, the findings of Thompson the carboxyl group of the glucuronic acid moiety is & Gross are suggestive but far from conclusive present in an ionized form. Such an interpretation evidence of the existence of two bound morphines. would necessitate the presence of a positive charge Woods (1954) also suggested that there must be at on the piperdine nitrogen. The titration curve of the least two bound forms of morphine excreted by the morphine conjugate gave two pK values which were dog, although his paper presents chromatographic consistent with the values predicted by Kumler evidence to the contrary. Woods based his con- (1955), who based his calculations on the assumption clusions instead on the results he obtained with his that morphine glucuronide exists as an ampholyte. experiments designed to isolate bound morphine. The question whether there is more than one He was successful in isolating a crystalline substance form of bound morphine is of considerable interest. from the urine which he demonstrated to be mor- Thompson & Gross (1941) reported that two forms phine-3-monoglucuronide. However, he found that of bound morphine existed in dog urine, one "easily" a far larger fraction of bound material was still hydrolysable and the other " difficultly " hydro- present which was amorphous, highly water soluble, 246 E. LEONG WAY & T. K. ADLER

and contained 30% glucuronic acid and 40% mor- Sung & Fujimoto, 1954), and mice (Kokka, Elliott phine. He suggested that the compound was a & Way, unpublished data) given morphine. The di-conjugated morphine, the alcoholic group being rate of conjugation of morphine in vivo did not differ conjugated as a glucuronide and the phenolic group materially among rats ranging in age from 2 days to as an ethereal sulfate. No data on sulfur analysis 32 days. However, newborn rats less than 8 hours of were given. age showed a somewhat lesser ability to conjugate Fujimoto & Way (1957), in their studies on urine morphine (Kupferberg & Way, unpublished data). obtained from addicts, found an amorphous, Although the crystalline compound was not isolated highly water-soluble morphine conjugate which in each instance, the conjugated product is very apparently resembled the amorphous bound mor- likely morphine-3-monoglucuronide. phine Woods found in dog urine. However, after A great deal of information on the sequences further purification and especially after removal of leading to the conjugation of morphine has been ninhydrin-reacting material, a crystalline substance gained from studies using isolated tissue preparations. was isolated which was identical with the morphine- The most extensive studies have involved the liver 3-monoglucuronide isolated by Woods (1954). and have resulted in considerable clarification of Since this crystalline monoglucuronide was entirely early observations that morphine " is altered " or derived from the amorphous material, it was con- " disappears " when perfused through the liver cluded that the crystalline and amorphous bound (Hatcher & Gold, 1929; Rink, Gray & Rueckert, morphine actually represented simply different states 1956) or incubated with liver mince or slices (Ko, of a single substance. Moreover, since the amor- 1937; Kuwahara, 1938; Inoue, 1940; Bernheim & phous conjugate was found in large amounts in the Bemheim, 1944, 1945; Fawaz, 1948; Deneau, urine and was the only bound morphine noted, it Woods & Seevers, 1953; Zauder, 1952). was concluded that only one form of bound mor- Inoue (1940) studied the ability of liver tissues of phine is excreted in the urine in any appreciable various animal species to metabolize morphine in an quantity. Further evidence supporting this con- attempt to arrive at some conclusion concerning the clusion that only one major bound form was present etiology of natural tolerance. He reported that when in urine was derived from experiments using counter- a 10-mg quantity of morphine hydrochloride was current distribution, paper chromatography and added to 10 g of liver from various animals and infra-red analysis. The paper chromatography incubated at room temperature for 30 minutes, the studies of Woods (1954) and of Seibert, Williams & proportion recovered was as follows: dogs, 52-82 %; Huggins (1954) were also cited in support of the cats, 51-73 %; rabbits, 48-61 %; guinea-pigs, 48-57 %; argument for a single main form of bound morphine. pigeons, 43-60 %; hens, 41-58 %. He concluded that Subsequently, Woods (personal communication) the difference in the ability of liver to alter morphine found that the water-soluble amorphous bound may be a cause (not necessarily the chief one) of the mcrphine in dog urine could also be changed into difference in the natural tolerance to morphine of the crystalline poorly water-soluble substance by various animal species. Inasmuch as a full descrip- manipulative techniques. Thus it would appear that tion of the experimental methods is not presented, morphine-3-monoglucuronide may be strongly asso- it is difficult to evaluate the findings. It appears, ciated with substances in urine which enhance its however, that even if the concept turns out to be water solubility and prevent it from crystallizing. correct, the experiments as presented are inadequate It is quite possible that the difference in phy- to establish the point. sical properties between the " easily " hydrolysable Bernheim & Bernheim (1944) found that when and the " difficultly " hydrolysable forms of bound morphine was added to rat liver slices, the compound morphine found in dog urine may be related disappeared under aerobic conditions. Kidney and to these factors of asseciation with extraneous brain slices were ineffectual. Liver cell suspensions substances. showed reduced activity. They concluded that Conjugation of morphine appears to be a meta- oxidation of morphine had resulted and ruled out bolic pathway common to many species. Increased conjugation as a possible mechanism. In a second amounts of morphine were found to have been study (Bernheim & Bernheim, 1945), however, they liberated after acid hydrolysis from the excreta or reported that their method for determining con- tissues of monkeys (Mellett & Woods, 1956), jugated morphine was inadequate. After applying rabbits (Hosoya, 1959), rats (Zauder, 1952; Way, the method of Gross & Thompson (1940) for bound BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 247 morphine, they concluded that conjugation and not MgCl2, and morphine, a marked reduction in free oxidation accounts for the disappearance of mor- phenol concentration occurred. It may be inferred phine when the compound is incubated with liver that synthesis of morphine glucuronide took place slices from the dog, cat, rat, and guinea-pig. The under these conditions, although the product was reaction is completely inhibited by M/1500 iodo- not isolated and identified. Further experiments acetic acid, M/500 sodium cyanide, or M/50 sodium indicated that UDPG is first oxidized to UDPGA fluoride. With respect to the cat, the disappearance by a dehydrogenase system present in the particle- of morphine in the presence of liver slices ought to free supernatant and that for every mole oxidized be explored more thoroughly, inasmuch as it has two moles of DPN+ were reduced. Strominger, been reported that cats do not form glucuronides Maxwell, Axelrod & Kalckar (1957) have purified in vivo (Robinson & Williams, 1958), presumably the enzyme from calf liver and Strominger & Map- because of the lack of glucuronyl transferase son (1957) have obtained a purified enzyme from pea (Dutton & Grieg, 1957). seedlings and report that it closely resembles the A fairly extensive store of knowledge has been one from the calf liver. The enzyme-catalysed attained in recent years which emphasizes the oxidation of UDPG is inhibited by sodium fluoride importance of carbohydrate metabolism in the but not by iodoacetate. Recently, Takemori (1960) formation of glucuronides by liver preparations. It has found that the UDPG dehydrogenase activity appears that the key substance in glucuronide syn- in male rat liver is increased often after only a single thesis is (UDPG) and injection of morphine. that this compound is derived from uridine triphos- The product of oxidation is not a substrate for phate and glucose-l-phosphate (Mills, Lockhead /-glucuronidase, indicating an a-linkage of glucu- & Smith, 1958). Since adenosine triphosphate ronic acid in UDPGA. An enzyme or enzymes (ATP) is required for both phosphate forma- present in the microsomes catalyse the transfer of tion and resynthesis of uridine triphosphate, the the glucuronic acid moiety of UDPGA to morphine. availability of carbohydrate substrates and the Several steps may be involved in this reaction since regeneration of ATP will greatly influence the ability the transferase has no 3-glucuronidase activity of liver slices or liver mince to conjugate morphine. (Isselbacher, 1956). Thus, an inversion of the Thus, with rat liver slices respiring in Krebs-Ringer a-linkage of glucuronic acid probably takes place solution the replacement of sodium by potassium just prior to or during the final coupling with mor- resulted in the concomitant delay of glycogenolysis phine. and of morphine conjugation, although the potas- Inscoe & Axelrod (1960), using o-aminophenol as sium had no effect on the conjugation of morphine the substrate, have shown that liver microsomes of when glucose was added to the medium (Marks & newborn rats and guinea-pigs have increased Huggins, 1959). It is quite possible that depletion glucuronyl transferase activity 24 hours after of carbohydrate stores, which, in turn, limits the injection of 3,4-benzpyrene. They have found also amount of UDPG formed, is primarily responsible that in adult rats the in vitro activity of the glu- for the impaired ability to conjugate morphine curonyl transferase system of liver microsomes is observed in liver mince obtained from traumatized sex-dependent and can be markedly enhanced by rats (Goldbaum, Gray, Rink, Rueckert & Ostash- previous in vivo treatment of the rat with androgens ever, 1956) or from tolerant rats during withdrawal or the carcinogenic polycyclic hydrocarbon, 2,3- (Deneau, Woods & Seevers, 1953). benzpyrene, or the activity can be as dramatically The importance of UDPG in the conjugation of reduced by pretreatment of rats with oestrogens. morphine was first suggested by the studies of During cold stress the reduction in glucuronyl Strominger, Kalckar, Axelrod & Maxwell (1954) transferase activity is probably due to a reduction following the demonstration by Dutton & Storey in total liver microsomal content as evidenced by the (1954) that uridine diphosphate glucuronic acid reduction in microsomal nitrogen. However, in (UDPGA) participates in glucuronide synthesis. chronically morphinized rats a reduction in micro- Strominger, Kalckar, Axelrod & Maxwell (1954) somal glucuronyl transferase activity occurs in the have shown that when the supernatant fluid from absence of any decrease in nitrogen content of the homogenates of guinea-pig or calf liver containing microsomes (Takemori, 1960). both microsomes and soluble enzymes was incubated These interesting changes in the in vitro activity with UDPG, diphosphopyridine nucleotide (DPN+), of the glucuronyl transferase system that result 248 E. LEONG WAY & T. K. ADLER

from pretreatment of the animal in vivo raise the over-all pharmacological action of morphine. question of whether one can predict the in vivo Despite this lack of correlation, conjugation of ability to conjugate morphine on the basis of the morphine must be viewed as a detoxication process in vitro liver microsomal activity. For example, one in so far as the product of conjugation is far less would expect to find a marked increase in urinary active than the parent substance. conjugated morphine in male rats or androgen- treated female rats as compared with normal females N-demethylation or oestrogen-treated males, and in benzpyrene- The second of the metabolic pathways outlined in treated rats as compared with untreated ones. Fig. 3-namely, N-demethylation-has thus far Conversely, a decreased urinary excretion of con- proved difficult to categorize as either a detoxication jugated morphine should occur during cold stress, or an enhancement process. Moreover, in neither or during chronic morphine administration. We in vitro nor in vivo experiments has the product of have found no published studies in which such demethylation, normorphine, been isolated in crys- comparisons have been made except in respect of talline form. While the in vitro experiments have had chronically morphinized rats and here the in vivo some measure of success in establishing normorphine reduction in urinary conjugated morphine appears as a morphine metabolite by providing evidence of to be correlated with the reduced glucuronyl trans- a product with the solubility characteristics of nor- ferase activity of the microsomes. However, the morphine, similar success in in vivo experiments has results obtained with liver slices suggest that the been obtained only in respect of the rat. The bulk of parallelism may be merely fortuitous, since several the experimental evidence available at present sug- experiments have shown that with liver slices from gesting normorphine formation in vivo are the morphinized rats there is no reduction in the ability observations that 14CO2 appears in the breath to conjugate morphine (Fawaz, 1948; Way, Sung & shortly after injection of morphine-N-14CH3 in Fujimoto, 1954) and possibly even an increase several species, including man. (Zauder, 1952). Moreover, the conjugating ability In rats (Wistar type) the pulmonary excretion of of liver slices obtained from rats receiving other 14CO2 after subcutaneous administration of 5 mg/kg kinds of treatment, such as adrenalectomy or ACTH of morphine-N-methyl-14C hydrochloride was found injections, provides no basis for predicting the to be greater in males than in females (March & comparable conjugating ability in vivo. Thus, liver Elliott, 1954). The excretion rate for both groups was slices from adrenalectomized rats are either unable found to be more rapid during the first two hours. to conjugate morphine at all (Zauder, 1952) or show Four male rats excreted nearly 5 % of the dose as no change from normal (Way, Sung & Fujimoto, radioactive CO2 within six hours, whereas the per- 1954), whereas adrenalectomized rats show higher centage excreted by seven females over the same time concentrations of both free and conjugated morphine interval was less than 0.5 %. Four female rats treated in the plasma than are found in normal rats (Adler, with a total of 45 mg of cyclopentyltestosterone pro- Elliott & George, 1957). The ability of liver slices pionate in subcutaneous doses over a 38-day from ACTH-treated rats to conjugate morphine is period prior to morphine administration exhibited a increased (Zauder, 1952), but plasma levels of con- pulmonary excretion curve almost identical with that jugated morphine in ACTH-treated rats are lower of the male rats. In male Sprague-Dawley rats after than those in normal rats (Adler, Elliott & George, subcutaneous injection of 10 mg/kg tritiated mor- 1957). phine the 24-hour urine contained some 5 % of the In general, extrapolation from liver slice in vitro dose as a free and conjugated radioactive metabolite experiments to in vivo predictions may be meaning- that was chromatographically homogeneous with less since, on the one hand, under in vitro conditions normorphine in two separate solvent systems the residual carbohydrate stores can profoundly (Misra, Mule & Woods, 1961). It has been reported affect the conjugation of morphine (see above) and, in a preliminary communication that paper chroma- on the other hand, under in vivo conditions tissues tographic evidence was obtained for the presence other than the liver may contribute to the total of two normorphine derivatives in the liver and amount of conjugated morphine excreted. Finally, brain of rats given morphine (Penna, Arevalo, Fer- as will be discussed in a later section, although many nandez, Navia & Mardones, 1959). An assessment conditions profoundly affect the conjugation mecha- of these findings must await the publication of the nism in vitro, there is no concomitant effect on the experiments in greater detail. BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 249

In the dog and the monkey, 24 hours after sub- phine in man about 75 % of the dose appears in urine cutaneous injection of 2 mg of morphine-N-14CH3 as an extremely labile conjugate plus a large propor- per kg, the exhaled labelled carbon dioxide repre- tion of free alkaloid (Sloan, Eisenman, Fraser & sented, respectively, 0.2% and 1.2% of the dose Isbell, 1958), a different fate may characterize the (Mellett & Woods, 1961). relatively small amounts which might be expected to In man, the pulmonary excretion of 14CO2 in five be released from a therapeutic dose of morphine in subjects given 10-15 mg of morphine-N-methyl-14C vivo. On purely speculative grounds the reviewers sulfate intramuscularly ranged from 3.5 % to 6% of suggest the following possibilities: (a) biosynthetic the injected dose in 24 hours (Elliott, Tolbert, Adler normorphine may be excreted preferentially in the & Anderson, 1954). No sex difference in the amount faeces; (b) biosynthetic normorphine may be further of expired 14CO2 was observed between the two male metabolized in an unknown manner; (c) biosyn- and three female subjects. The peak rate of 14CO2 thetic normorphine may be retained by tissues for a excretion in the breath was found to be between 30 fairly prolonged period of time. Although the last and 90 minutes after drug administration. A plateau suggestion is compatible with the pharmacology of in the rate of excretion occurred after six hours. normorphine in that marked cumulative effects of Measurable amounts of radioactivity were found to the drug are seen after multiple doses (Fraser, be present in two subjects 4-5 days after drug admini- Wikler, Van Horn, Eisenman & Isbell, 1958), these stration. It was suggested as a consequence that suggested possibilities can be placed in proper per- some transfer of 14CH3 groups from the morphine spective only when data are furnished regarding the molecule may occur from which 14CO2 is slowly excretion of normorphine after injection of small liberated by catabolic processes. doses of this compound. In none of these early '4C studies was there an That morphine may be demethylated in vitro was attempt to identify any of the morphine metabolites first suggested by the work of March & Elliott (1954). other than carbon dioxide. However, since N-de- Using rat liver slices and morphine-N-14CH3 as methylation with the release of the corresponding substrate, these authors showed that under aerobic nor-compound has been established as an in vivo conditions there is a release of 14CO2. Subsequently, metabolic pathway for such closely related con- Axelrod (1955a, 1956a) found that an oxidative geners as codeine and pethidine (Adler, Fujimoto, mechanism located in liver microsome preparations Way & Baker, 1955; Plotnikoff, Elliott & Way, 1952; is responsible for the oxidation of the methyl group Plotnikoff, Way & Elliott, 1956) the inference that a to formaldehyde. Qualitative evidence for normor- similar process occurs with morphine is easy to make. phine formation was obtained by paper chromato- It can be noted that the amount of 14CO2 obtained in graphy and it is assumed that the amount of the breath in man after injection of morphine-N- formaldehyde formed during the reaction bears a 14CH3 is about the same as that obtained in the breath stoichiometric relationship to the amount of nor- in man after injection of codeine-N-"4CH3 (Adler, morphine released. Fujimoto, Way & Baker, 1955) and accordingly, if The demethylating system described by Axelrod the N-demethylation processes are analogous, some resembles the system responsible for oxidative 10% of a therapeutic dose of morphine ought to be demethylation of methylated aminazo dyes, first found in a 24-hour urine sample as free and bound observed by Mueller & Miller (1953) in liver homo- normorphine. However, attempts to show that any genates and later (Conney, Brown, Miller & Miller, normorphine is present in urine have met with no 1957) located in the microsomes. Although the two success, and the recent work of Rapoport (personal demethylating systems are not identical (Takemori communication) indicates that the total amount of & Mannering, 1958), both enzyme systems require normorphine excreted in 72 hours in the urine of reduced triphosphopyridine nucleotide (TPNH) and man must be less than 0.5 % of the dose of morphine. oxygen. The work of Gillette, Brodie & LaDu The absence of normorphine from the urine after (1957) has shown that oxidative dealkylation of morphine administration is, of course, no proof that mono-methyl-4-aminoantipyrine occurs when there the metabolite is not formed in vivo. Any one of is a concomitant oxidation of TPNH by a specific several explanations could account for a failure to TPNH-oxidase and not when TPNH is oxidized by detect normorphine even if small amounts were the cytochrome system. The same authors have released in the body. Although it has been shown further shown that the reaction of TPNH with that after very large doses (75-150 mg) of normor- TPNH-oxidase yields " organic peroxides " even in 250 E. LEONG WAY & T. K. ADLER

the absence of drug substrates. This suggests that demethylation. This inference is based on Axelrod's the initial oxidative attack at the N-methyl position finding that the only reaction catalysed by a dialysed of morphine may depend on the formation of a preparation of rabbit liver microsomes when in- specific oxidant, possibly an " activated " peroxide. cubated with codeine is the formation of morphine Apparently H202 per se does not function in the and formaldehyde in equimolar amounts (Axelrod, microsome system but can function in an iron- 1955b). It appears to the reviewers that if such a containing model system since generation of H202 dialysed preparation were capable of effecting by the glucose/glucose-oxidase system was effective N-demethylation an additional amount of form- in promoting oxidative dealkylation of N-alkyl ami- aldehyde would be formed from the N-methyl of nes in the model system but not in the microsome the released morphine as well as from the N-methyl system (Gillette, Dingell & Brodie, 1958). If the of the otherwise unmetabolized codeine. Another initial reaction between an " activated peroxide " example of the importance of co-factors in species and morphine results in the formation of an N- difference is the finding that rabbitlivers donot, butrat oxide, the subsequent steps leading to formaldehyde livers do, contain a heat-labile inhibitory factor local- release need not necessarily depend on enzymatic ized in the nuclei and mitochondria (Axelrod, 1956a). reactions. It has been shown that rearrangement of The early observations made by March & Elliott tertiary amine N-oxides to a carbinol compound (1954) of the large sex difference in the ability of rats followed by hydrolysis to formaldehyde and the to form 14CO2 from morphine-N-'4CH3 both in vivo secondary amine can occur under mild chemical and with liver slices, and the marked effect of pre- conditions (pH 5-7, 38°C) when catalysed by a treatment of the animal with either androgens or ferric-ion-tartrate complex (Fish, Johnson, Law- oestrogens, were confirmed and extended by Axelrod rence & Homing, 1955; Fish, Sweeley, Johnson, (1956a) to rat liver microsomal preparations. Lawrence & Horning, 1956). Under such conditions Furthermore, Axelrod & Cochin (1957) report a one would expect a secondary reaction to occur- marked (non-competitive) inhibition of microsomal namely, reduction of some of the N-oxide by form- enzymatic N-demethylation of morphine by added aldehyde leading to regeneration of the tertiary nalorphine. Although March & Elliott (1954) found amine and oxidation of formaldehyde to formic acid. no such inhibition in their liver slice experiments, we This secondary reaction would be minimized in a feel that this apparent discrepancy may possibly be system containing a formaldehyde-trapping re- related to the fact that nalorphine is rapidly conju- agent. If morphine-N-oxide is, indeed, formed gated in liver slices (Seibert & Huggins, 1953) and, during the demethylation process it cannot be ignored hence, may not reach the demethylating enzymes as a possible contributor to some of the morphine within the microsomes. effects, since after injection it exerts certain central In 1956 Axelrod found that in chronically mor- depressant effects such as respiratory depression phinized male rats there was a marked reduction in and antitussive action, although it apparently has the ability of the liver microsomes to demethylate no analgesic properties (Kelentey, Stenszky, Czollner, dilaudid or Szlavik morphine, meperidine; cocaine, on the & Meszaros, 1957). other hand, was N-demethylated exceedingly well Axelrod (1956a) has found that microsomes by microsomes from either morphine-treated or prepared from livers of the rat, rabbit, and guinea- untreated rats. This interesting finding has served as pig, but not the mouse, are capable of catalysing the the basis for a provocative hypothesis concerning the formation of formaldehyde from morphine. The mechanism of tolerance which assumes that the liver inability of mouse microsomes to catalyse the reac- N-demethylating enzyme and the receptors in the tion is apparently restricted to certain strains, since central nervous system are closely related (Axelrod, Takemori & Mannering (1958) have found consider- 1956b, Cochin & Axelrod, 1959). A detailed dis- able N-demethylating activity in mouse liver micro- cussion of the evidence for, and the implications of, some preparations. Microsomes prepared from the this hypothesis will be found in a later instalment. kidney, brain, muscle, or spleen of male rats are An equally provocative hypothesis relating N- inactive in .this respect (Axelrod, 1956a). Species demethylation to analgesia has been advanced by differences in activity of the enzyme system occur, Beckett and his colleagues (Beckett, Casy & Harper, but these may be related to the presence or absence 1956; Beckett, Casy, Harper & Phillips, 1956). This of co-factors. For example, rabbit livers appear to hypothesis, which would assign a prominent role in contain a dialysable co-factor necessary for N- initiating analgesia to the N-demethylation process, BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 251 will also be discussed in detail in the final instalment bic conditions. Under these conditions there was a of this series of papers. Both of these hypotheses concomitant reduction in the formation of morphine have stimulated much excellent experimental work glucuronide which normally takes place in vitro in on the examination of factors affecting N-de- the absence of added cytochrome c. These authors, methylation of morphine in both tolerant and non- however, were unsuccessful in their attempts to tolerant animals (Axelrod & Cochin, 1957; Lockett demonstrate the presence of the compound in rat & Davis, 1958; Mannering & Takemori, 1958; liver after morphine administration in vivo. Inas- Takemori & Mannering, 1958; Chernov, Miller & much as chromatographic homogeneity in any one Mannering, 1959; Cochin & Axelrod, 1959; Cochin solvent system does not constitute proof of identity, & Economon, 1959; Herken, Neubert & Timm- it is possible that the compound obtained by Hosoya ler, 1959; Horlington & Lockett, 1959; Cochin & & Brody is something other than pseudomorphine. Sokoloff, 1960; Elison, Rapoport & Elliott, to be It is interesting to note that the chromatographic published). Suffice it to say at this time that attempts behaviour of this compound resembles that of an to classify N-demethylation of morphine either as unknown morphine metabolite present in the urine an activation or as a detoxication process are thwarted of a strain of rats showing low urine and plasma by the complex nature of the pharmacological concentrations of bound morphine after injection of properties of normorphine. morphine-N-14CH3. The metabolite was absent from the urine of another strain of rats showing twofold to Oxidation threefold higher urine and plasma values of bound Since oxidation of morphine to pseudomorphine morphine (Adler, Elliott & George, 1957). The (also known as dehydromorphine, oxydimorphine unknown metabolite, although chromatographically or 2,2'-bimorphine} occurs very easily under mild homogeneous with pseudomorphine, differed mar- chemical conditions (Bentley & Dyke, 1959), it is not kedly from pseudomorphine in its spectrophoto- surprising that this has been presumed to occur metric properties (Adler, unpublished data). It is under biological conditions as well. Although thus apparent that a small part of the dose of mor- pseudomorphine exhibits some striking pharmaco- phine is metabolized in an unknown manner pro- logical properties following intravenous injection, bably not by oxidation to pseudomorphine. especially on the cardiovascular system where it An " oxidized morphine " has recently been pro- produces intense depressor effects (Travell, 1932; duced by partial chemical oxidation of morphine; Schmidt & Livingston, 1933), it is of special interest this compound is different from pseudomorphine because it has long been considered to be responsible and is pharmacologically more active than morphine for some of the effects ascribed to morphine, particu- in many respects (Woods, Daly, Haggart & Seevers, larly the withdrawal syndrome. This hypothesis, 1952). However, a full account of the work has not however, has received practically no support from been published and the preliminary announcements experimental evidence in the past, as can be judged give no indication that the compound can be derived from Eddy's review of the conflicting and incon- from morphine by biotransformation. clusive findings (Krueger, Eddy & Sumwalt, 1941). More recent attempts to establish the presence of 3-0-methylation pseudomorphine in tissues after morphine admini- The most recently discovered metabolic pathway stration have been unsuccessful. Fichtenberg (1951) for the biotransformation of morphine indicates that has reported that pseudomorphine is absent from codeine is a metabolite of morphine. Qualitative blood or muscle of normal rats or from blood, evidence of the presence of a small amount of muscle, or liver of habituated rats after injection of codeine in pooled urine samples of male rats given large doses of morphine. Her conclusions are based 5 mg/kg morphine-N-14CH3 was obtained by paper on the results of a bio-assay procedure reported to be partition chromatography. Evidence of codeine for- sensitive to quantities of pseudomorphine of the mation from morphine-N-14CH3 in in vitro experi- order of 20-50 [kg/ml of extracted solution. Hosoya ments using liver homogenates or the liver soluble & Brody (1954) have demonstrated the formation of fraction was obtained by counter-current distribu- a compound in vitro, indistinguishable by chromato- tion analysis of radioactivity and basic amines. The graphic analysis from authentic pseudomorphine, by experiments showed that methionine is not the rat liver homogenates when fortified with cyto- methyl donor for the methoxy group of codeine chrome c and incubated with morphine under aero- (Elison, 1961). 252 E. LEONG WAY & T. K. ADLER

EXCRETION reported by different workers are in good agreement. The variation in the amount of morphine excreted in With certain quantitative differences, various the urine can be attributed chiefly to variation among species excrete morphine more or less in a similar individuals, but even the same individual varies con- fashion. Most of a given dose of morphine can be siderably in his ability to excrete morphine from day accounted for, largely in the urine and to a lesser but to day (Fry, Light, Torrance & Wolff, 1929; Deckert, significant extent in the bile and faeces. Other 1936a, 1936b). The amount of free morphine ap- fluids, such as saliva, milk, and perspiration, appear pearing in the urine is dependent in part on the to play only a very minor role in the excretion of dosage of morphine administered. In studies on non- morphine. The forms in which administered mor- addicts, less than 1 % of the dose can be recovered in phine have been isolated in the crystalline state from the urine 24 hours after subcutaneous administration urine are free morphine and morphine-3-mono- of 5 mg of morphine (Paerregaard, 1957b). With a glucuronide (bound morphine). Free morphine is dose of 10 mg, the relative amount of morphine excreted in the urine by all species in small but excreted is increased, but further increases in the significant amounts, the average percentage urinary dose of morphine generally result only in increased excretion of free morphine being lowest in man amounts of free morphine commensurate with the (about 5 %) and highest in rats (about 20 %). Bound dosage administered. Oberst (1940) found little morphine represents the main form in which mor- difference in the relative amounts of free morphine phine appears in urine in the various species which excreted in the urine using doses of morphine have been studied, the amount excreted usually being ranging from 30 to 3317 mg. twofold to sevenfold greater than that of free mor- A few studies of factors which may influence the phine. Normorphine and codeine may be excreted excretion of morphine have been reported. It is in minor amounts as biotransformation products of claimed that administration of lecithin and glucose morphine, but the evidence is preliminary and has accelerates the excretion of free morphine (Chopra, not been established with certainty in higher species. Chopra & Roy, 1941). Neostigmine does not produce any marked change in the excretory pattern Renal of morphine (Himmelsbach, Oberst, Brown & Man. Morphine has been accounted for in the Williams, 1942). urine largely as a glucuronide conjugate and in lesser Morphine monoglucuronide constitutes the chief amounts as free morphine. The available evidence metabolic product of morphine and is excreted suggests that unknown metabclic products are also almost exclusively in the urine. The amounts of mostly excreted by way of the kidneys. urinary bound morphine reported by various in- Free morphine constitutes only a minor fraction of vestigators are summarized in Table 3. The values the total dose of morphine which can be accounted show a range of 11-45 % of the dose. for in the urine. While most of the studies have been Recent work by Rapoport (personal communica- carried out on addicts and post-addicts, a recent tion) on non-addicts indicates that conditions for study by Paerregaard (1957b) on non-addicts indi- hydrolysing bound morphine in the earlier studies cates no striking differences in the excretory pattern. may not always have been optimal. Using more The excretion of free morphine is quite prompt. vigorous hydrolytic conditions he and his associates Nearly all the excreted free morphine can be found found that more than half the administered dose of in urine within 8 hours after intravenous or sub- morphine-N-14CH3 could be attributed to a morphine cutaneous administration (Paerregaard, 1957b), but conjugate. Their data on the 24-hour excretion of only negligible amounts are excreted at 3 hours total morphine, that is, free and bound morphine, (Eisenman, 1945). The excretion of free morphine, indicate that approximately 70% of the dose of however, may persist in trace amounts for several morphine can be accounted for by these two com- days (Fry, Light, Torrance & Wolff, 1929; Oberst, pounds. The urinary excretion of 14C continues for 1940; Paerregaard, 1957b). several days beyond the initial 24-hour period and The average amount of free morphine excreted in virtually all of the dose of radioactivity can be the urine within a 24-hour period is approximately recovered from urine within 72 hours after injection 7 %, the range being 1-14%. These mean values were of morphine-N-14CH3. No evidence has been found calculated from those data in Table 3 which were for the excretion of other forms of bound morphine considered to be reliable. In general, the values nor for the excretion of free or bound normorphine. BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 253

TABLE 3 URINARY EXCPETION OF MORPHINE IN MAN

Percentage of No. morphine excreted Subjects exam- Dose Route a in 24 hours Remarks Reference ined (mg)__ Free |Bound

Addicts stabilized Rate not influenced by age, Fry, Light, on morphine 5 972 SC 9(7-10) weight, urine volume, length or Torrance & Wolff degree of addiction. (1929)

Addicts 3 20-120 SC 14(2-24) Considerable variation found in same individual from day to Deckert (1936a) day.

Opium addicts 11 40-300 Oral 27(23-31) Opium addicts 10 10-100 Oral 29(18-37) -valuesMethod apparentlytoo high. not reliable To & Ri (1938) Morphine addicts 3 21-50 SC 57(45-72)

Addicts stabilized 12 30-90 SC 7(3-12) 18(11-20) on morphine Percentage excreted after oral 22 101-371 SC 6(4-12) 22(14-30) doses about half that after SC Oberst (1940) doses 28 524-3317 SC 3(1-9) 32(27-45)

Addicts stabilized 26 120-600 SC 6 33 Collection time not given. Oberst (1941) on morphine

Addicts stabilized 13 8-2072 SC 6 30 Data on 5 non-tolerant subjects Oberst (1942) on morphine included.

Patient 1 25-30 SC 10 30 Excretion also studied in opium Oettel (1950) smokers.

Addicts stabilized 6 260 SC 4(2-6) 20(16-37) Fujimoto & Way on morphine (1957)

Volunteers 6 5 SC 1 38(37-42) Two-thirds of total excretion Paerregaard Psychiatric patients 6 10 SC 8(4-10) 36(28-42) occurs within 8 hours. Traces (1957b) still detectable after 40 hours. Surgical patients 3 20 IV 9 39

a SC = subcutaneous; IV = intravenous.

Following its formation, the excretion of bound mor- Normorphine and its conjugate have not been phine appears to be rapid. Paerregaard (1957b) reported to be present in urine after morphine reported that over 50%. of the total amount to be administration, and, according to Rapoport (per- excreted in the urine appears within 8 hours, and by sonal communication), if they are present the total ex- 24 hours the value is roughly 90%. Traces of bound cretion would be less than 0.5 % of the injected dose. morphine are still detectable, however, after 48 hours. Monkey. The excretion of morphine in the urine There is considerable variation in the amount of by monkeys (Macaca mulatta) was reported by bound morphine excreted by different individuals Mellett & Woods (1956). Excretion of free mor- and Paaeregaard's results indicate that the same phine is rapid, with detectable amounts appearing individual on a constant dosage regime of morphine in bladder urine within 30 minutes after subcuta- varies considerably from day to day in the amount neous administration of a single 30 mg/kg dose of of bound morphine he excretes. morphine. Roughly 10% of the dose was accounted 254 E. LEONG WAY & T. K. ADLER for in the 24-hour urine of 4 non-tolerant animals Haggart, Woods & Seevers, 1954; Paerregaard & receiving 15 mg/kg morphine twice daily, but there Poulsen, 1958). This range also defines the variation was considerable variation in the amounts excreted which an individual animal may exhibit from day to from day to day even by the same animal, the day (Pierce & Plant, 1932). standard deviation being 15%. Tolerant animals Various factors which may influence the excretion excreted an average of 7 % ± 3 % of the morphine of free morphine have been studied. The dosage of dose as the free alkaloid. Conjugated morphine, morphine does not appear to be an important-factor presumably the glucuronide, was also rapidly ex- in affecting the proportion excreted as free morphine, creted, appearing in bladder urine within 30 minutes. since animals receiving a subcutaneous dose of The 24-hour excretion averaged 67% ± 25%. In morphine as low as 0.2 mg/kg (Paerregaard & tolerant animals the 24-hour excretion of bound Poulsen, 1958) or as high as 200 mg/kg (Fry, Light, morphine averaged 59% ± 11 %. The total mor- Torrance & Wolff, 1929) excrete free morphine phine recovered in the 24-hour urine exceeds that within the percentage range given. A questionable found in dogs, suggesting a more rapid rate of slight increase in the total amount of free morphine elimination of morphine by monkeys. excreted in the urine may be induced by increasing Dog. In the dog the major fraction of the mor- the daily water intake of dogs by 200 ml (Pierce & phine dose can be accounted for within 24 hours in Plant, 1932). The excretion of free and bound the urine. About 15 % appears as free morphine and morphine is not greatly influenced by neostigmine generally over 50 % is excreted as a conjugated pro- methylsulfate, 0.1 mg/kg (Slaughter, Treadwell & duct of glucuronic acid. Gales, 1941). Free urinary morphine excretion is The urinary excretion of free morphine begins increased appreciably by liver damage produced with promptly. The compound appears in bladder urine carbon tetrachloride (Gross, 1942) or chloroform within 15-30 minutes after parenteral administra- (Gross, Plant & Thompson, 1938). tion (Cochin, Haggart, Woods & Seevers, 1954). Several studies comparing the excretion of free After subcutaneous injection over 80% of the total morphine in tolerant and non-tolerant dogs have amount to be excreted in the urine can be accounted been carried out. Despite varying conditions for the for within 8 hours (Wolff, Riegel & Fry, 1933; production of tolerance, there is general accord that Paerregaard & Poulsen, 1958). The rate of elimina- the excretion of free morphine is not altered by the tion of free morphine is greatest within the first two development of tolerance to morphine (Pierce & hours, when blood levels are elevated (Pierce & Plant, 1932; Wolff, Riegel & Fry, 1933; Gross & Plant, 1932). The renal clearance of morphine was Thompson, 1940; Woods, 1954). found to be about the same as that of inulin. On the The increase in cellular metabolism that followed assumption that protein binding of morphine by the administration of small doses of dinitrophenol or plasma was 25 %, it was concluded that morphine is thyroid feeding decreased the excretion of free cleared primarily by glomerular filtration and only to morphine in non-tolerant dogs but not in tolerant a very minor extent by tubular secretion (Baker & animals (Plant & Slaughter, 1936, 1938). It was Woods, 1957). Traces of the morphine continue to concluded that there is a marked difference between be excreted for several days (Pierce & Plant, 1932). tolerant and non-tolerant dogs in the manner in This residual morphine probably has its origin in the which morphine is handled in the tissue. These morphine stored in the gall-bladder (Woods, 1957). studies were conducted prior to the establishment of The delayed appearance of morphine in the urine bound morphine as a major morphine metabolite. may be due to re-absorption of free morphine either Since free morphine excretion represents but a minor secreted into the gastro-intestinal tract via the bile fraction of the morphine dose and the consequences or formed by hydrolysis of biliary bound morphine of dinitrophenol or thyroid feeding are complicated, in the gut. it is difficult to evaluate the significance of these Approximately 15% of the dose of morphine is findings. excreted as free morphine in the urine within The major urinary product of morphine is mor- 24 hours. The data reported by various investigators phine-3-monoglucuronide. Slightly more than one- on urinary free morphine after subcutaneous ad- half of the morphine dosage can be accounted for ministration indicate general agreement, virtually within 24 hours as morphine monoglucuronide, all values falling within the range 9-20% (Pierce & most values reported for the morphine conjugate Plant, 1932; Wolff, Riegel & Fry, 1933; Cochin, falling between 38 % and 75 %. The fluctuation of BIOLOGICAL DISPOSITlON OF MORPHINE AND ITS SURROGATES. 1 255 values is due to variation among dogs, daily in- injection of 20 mg/kg morphine for one or more dividual variations, dosage of morphine adminis- years was 35-66%, whereas, in non-tolerant dogs, tered, and perhaps to conditions which may not have the total morphine excretion was 80-92%. Since been optimal for complete hydrolysis of bound the free morphine excretion in both groups was morphine. With regard to variations due to dosage, approximately the same (between 10% and 20 %), the it was shown that the proportion of conjugated difference between the two groups was attributed morphine appearing in the urine of dogs after a dose to a decrease in bound morphine excretion by the of 2 mg/kg morphine was approximately 36 % of the tolerant animals. On the other hand, Cochin, dose, whereas after a dose of 10 mg/kg the propor- Haggart, Woods & Seevers (1954) reported that the tion of conjugated morphine in the urine of the same urinary excretion of bound morphine in dogs made animals was increased to 60% of the dose. During tolerant to 30 mg/kg morphine by daily hypodermic this same interval, the percentage excretion of free injections of gradually increasing doses of morphine morphine did not change (Paerregaard & Poulsen, over a period of 2-3 months was the same as that for 1958). non-tolerant animals; the mean value for both The formation and excretion of bound morphine groups was found to be between 50% and 60% of in the dog is rapid. Its appearance in bladder urine the injected dose. Woods (1954), on the basis of his was detected within 15-30 minutes after parenteral findings that biliary excretion of morphine is an administration (Cochin, Haggart, Woods & Seevers, important pathway for the disposal of morphine, 1954). Approximately one-fifth of the morphine offered the explanation that the reduced urinary dosage after a subcutaneous injection of 20 mg/kg excretion of bound morphine found in tolerant dogs can be accounted for in the urine as conjugated by Gross & Thompson (1940) was most likely product(s) at the end of two hours (Thompson paralleled by an increase in faecal excretion. This & Gross, 1941). It can easily be calculated from the would mean that between 34 % and 65 % of the dose data reported by Paerregaard & Poulsen (1958) for ought to be found in the faeces. Although such an the total and free morphine excreted over an 8-hour explanation is not without plausibility, the question period that between 57% and 77% of the total arises why there should be a greatly increased faecal amount of conjugated morphine to be excreted excretion in this group of tolerant dogs over that appears within this interval. The clearance of bound found by Woods in his tolerant animals. Woods morphine was reported to be the same as that of suggests that the difference may be due to a difference free morphine, the values given being 66 ml/min. in the amount of exercise allowed the two groups of (Baker & Woods, 1957) and 80 ml/min. (Cochin, dogs from the two laboratories. Haggart, Woods & Seevers, 1954). Since these It seems to the reviewers that more important values were found to be similar to that for inulin, differences have been overlooked and that the two and since binding of morphine monoglucuronide groups of tolerant dogs are not strictly comparable by plasma was considered to be less than a few per since one group had been treated with morphine cent. (Blaney & Woods, 1956), it was concluded for one or more years while the other had been that conjugated morphine is cleared essentially treated for only 2-3 months prior to the study. It by glomerular filtration (Baker & Woods, 1957). may well be that, in contrast to the tolerance pro- Despite the initial rapid rate of excretion of conju- duced to the central effects, the long-term administra- gated morphine, its presence in the urine may be tion of morphine has progressively disruptive effects detected for several days, the prolonged excretion of on the mechanisms responsible for re-absorption conjugated morphine persisting to a greater degree of bound morphine from the gut. This would be of the higher the dose of morphine (Paerregaard considerable importance in the dog and the rat, in & Poulsen, 1958). The bound morphine which which there is extensive biliary excretion of morphine appears in the urine of dogs later than 24 hours metabolites. Another possibility with the long-term after injection stems from the compound stored in administration of morphine is that there is a depres- the gall-bladder (Woods, 1954). sion of the processes involved in the conjugation of Studies comparing the excretion of bound mor- morphine, with the resultant shunting of morphine phine in tolerant and non-tolerant dogs are con- to a metabolic pathway which is still unknown. flicting. Gross & Thompson (1940) reported that These hypotheses, as well as the one implicating the total (free and bound) morphine excreted in the exercise as an important factor in the disposition urine of animals made tolerant by daily subcutaneous of morphine in tolerant dogs, cannot be other than 256 E. LEONG WAY & T. K. ADLER tentative in the face of the inadequate data available administration. Pretreatment of rabbits with sodium at present. glucuronate or glucuronlactone before morphine In a second report, Thompson & Gross (1941) injection resulted in the earlier appearance and in carried out kinetic studies on the hydrolysis of larger amounts of bound morphine in the urine; combined morphine excreted by tolerant and non- free morphine excretion was increased sometimes tolerant dogs. On the basis of these studies they by the same treatment. further classified the combined morphine into two Rat. Free morphine excretion in the urine of the fractions-(a) an " easily " hydrolysable fraction, rat appears to be higher than in most species. The excreted in larger amounts in the non-tolerant dog, values cited by various authors, using different doses and (b) a "difficultly" hydrolysable fraction, excreted of morphine and strains of rats, indicate a 24-hour in greater quantities in the tolerant dog. We have urinary excretion of between 10% and 30% of the already pointed out in the section on metabolism dose of morphine injected, with an average close to our objection to interpretations based on kinetic 20%. Bound morphine excretion is more variable evidence obtained on systems as complicated as and appears to be related in part to strain differences. urine and we have also cited arguments for the The amount excreted within 24 hours usually falls existence of only one form of bound morphine. between 20 % and 50% of the dose injected. If another bound product is excreted in urine, the Free and bound morphine appear in the urine amount present would be considerably less than the shortly after parenteral administration of morphine. 8-16% cited by Thompson & Gross for the form of Within 30 minutes after subcutaneous injection of bound morphine which was excreted in the lesser 8 mg/kg of labelled morphine in a Long-Evans rat, quantity-namely, the " easily " hydrolysable frac- both free and bound morphine were detected in the tion. Moreover, assuming the validity of their urine (Adler, Elliott & George, 1957). In Sprague- experiments, the difference between tolerant and Dawley or Long-Evans rats receiving 2 mg/kg of non-tolerant animals in respect of the excretion of labelled morphine subcutaneously or via the popli- the " easily " hydrolysable fraction does not appear teal space, some 14 % of the dose appeared as free to be at a level of statistical significance. morphine in the urine at 1 hour in both strains; Rabbit. There have been numerous studies made at the same time, the bound morphine in the urine on this species, but most of the reports antedate the was 18-40% of the dose in the Sprague-Dawley development of reliable methods for the estimation rats and 10-12% of the dose in the Long-Evans rats of morphine and the establishment of morphine (Adler, Elliott & George, 1957). In rats given glucuronide as a major biotransformation product 5 mg/kg of labelled morphine subcutaneously, 18% of morphine in the urine. More recent work indi- of the dose appeared as 14C in the urine at 1 hour, cates that the urinary excretion pattern ofmorphine in 64% at 6 hours and 52% at 24 hours (March & rabbits is more or less similar to that in other species. Elliott, 1954). Keeser, Oelkers & Raetz (1933), after giving Zauder (1952) studied the urinary excretion of doses ranging from 77 to 186 mg/kg, recovered morphine over a 9-week period in 12 Wistar between 3% and 12% of the dose as morphine rats. For the first 6 days the animals were injected (free) in the 48-hour urine. Similarly, Yoshikawa each day with 12 mg/kg. During this period the (1940) found that, after subcutaneous administration 24-hour excretion of free morphine averaged 28 %, of 0.05-0.2 mg/kg morphine hydrochloride, 6-10% of and the bound morphine 36%. As the dose of the dose was excreted within one or two days in the morphine was increased by one-half over each 10-day urine and faeces, " but far more in urine than in period and the animals developed tolerance, the feces". He compared the excretion of morphine average excretion offree morphine for the subsequent in the cat and the hen under similar conditions and 8 weeks remained fairly constant at about 19% of concluded that natural tolerance cannot be satis- the dose administered. In contrast to this the per- factorily explained on a basis of different capacity centage of conjugated morphine in urine at first to destroy morphine. To the reviewers, the excretion increased from 36 % to 60% and remained elevated data alone appear to be insufficient to support or for 3 weeks, but with each succeeding week there- refute this hypothesis. after it gradually decreased until after 9 weeks the Hosoya (1959) and Otobe (1960) found consider- bound morphine excretion was 38 %. In terms of able free and bound morphine appearing in the urine absolute amounts there was a gradual increase in the of a rabbit within 30 minutes after intravenous amount of conjugated morphine excreted, although BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 257 this increase was not commensurate with the increase free morphine within 1-2 days and the hen 10-13% in morphine dosage. of the dose. The excretion of free and conjugated morphine was studied in 12 Long-Evans rats receiving 30 Gastro-intestinal or 45 mg/kg subcutaneously in two divided doses The gastro-intestinal tract has been generally (Way, Sung & Fujimoto, 1954). The 24-hour urine considered to be a minor route for elimination of showed wide variations in the amount of free and morphine, because only small amounts have been conjugated morphine excreted. The excretion of free found in faeces and because the major fraction of morphine ranged from 8 % to 29 % on the first day, the morphine dose is recovered from the urine. Only with a mean of 18 %, and the excretion of conju- recently has the importance of the biliary pathway gated morphine ranged from 3% to 31 %, with a for the disposal of morphine been established. It mean of 14 %. Continued administration of the same now appears that a significant fraction of morphine dose of morphine for 3 consecutive days resulted in can be accounted for in the bile, primarily as conju- an increase in free morphine and some decrease in gated morphine, and that biliary excretion is chiefly conjugated morphine excretion. responsible for the morphine appearing in the faeces. In rats receiving 200 mg/kg morphine hydro- Man. Surprisingly few studies have been carried chloride subcutaneously, the average urinary excre- out on the gastro-intestinal excretion of morphine tion after 72 hours was found to be 48 % of in man. The recovery of free morphine from the the morphine injected; 12% of the dose was free faeces of addicts on a total daily dose ranging from morphine and 36 % bound morphine. In rats 972 to 3888 mg of morphine was less than 5% receiving three successive subcutaneous injections (Fry, Light, Torrance & Wolff, 1929). The study of 100 mg/kg morphine hydrochloride daily and of Oberst (1942) is of interest. In 9 samples exa- 200 mg/kg on the fourth day, the 72-hour urinary mined from an addict receiving 283-489 mg daily, he excretion was 18% in the free form and 35% in found that the faecal excretion of free morphine the bound form. Over a period of from 3 to 12 did not exceed 1 %. In another addict, the amount weeks, the rats were gradually made tolerant with of free morphine appearing in faecal material was doses of morphine hydrochloride increasing from less than 3 % after a daily dose of 2455 mg. In both 100 to 500 mg/kg. The average excretion of free individuals the amount of conjugated morphine morphine did not change materially with this treat- excreted was even lower, the concentration being ment, fluctuating between 10% and 20% of the dose below the sensitivity of the method. No free mor- administered. The bound morphine excretion did phine and only a low concentration (0.7 jg/ml) of not vary much for 3 weeks, remaining at levels conjugated morphine was detected in the bile between 35% and 40%, but after 7 weeks of daily collected in a test-tube in the common duct of a injection of morphine hydrochloride, the percentage non-tolerant patient who had had a cholecystectomy excretion of bound morphine was reduced to 4-16% and who had been receiving 15 mg of morphine of the dose (Fichtenberg, 1951). sulfate every 4 hours for 3 days. In four specimens Mouse. Fairly rapid urinary excretion of 14C which were examined, the gastric contents showed occurred in the mouse after injection of 3.5 mg/kg free morphine concentrations ranging from 0.2 to biosynthetically labelled morphine. Between one- 0.7 ,ug/ml and bound morphine concentrations third and three-quarters of the dose was found in the ranging from 0 to 1.3 ,ug/ml. No morphine was urine within 6 hours after injection, most of this found in the vomitus of two morphine addicts on the excretion occurring from the fourth to the sixth second day of withdrawal. Unfortunately, no hour. Intraperitoneal injection of 25 mg/kg nalor- attempts were made to measure bound morphine phine following the morphine injection resulted in in this study. a somewhat enhanced urinary excretion during the After intramuscular administration of 10-15 mg first 6 hours, the greater part of the radioactivity of labelled morphine sulfate in 5 human subjects, being excreted during the second hour. These time the faecal excretion of radioactivity over 72 hours differences observed are very probably related to the accounted for 7-10 %. of the dose. In one of the sub- nalorphine antagonism of the morphine depression jects a duodenal tube was passed, and 14C was of micturition (Achor & Geiling, 1953). measured at various time intervals. The radio- Other species. Yoshikawa (1940) reported that activity in the duodenal secretions was highest the cat excreted 11-27 % of the dose in the urine as between 1l/2 and 6 hours after administration of the 258 E. LEONG WAY & T. K. ADLER

dose. On the assumption that the radioactivity in for both groups was between 2% and 3%. There the duodenal samples originated in the bile and that was less variation in the daily excretion of an indivi- the 24-hour bile output was 500 ml, it was estimated dual animal than between animals, but in neither that 7.4% of the injected dose was potentially case did the amount of free morphine appearing in available for excretion by the intestinal route. Since the faeces on any given day exceed 10% of the this figure approximated that obtained in the administered dose (Plant & Pierce, 1932). Wolff, faecal excretion studies, it was concluded that the Riegel & Fry (1933) reported no significant difference total faecal output arises from biliary excretion. It between tolerant and non-tolerant animals in respect was also concluded that relatively little re-absorption of the amount of free morphine appearing in the of biliary morphine occurs, since 24-hour retention faeces. They gave figures averaging between 5 % and of faeces by one subject did not result in a de- 6 %, but felt that their values were on the high side creased amount of radioactivity excreted in the because their analytical procedure for estimating faeces as compared with other subjects (Elliott, morphine in faeces was deemed to be inaccurate. Tolbert, Adler & Anderson, 1954). This single study Cochin, Haggart, Woods & Seevers (1954) found is not consistent with the findings on other species about 6% free morphine and 1 % bound morphine discussed below. While biliary excretion may not in 4 non-tolerant dogs receiving 30 mg/kg sub- be an important route for the disposal of morphine cutaneously. Significantly higher amounts of free in man, it should be pointed out that the duodenal morphine but not of bound morphine were observed samples may not reflect accurately the total amount in 4 tolerant dogs (stabilized on 30 mg/kg mor- of morphine capable of being excreted via the bile. phine for 60-90 days) than in non-tolerant animals, the average free morphine excretion for the tolerant Monkey. The bile of non-tolerant and tolerant animals being 14%. They found a greater variation monkeys receiving 30 mg/kg morphine subcutane- in the latter and felt that the was to group low faecal ously found contain significant amounts of excretion in one of morphine; over 99 morphine the 4 animals in this % of the amount present in both series was attributable to groups was in the decreased faecal bulk conjugated form. The total resulting from a meat diet. The faecal excretion of morphine excretion in the bile of a non-tolerant monkey at 90 minutes conjugated morphine in tolerant and non-tolerant was equivalent to 19.7% of animals did not exceed 2 % of the dose. the dose. In another non-tolerant monkey the total biliary morphine at 4 hours was equivalent to 11.5 % Studies on the biliary excretion of morphine in of the dose. A bound morphine concentration of 10 non-tolerant dogs at various time intervals after 300 ,ig/ml was noted in the bile of an animal sacri- administration of the drug indicated that a large ficed at 24 hours. The percentage excretion of one fraction of the morphine dose (30 mg/kg subcuta- tolerant monkey at 90 minutes was 5.7 % and of neously) was excreted in the gall-bladder bile within another tolerant monkey at 4 hours was 5.6 % of the 24 hours, almost entirely as conjugated morphine dose. Apparently re-absorption and partial hydro- (Woods, 1954). In general, the conjugated morphine lysis of biliary bound morphine occurs, since only accounted for more than 99 % of the total morphine small quantities of morphine appear in the faeces, excreted in the bile, although low concentrations of chiefly in the free form (Mellett & Woods, 1956). free morphine (11-65 ug/ml) were detectable at all Four non-tolerant monkeys receiving hypodermic time intervals studied. At 90 minutes the total mor- injections of 15 mg/kg morphine twice daily excreted phine recovered was in excess of 10% of the dose. only 2% ± 1.5% free morphine in the faeces; At various time intervals between 4 and 24 hours, 5 tolerant monkeys excreted 4% ± 3 %. The approximately one-quarter to one-third of the dose average concentration of bound morphine excreted was recovered in the bile. The amount declined to in tolerant and non-tolerant animals was found to be about 15% at 48 hours and to less than 1% at less than 1 % (Mellett & Woods, 1956). 72 hours. Three tolerant dogs similarly excreted large amounts of conjugated morphine in the bile. Dog. In the dog the faecal excretion of free Indeed, at 12 hours the gall-bladder of the tolerant morphine is considerably less than the urinary animals was found to contain 48% of the injected excretion. In studies on 11 tolerant and 7 non- dose and practically all of this was present in the tolerant dogs on daily doses of 10-50 mg/kg mor- conjugated form (Woods, 1954). phine sulfate, the average amount of morphine Evidence was also furnished that the morphine excreted daily in the faeces over a period of 6-14 days present in faeces arises almost exclusively from the BIOLOGICAL DISPOSITION OF MORPHINE AND ITS SURROGATES. 1 259 bile. In an animal with a ligated cystic duct and a fraction. Since the intact animal ex9retes a much common bile duct fistula, no free or conjugated greater percentage of the dose in the urine over the morphine was detectable in the faeces up to 96 hours same time interval, it was suggested that there is and yet 98 % of the dose was recovered within evidence for an entero-hepatic circulation of mor- 24 hours in the excreta as follows: urinary free phinie or its metabolites following initial excretion by morphine, 20 %; urinary conjugated morphine, 43 %; liver in the bile. biliary free morphine, 1 %; and biliary conjugated morphine, 34% (Woods, 1954). Other routes While renal and alimentary excretion constitute Rat. Morphine elimination via the gastro-intes- the major pathways for the elimination of morphine, tinal tract is more important in the rat than in most other body fluids, such as saliva, tears, sweat and species. Although the amount of morphine appear- milk, may be expected to carry at least traces of the ing in the faeces is considerably less than that ap- drug. That the presence of morphine has not always pearing in the urine, a rather large fraction of the been detected in these fluids by chemical tests in- morphine dose appears initially in the bile, chiefly in dicates that the amount of morphine excreted by the bound form. However, only a fraction of this is such routes is very low indeed. There has been excreted in the faeces owing, undoubtedly, to re- very little information to supplement that found in absorption of the compound. Generally, less than the review by Krueger, Eddy & Sumwalt (1941). 2 % of the injected dose is found in the faeces as free Unequivocal chemical evidence of the presence of morphine and less than 20 % as bound morphine. morphine in milk has still not been furnished. In rats given 200 mg/kg morphine hydrochloride In contrast to claims made in two reports cited by subcutaneously, about 1-2 % of the dose can be Krueger, Eddy & Sumwalt (1941), Oberst (1942) was recovered in the faeces within 72 hours as free unable to detect free or bound morphine in the saliva morphine and about 11 % as bound morphine. In of addicts on a daily dosage of between 105 and animals made tolerant to increasing doses of mor- 4200 mg of morphine, using a technique sensitive to phine administered over a period of 12 weeks, the 0.2 ,ug/ml. However, according to Peterson (1955), free morphine faecal excretion remained fairly the presence of morphine in the saliva and urine of constant and represented generally less than 5 % horses after parenteral administration has been of the injected morphine. Bound morphine excre- noted in several laboratories. tion was more variable but after an initial increase, Traces of free morphine (0.2-5 ,ug/ml) were found as with the urinary excretion, the bound morphine in the perspiration of addicts after a daily 300-mg level fell from a peak of 28 % at the end of 3 weeks dose of morphine (Oberst, 1942). to 8 % after 7 weeks (Fichtenberg, 1951). March & Elliott (1954), using morphine-N-14CH3, Other animals found 25 % of the radioactivity in the faeces within Comparatively little work has been devoted to 24-48 hours after subcutaneous injection of 5 mg/kg. studies on the excretion of morphine in animal In one rat they recovered 63% of the dose in bile species other than those already mentioned. Krueger, collected during the first 6 hours after injection, as Eddy & Sumwalt (1941) cite earlier studies which compared with 18 % in the urine during this period. have been reported on the cat, guinea-pig, goat, cow, The material secreted in the bile was stated to be free chicken, dove, frog and toad. We have not encoun- and bound morphine, with the latter being the major tered any recent studies on these species.

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