Academy of Pharmaceutical Sciences Symposium, 1971.
`(152) M. W. Anders and J. P. Latorre, Anal. Chem., 42, 1430
`( 1970).
`(153) C. A. Burtis, W. C. Butts, and W. T. Rainey, Jr., Amer. J.
`Clin. Puthol., 53, 769( 1970).
`(154) C. D. Scott, J. E. Attril, and N. G. Anderson, Proc. Soc.
`Exp. Biol. Med., 1967, 181.
`(155) C . D. Scott, Clin. Chem.. 1968, 521.
`(156) C. A. Burtis and K. S. Worren, ihid., 1968, 290.
`(157) C. D. Scott, K. L. Jolley, W. W. Pitt, and W. F. Johnson,
`Amer. J. Clin. Putliol., 53, 701(1Y70).
`(158) W. W. Pitt, C. D. Scott, and W. F. Johnson, Cliti. Chern.,
`1970, 657.
`(159) J. E. Mrochek, W. G. Butts. W. T. Kainey, and C. A.
`Burtis. ibid., 17, 72( 1971).
`(160) D. S. Young, Amer. J. Clin. Puthol., 53, 803(1970).
`(161) E. F. Walborg, Jr., and L. E. Kondo, A d . Biochem., 51,
`320(1970).
`(162) A. Floridi, J. Chromatogr., 59, 61(1971).
`(163) M. W. Anders and J. P. Latorre, Pharniucologi.~f, 1970,
`180.
`(164) M. W. Aiiders and J. P. Latorre, J . Chroniatogr., 55, 409
`( 197 I).
`(165) R. A. Henry, J. A. Schmit, and J. F. Dieckman, J . Chro-
`mutogr. Sci., 9, 513(1971).
`(166) J. A. Mollica, C. R. Rehm, J. B. Smith, and R. F. Strusz,
`APHA Academy of Pharmaceutical Sciences Symposium, 1971.
`(167) R. Henry and J. A. Schmit, Chromutogruphiu, 1970, 116.
`(168) R. L. Stevenson and C. A. Burtis, J. Chromarogr., 61, 253
`(1971).
`(169) J. C. Wolford, J. A. Dean, and G. Goldstein, ihid.. 62, 148
`(1971).
`(170) G. Ertingshausen, H. J. Alder, and A. S. Reichler, J.
`Chromuiogr., 42, 355( 1969).
`
`(171) C. A. Burtis, ihid., 51, 183(1970).
`(172) F. Murakami, S. Rokushika, and H. Hatano, ihid., 53,
`584( 1970).
`(173) H. W. Shmukler, J. Chromatogr. Sci., 8, 581(1970).
`(174) Ihid., 8, 653( 1970).
`(175) W. P. Kennedy and J. C. Lee, J. Chromatogr., 51, 203
`(1970).
`(176) P. R. Brown, ibid., 52, 257(1970).
`(177) T. K. Gabriel and J. Michaleusky, ihid.. 67, 309(1972).
`(178) G . Goldstein. A d . Biochem., 20, 477( 1967).
`(179) G. Brooker, Anal. Chem., 42, 1108(1970).
`(180) R. B. Poet and H. H. Pu, APHA Academy of Pharmaceu-
`tical Sciences Symposium, 1971.
`(181) G. R. Gordon and J. H. Peters, J. Chroniuiogr., 47, 269
`(1970).
`(182) J. Churacek and P. Jondera, ibid., 53, 69(1970).
`(183) H. W. Lange, H. F. K. Mannl, and I(. Hempel, Anal.
`Biochem., 38, 98(1970).
`(184) H. W. Lange and K. Hempel, J. Chruuiutogr., 59, 53
`(1971).
`(185) R. H. Stehl, Anul. Chem., 42, 1802(1970).
`(186) M. Popl, J. Mostecky, and Z. Havel, J. Chrotnuiogr., 53,
`233( 1970).
`(187) W. F. Beyer, Anal. Cliem., 44, 1312(1972).
`(188) L. I-. Krzeminski, L. C. Byron, and A. W. Neff, ibid.. 44,
`126( 1972).
`
`ACKNOWLEDGMEhTS AND ADDRESSES
`
`Received from the Phtirmticy urrd A/rulyticul Research Depart-
`nient, Research Dioisiotr, Suiidoz- Warider, h c . , Eusr Hunocer, NJ
`07936
`A To whom inquiries should be directed.
`
`HESEAI<CII A R T I C L E S
`
`Absorption and Distribution of Naloxone in
`Rats after Oral and Intravenous Administration
`
`S. H. WEINSTEIN', M. PFEFFER, J. M. SCHOH, L. FRANKLIN,
`M. MINTZ, and E. R. TUTKO
`
`Abstract 0 The effect of route of administration on the absorption
`and distribution of naloxone, a narcotic antagonist, was investigated
`in rats. Plasma levels were determined by GLC. Five minutes after
`intravenous administration of 1 mg./kg., the plasma concentration
`was 258 ng./ml. I'lasma levels after low oral doses were undetect-
`able; but after 100 mg./kg. orally, the peak level of unchanged drug
`was almost 5000 ng./nil. In terms of percent of administered dose,
`the maximum amount of naloxone in the calculated plasma volume
`is 1.04% of the intravenous dose wrsus 0.197; of the oral dose.
`Pharmacokinetic parameters were generated with a computer pro-
`gram; the models constructed are of a rapidly absorbed and rapidly
`
`excreted and/or metahohzed drug. These results, together with
`results from absorption studies with
`ligated
`intestinal
`loops,
`indicate that poor abborption of naloxone is not the cause of its
`relatively low oral potency. Iir rirro metabolic studies with rat liver
`slices confirmed rapid naloxone metabolism, suggesting that the
`lower potency of oral naloxone compared to parenteral naloxone
`is due to rapid first-pass liver metabolism.
`Keyphrases 0 Naloxone hydrocl~loride-absorption and distrtbu-
`tion aftcr oral and intravenous administration. rats i: Absorption
`and distribution. naloxone hydrochloride - alter oral and intra-
`venous administration, plasma levels, liver metabolism, rats
`
`[( -) - N - ally1 - 14 - hydroxynordihydro-
`Naloxonc
`morphinonc] is a potent narcotic antagonist upon
`
`pareliteral adniinistration to laboratory animals (1, 2)
`or man (31, but it is approximately one-fiftieth as po-
`
`1416 0 Journul of Pharmuceuricul Sciences
`
`Nalox1239
`Nalox-1 Pharmaceuticals, LLC
`Page 1 of 4
`
`

`

`tent when administered orally in human subjects (3).
`A similar relationship was reported for rats and mice
`(4).
`The reduced oral effectiveness of a drug may be due to
`poor or incomplete absorption from the GI tract or to
`rapid first-pass metabolism
`in the liver since orally
`administered drugs enter the systemic circulation uiu
`the hepatic portal system. To determine the extent of
`absorption of naloxone from the intestine, in uiuo in-
`testinal loop experiments were performed in rats. This
`technique has been used to study the absorption of
`many compounds including quaternary ammonium
`compounds ( 5 ) and digitalis glycosides (6).
`1)z uitro rat liver slice experiments were performed to
`determine the extent of naloxone metabolism in the
`liver. This method was previously applied to the study
`of morphine metabolism (7). Naloxone has been re-
`ported
`to undergo glucuronidation, N-deal kylation,
`and reduction of the 6-0x0 group (8,9).
`Plasma levels of naloxone in rats were determined
`following oral and intravenous administration. Ab-
`sorption and elimination rate constants, as well as
`other pharmacokinetic parameters, were calculated
`to gain insight into the fate of naloxone following ad-
`ministration by the two routes.
`
`EXPERIMENTAL
`
`Analytical Methods-Extraction of naloxone from rat plasma was
`performed as described by Mule (10). except that chloroform con-
`taining 1 :/; isopropanol was used as the solvent and was back-ex-
`, tracted with 1.3 ml. of 0.1 N HCI. A I-ml. portion of the final hydro-
`chloric acid extract was evaporated to dryness'. An internal standard,
`500 ng. of tetraphenylethylene (25 pl. of a 20 mcg./ml. methanol
`solution), was then added. The samples were again evaporated to
`dryness, and 25 pl. of bis(trimethy1silyl)trifluoroacetamide con-
`taining 1 % trimethylchlorosilane2 was added. Wilkinson and Way
`(I I ) demonstrated the efficacy of bis(trimethylsily1)trifluoroacet-
`amide as a silylating agent for morphine, and it was equally useful
`for naloxone. The tubes were flushed with dry nitrogen and closed
`with ground-glass stoppers, and the silylation reaction was carried
`out for 30 min. at 60-65" in a dry heating blockJ. One-microliter
`aliquots of the silylated samples were injected into a gas chromato-
`graph', with carbon tetrachloride as a solvent Rush. Naloxone was
`measured by comparing the peak height ratio of naloxone-tetra-
`phenylethylene in the experimental samples to the peak height
`ratio obtained from standards extracted from plasma.
`Naloxone was extracted from the liver and intestine by homog-
`enizing the tissue in a total of 4 ml. of water (including the volume
`of water used to rinse the homogenizer), and the protein was pre-
`cipitated by addition of 0.5 ml. of 10% ZnSO, and 0.5 ml. of 0.5 N
`NaOH. This was centrifuged 15 min. at 3000Xg, and 3 ml. of the
`protein-free supernate was diluted to I5 ml. with 0.5 M phosphate
`buffer, pH 7.4. The diluted supernate was then sequentially ex-
`tracted with three 15-ml. portions of chloroform. The pooled
`chloroform extracts were evaporated to dryness in a rotary evapo-
`ratorb, and the residue was quantitatively washed out of the evap-
`orating flask with 5 ml. of chloroform into a conical centrifuge
`tube. This chloroform was removed by evaporation to dryness'. The
`residue was redissolved in 0.1 ml. chloroform containing 300 ng.
`of N - cyclobutylmethyl - 7.8 - dihydro - 14 - hydroxynormorphine
`(nalbuphine. 3 mcg./ml.) as the internal standard. A portion of this
`chloroform solution was injected into the gas chromatograph, and
`the naloxone was measured by comparing the peak height ratio of
`
`1 Evaporation pcrformod in 5-1111. conical ccntrifuyc tubes with ti
`Buchlrr Evapo-mix.
`Rcyisil, Regis Chemical C o .
`a TEMP-BLOK Heater. Lah-Line Instrunicnts.
`4 Hcwlctl-Packard, F & M 402.
`5 Rinco rotary ewporator.
`
`naloxone-nalbuphine to the peak height ratio obtained for standards
`also extracted from intestinal loops or rat liver slices. Metabolites
`such as noroxymorphone and naloxone glucuronide are not ex-
`tracted under these conditions.
`The gas chromatograph was equipped with a flame-ionization
`detector. The column was 3.8% UC-W98 on high performance
`Chromosorb W, 80-100 mesh. Conditions used were: oven. 245";
`detector, 310"; Rash heater, 300"; hydrogen Row rate. 37 ml./min.;
`helium, 75 ml./min.; and air, 350 ml./min.
`Animal Studies--Three
`to five fasted, male, CFN rats (average
`weight 397 g.) were used for each time interval of the intravenous
`study. Naloxone hydrochloride ( 1 mg./ml.. aqueous solution) was
`administered riu the tail vein at a dose of 1 mg./kg. while the rats
`were under light ether anesthesia. At 5.10, 15,22,30,38,45,50, and
`60 min. after dosing, blood was obtained by cardiac puncture with
`heparinized syringes. The plasma was separated by centrifugation
`at 3000Xg for 20 min.
`For the oral studies, naloxone hydrochloride was administered to
`fasted rats (average weight 375 9.) in aqueous solution (100 mg./ml.)
`cia stomach tube at a dose of 100 mg./kg. At 2, 3.5,5, 15.22, 30, 35,
`45, 50, and 60 min. after dosing, blood was obtained as already
`described. Four to I I rats were used for each time interval.
`Intestinal loops were prepared, while fasted rats were under ether
`anesthesia, by exposing the intestine and locating a section sup-
`plied by three blood vessels, approximately 20-22 cm. from the
`pyloric sphincter. One end of a segment approximately 2.54 cm.
`(I in.) in length was securely ligated with thread. The other end of
`the segment was ligated with a 22-gauge needle entering the end
`through the center of the tightened loop of thread. Five hundred
`micrograms of the drug in 0.5 ml. of saline solution was injected
`and, as the needle was removed, the ligature was completed. The
`midline incision made to expose the intestine was then closed. At 30,
`60, or 90 min. after treatment, the animals were again anesthetized
`and the previously tied section of gut was removed. for analysis as
`already described. Controls for these intestinal loop experiments
`consisted of the incubation of 500 mcg. of naloxone in gut sections
`in rirroat 37" in normal saline for a time interval equal to that of the
`irr ciro incubation. Loss of drug by absorption was determined by
`comparing the naloxone recovered from iu cico intestinal loops to
`the naloxone recovered from iir citro loops. Loss of drug due to
`metabolism by intestinal tissue itself was determined by comparing
`the naloxone recovered after incubation in cirro in intestinal loops
`to the naloxone recovered from ill citro loops that were not in-
`cubated but were homogenized and extracted immediately after
`the drug was placed within the loop.
`For liver slice experiments. ether-anesthetized rats were killed
`and the livers were immediately removed. The livers were washed
`with ice-cold normal saline solution and sliced with a hand micro-
`tornee. Three hundred milligrams of liver slices was placed in
`25-1111. conical flasks containing 1.5 ml. of Krebs-Henseleit soh-
`tion. Then 0.5 mg. of naloxone hydrochloride in 0.5 ml. of Krebs -
`Henseleit solution was added, giving a total incubation volume of
`2.0 ml. ffior to use, the Krebs solution was saturated with 95%
`01-5% COI (7).
`The incubations were carried out in duplicate on a shaking
`incubator'at 37" under a 95% O2 5 % CO, atmosphere delivered at
`a rate of 5 f ~ . ~ / h r .
`After 30. 60, and 120 min., duplicate incubation mixtures were
`homogenized. Extraction of naloxone from the homogenized in-
`
`~
`
`-
`
`~
`
`~~
`
`Stadic-Riggs halid microtomc.
`7 Dubnoff.
`
`Vol. 62, No. Y, Srpti,nthcr 1973 0 1417
`
`Nalox1239
`Nalox-1 Pharmaceuticals, LLC
`Page 2 of 4
`
`

`

`-t
`
`volume of distribution
`K J b , = 0.96 min.-'
`
`K,I = 0.16 min.-'
`
`ti:, = 0.72 min.
`lag time = 1.98 20.0 min. I./kg.
`100 p.0. rng./kg.
`
`11,'~ = 4.25 min.
`
`Scheme 11-One-compartment open model for orally administered
`naloxone
`
`cubation mixture was carried out as previously described. The ex-
`tracts were analyzed for unmetabolized naloxone by GLC. The
`peak height ratio (naloxone-nalbuphine) of naloxone obtained
`from extracts of incubation mixtures was compared with peak
`height ratios obtained from extracts of unincubated liver-naloxone
`mixtures. Duplicate determinations were performed for each
`sample.
`As controls, to determine whether loss of naloxone was an assay
`artifact or due to liver metabolism, incubations were also carried
`out with boiled liver slices.
`computer program (COMPT)
`Phannacokinetic Calculations-A
`for optimizing the solution of integral nonlinear cornpartmental
`models of drug distribution, written in extended BASIC for use in
`time-sharing computer systems, was used to generate pharmacoki-
`netic parameters (1 2).
`The mathematical formulations for compartmental models
`(Schemes I and 11) reduce, in their most general form, to sums of
`exponential terms :
`
`M
`
`Ni exp (-aiT)
`f ( T ) =
`8 - 1
`The formulations for the one- and two-compartment models are,
`respectively :
`
`(Eq. 1)
`
`'
`
`1 0 1
`
`.?
`10
`
`'
`
` 1
`
`1
`20
`
`1
`30
`MINUTES
`Figure I-Semilog plot of rraloxorre pbsmu coricentraiions in rats
`following I mg./kg. i.v.
`
`'
`
` 1
`40
`
`' ' 1
`50
`
`'
`60
`
`1418 0 Journul of Plrarmaceirtical Sciences
`
`t
`
`1 1 ' 1
`10
`
`' ' '
`20
`
`1
`
`' ' ' '
`40
`50
`
`30
`MINUTES
`Figure 2-Semilog plot of naloxone plasma concentrations in rals
`following 100 mg./kg. p.0.
`
`Ni and u, are obtained from the line projected from the terminal
`portion of the log C cersus Tcurve. Calculation of pharmacokinetic
`parameters for these models was extensively discussed by Wagner
`(13).
`
`RESULTS
`the described chro-
`Gas Chromatography and Extractions-Under
`matographic conditions, tetraphenylethylene had a retention time of
`2.2 min. and the trimethylsilyl derivative of naloxone had a retention
`time of 4.5 min. Underivatized naloxone and nalbuphine had reten-
`tion times of 3.6 and 7.8 min., respectively.
`The use of parallel extraction standards as controls was necessary
`because the recovery of naloxone from plasma was 50-6OX.
`There was a linear, reproducible relationship between GLC response
`and plasma concentration of naloxone.
`Preliminary experiments showed that naloxone was recovered
`from intestinal loops and liver slices in quantities sufficient for GLC
`analysis without silylation and that there was a linear relationship
`between naloxone concentration and GLC response.
`Experiments in which naloxone was incubated in uitro in sections
`of gut showed that the gut itself did not metabolize naloxone.
`Therefore, disappearance from in cico loops a n n o t be attributed
`to destruction within the loop but must be ascribed to absorption
`from the loop. When naloxone was incubated with boiled liver
`slices, the naloxone was recovered intact, indicating that the disap-
`pearance found with unboiled slices was due to metabolism. Me-
`tabolites such as naloxone glucuronide or noroxymorphone are not
`extracted under the conditions used.
`Plasma Levels of Naloxone-Figure 1 is a plot of log of rat plasma
`levels of naloxone cersus time after intravenous administration.
`The increases in drug levels between 38 and 52 min. may be due
`to biliary recycling. The curve shown is the best fit computed with
`the COMPT program, neglecting those points attributable to biliary
`recycling.
`Initially, an oral dose of 10 mg./kg. was administered, but
`naloxone was not detectable in plasma at this level. At a dose of
`
`Nalox1239
`Nalox-1 Pharmaceuticals, LLC
`Page 3 of 4
`
`

`

`Table I-Absorption of Naloxone from In Viuo
`Intestinal Loop in Rata
`
`Table II-Percent Naloxone Remaining after Incubation with
`Rat Liver Slices
`
`Minutes
`
`30
`60
`90
`
`Number of
`Animals
`
`5
`8
`4
`
`Naloxone Remaining in
`Loop, % + SD
`55.5 f 17.9
`18.8 f 12.5
`4.73 f 3.6
`
`Minutes
`
`30
`60
`I 20
`
`----Naloxone
`Experiment 10
`
`Remaining, 7;-
`Experiment 2
`
`48.5
`23.6
`13.5
`
`73.5
`42.2
`13.4
`
`Five hundred micrograms injected into ligated intestinal loop in
`cico and assayed by GLC after indicated time. See text for details.
`
`a Experiments 1 and 2 reprcsent two separate expcrinients, each per-
`formed in duplicate.
`
`~
`
`100 mg./kg., detectable levels were obtained. Figure 2 is a plot of
`log of rat plasma levels of naloxone versus time after oral adminis-
`tration. The curve shown is the best fit computed with the COMPT
`program.
`Absorption from Ligated Intestinal Loop-The percent of adminis-
`tered naloxone absorbed from in sifu ligated intestinal loops at var-
`ious time intervals is presented in Table I. Naloxone is well ab-
`was absorbed at 90 min.
`sorbed; 95.3
`Metabolism by Rat Liver Slices-Table
`I1 presents the results of
`the incubation of naloxone with rat liver slices. After 2 hr., almost
`90% of the added naloxone was metabolized. Earlier time intervals
`showed some variations; but in 30 min., 25-50 7; of the naloxone was
`metabolized.
`Pharmacokinetics-The plasma level data obtained for intra-
`venously administered naloxone (Fig. 1) fit a two-compartment open
`model (Scheme I). The data for orally administered naloxone (Fig.
`2) fit a onecompartment model with first-order absorption (Scheme
`11).
`
`DISCUSSION
`
`The pharmacokinetic model (Scheme I) for intravenous naloxone
`yields a very rapid elimination rate ( f = 16 min.). The relatively
`large volume of distribution in Compartment 1 could indicate either
`extensive tissue binding or rapid metabolism, because the model is
`simply attempting to account for the absence of a large fraction of
`the administered dose as unchanged drug in the plasma. The small
`values of KI2 and V2 and the larger KS1 value indicate slow entry
`into, little binding in, and rapid removal from a peripheral compart-
`ment, respectively.
`The onecompartment model for naloxone plasma levels after
`oral administration (Scheme 11) satisfies the available data. If a
`more sensitive analytical method were available, a leveling off of
`plasma concentrations probably would have been observed after
`45 min., and the use of a two-compartment model would be in-
`dicated. The observed terminal straight-line portion of the curve in
`Fig. 2 is probably really the distributive phase. The computer-fitted
`curve does, however, show a very rapid absorption rate ( f I i2 =
`0.72 min.) for naloxone. The large volume of distribution may in-
`dicate either extensive tissue binding or rapid metabolism. The
`pharmacokinetic models of naloxone are, therefore, those of a
`rapidly absorbed and rapidly excreted and/or metabolized drug.
`Figures 1 and 2 show the plasma concentrations of naloxone
`after intravenous and oral administration. The peak concentration
`after oral administration, 4856 ng./ml., occurs at 5 min. In terms of
`percent of the administered dose, calculated from the average plasma
`volume of rats (40.4 ml./kg.) and the average weight of the rats
`used, the maximum amount of naloxone found in plasma is 0.19%
`of the oral dose. Five minutes after intravenous administration, the
`plasma concentration is 258 ng./ml. or 1.04% of the dose. This
`could indicate that a large portion of the oral dose is either not
`absorbed or is metabolized before reaching the systemic circula-
`tion.
`The rapid absorption rate observed for orally administered
`naloxone is in agreement with the results of the ligated intestinal
`loop experiments. At 30 min., 45% of the drug is absorbed and
`after 90 min. absorption is virtually complete (Table I). In these
`experiments the entire intestinal loop was analyzed for naloxone,
`eliminating the possibility of naloxone loss through binding to
`
`some component of intestinal tissue, as reported by Levine et a/.
`( 5 ) for quaternary ammonium compounds.
`These results lead to the conclusion that the lower therapeutic
`effectiveness of orally administered naloxone is not the result of
`poor absorption from the intestine. The remaining factor, rapid
`first-pass liver metabolism, therefore, appears to be the mechanism
`by which efficacy of orally administered naloxone is diminished.
`This conclusion is strongly supported by the rapid metabolism
`of naloxone by rat liver slices (Table 11). To be sure, plasma con-
`centrations of naloxone in rats are erratic after oral administration
`(coefficient of variation 1.10 compared to 0.25 for intravenous
`administration), but this may be due to the well-known biological
`variation in drug-metabolizing enzymes rather than to erratic ab-
`sorption from the GI tract.
`The data presented show that low oral effectiveness of naloxone
`cannot be attributed to poor absorption. I’eiitazocine’s low oral
`etfectiveness (compared to parenteral dosing) has been attributed
`to poor absorption (14). but this is now questionable Gnce Berko-
`witz et al. (15) reported finding an additional metabolite in plasma
`following oral administration which has not been found after
`intramuscular administration. This
`indicates route-dependent
`metabolism of pentazocine.
`
`REFERENCES
`
`(1) H. Blumberg, H. B. Dayton, and P. S. Wolf, Fed. Proc., 24,
`67M 1968).
`(2) H. Blumberg, P. S. Wolf, and H. B. Dayton, Proc. Soc.
`Exp. Biol. Med., 118,763(1965).
`(3) M. Fink, A. Zaks, R. Sharoff, A. Mora, A. Bruner, S. Levit,
`and A. M. Freedman, C h . Pliormncol. Tlrer., 9, 568( 1968).
`(4) H. B. Dayton and H. Blumberg, Fed. Proc., 29.686(1970).
`( 5 ) K. M. Levine, M. R. Blair, and B. R. Clark, J. Plrurmucol.
`Exp. Tlier., 114, 78(1955).
`(6) M. J. Greenberger. R. P. MacDerniott, J. F. Martin, and S.
`Dutta, ;bid., 167, 265(1969).
`(7) F. Bernheim and M. L. C. Bernheim, ibid., 82, 85(1915).
`(8) S. H. Weinstein, M. Pfetfer, J. M. Schor, L. Indindoli, and
`M. Mintz, J. Pliurm. Sci., 60, 1567(1971).
`(9) J. M. Fujimoto, Proc. Soc. ESP. Bid. izfed., 133, 317(1970).
`(10) S. J. Muli, Anul. Clrem., 36. 1907(1964).
`(11) G. K. Wilkinson and E. L. Way, Bioclreni. Plrurnrucol., 18,
`133% 1969).
`(12) M. Pfeffer, J. PlrurniucoAirr. Bioplurni., 1, 138(1973).
`(13) J. G. Wagner, “Biopharmaceutics and Relevant Pharma-
`cokinetics,” 1st ed.. Drug Intelligence Publications, Hamilton, 111..
`1971.
`(14) A. H. Beckett, J. F. Taylor, and P. Kourounakis, J. Plitrriii.
`Plitirmacol., 22, 123( 1970).
`(15) B. A. Berkowitz, J. H. Asling, S. M. Snider, and E. L. Way,
`Clin. Plrarmucol. Tlier.. 10, 320( 1969).
`
`ACKNOWLEDGMENTS AND ADDRESSES
`
`Received December 11, 1972, from the Deprrrniurr o/ Bio-
`chemistry, Endo Luborutories, Gtirde/r Ciry, N Y 11530
`Accepted for publication April 6, 1973.
`A To whom inquiries should be directed.
`
`Vol. 62, No. Y, Seplember 1973 0 1419
`
`Nalox1239
`Nalox-1 Pharmaceuticals, LLC
`Page 4 of 4
`
`