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Improvement of the Oral Bioavailability of Naltrexone in Dogs: A Prodrug Approach
Improvement of the Oral Bioavailability of Naltrexone in Dogs: A Prodrug Approach MUNIRA. HUSSAIN’,CHRISTOPHER A. KOVAL,MELVYN J. MYERS,ELIEG.SHAM,AND ELI SHEFTER Received January 21,1987,from E. 1. du Pont de Nemours 8 Company, Medical Products Department, Pharmaceuticals RbD Division, Experimenfal Station, Building 400, Wilmington, DE 19898. Accepted for publication April 2, 1987. ~
.
Abstract 3 In an effort to improve the oral bioavailability of naltrexone [ 17-(cyclopropylmethyl)-4,5a-epoxy-3,14-dihydroxymorphinan-
6-one;l],a number of prodrug esters on the 3-hydroxyl group were prepared: the anthranilate (2), acetylsalicylate (3), benzoate (4), and pivalate (5). The oral bioavailability of these prodrugs was determined in dogs. Compounds 2 and 3 exhibited the greatest enhancement of naltrexone bioavailability (45and 28 times greater than 1, respectively). No correlation was found between the rates of plasma hydrolysis and bioavailability. Naltrexone-3-acetylsalicylate hydrolyzed in human and dog plasma with a fast deacetylation step to naltrexone salicylate followed by a slower hydrolysis step to naltrexone.
tal animal in the study of the central nervous system consequences of narcotic antagonists.14 This article describes the evaluation of four such prodrugs in dogs.
Experimental Section Materials-Naltrexone hydrochloride [17-(cyclopropylmethyl)4,5a-epoxy-3,14-dihydroxymorphinan-6-one; 11 and nalbuphine hydrochloride [17-(cyclobutylmethyI)-4,5a-epoxymorphinan-3,6a,l4-
trio11 were obtained from Du Pont Pharmaceuticals. Isatoic anhydride and 4-dimethylaminopyridine were obtained from Fluka -~~~ .. .. .~ __ Chemical Corporation. NJV-Dimethylformamide and petroleum ether were obtained from J. T. Baker Chemical Company. Triethylamine, acetic acid, and phosphoric acid were purchased fmm E. M. Naltrexone hydrochloride Il7-(cyclopropylmethyl)-4,5a- Science. Methylene chloride, acetonitrile (HPLC grade), tetrahydmfuran (HPLC grade), sodium acetate, isopropyl alcohol, and toluene epoxy-3,14-dihydroxymorphinan-6-one; Trexan; Du Pont were obtained from Fisher Scientific Company. d,-Deuterated diPharmaceuticals] is currently used for the treatment of methyl sulfoxide was purchased from Columbia Organic Chemical opioid addiction. It is nonaddicting’ and has minimal side Company. Trimethylacetyl chloride, acetylsalicyloyl chloride, and effects.2.:’ Naltrexone has been shown to be well absorbed benzoyl chloride were purchased from Aldrich Chemical Company. Sodium heptanesulfonate was obtained from Sigma Chemical Comfrom the gastrointestinal tract.4 However, it undergoes extensive first-pass metabolism when given orally, being rapidpany. Melting points were determined on a FisherJohns melting point ly cleared by the gut and/or liver. This results in a relatively apparatus and are uncorrected. Proton NMR spectra were recorded low systemic bioavailability for t h e drug. Systemic bioavailwith a Bruker, WP2OOSY NMR spectrometer (IBM). ability of a single oral dose to healthy volunteers was Synthesis-”ltrexone-3-Anthranilate (2); (See Scheme I)-A estimated to be 5-6% in one study,6 and 20-22% after acute mixture of naltrexone (3.4 g, 0.01 mol), isatoic anhydride (2 g, 0.012 or chronic dosing i n another study.6 The major naltrexone mol), and 4-dimethylaminopyridine (1.2 g, 0.01 mol) in NJV-dimethmetabolites in humans are naltrexol and conjugated naltrexylformamide (25 mL) was heated at 80 “C for 4 h. After cooling, 50 one and naltrexol. It is not known whether first-pass metabomL of water was added and the mixture was stirred at ambient lism is selective for reduction to naltrexol or conjugation. temperature for 0.5 h. The precipitate was collected by filtration, Plasma levels in humans of conjugated naltrexone were higher than naltrexol levels after a single oral 50-mg dose,‘ 1 Rx H but urinary recovery of naltrexol was higher t h a n that of conjugated n a l t r e ~ o n e . ~ Through the use of prodrugs, i t is sometimes possible to reduce the extent of first-pass metabolism and improve the t R : oral bioavailability of a drug. This approach has been successfully used for phenols and catechols, such as terbutaline8 and d dopa.^ The bioavailability of methyldopa in rats and humans was enhanced when administered as the pivaloyloxyethyl ester.Lo,llThere are very few published reports discussing the use of prodrug derivitization of phenolic opioids for enhancing their oral bioavailability. A number of ester derivatives on the 3-phenolic hydroxyl group of naltrexone were examined for their ability to overcome the extensive first-pass metabolism of the parent drug. In dogs administered iv naltrexone, naltrexol was not a metabolic product.12 Specifically, the goal of this study was to block conjugation during absorption. The dog is a valid animal model for initially testing the hypothesis of using prodrugs to block naltrexone conjugation because conjugation is the major metabolic route in this species. Hydrolysis 5 a: rates of prodrugs in dog plasma were expected to be similar to CH, rates in human plasma, based on previous data obtained with nalbuphine p r o d r u g ~ ,in ~ ~contrast to those in rats and monkeys. In addition, the dog is a commonly used experimenScheme CNalfrexone (1) and nalfrexone prodrugs (2-5).
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washed with water, and air dried. The product was dissolved in methylene chloride and treated with charcoal to remove the tan color. The methylene chloride solution was evaporated and the resulting product was a white solid recrystallized from ethyl acetate, mp 179-181 "C, 75% yield; 'H NMR (Me2SO-d,): 60.13-3.23 (m, 18, aliphatic), 4.93 ( 8 , 1, C-5), 5.17 ( 8 , 1, OH), 6.53-6.66 (m, 2, aromatic), 6.73 (8, 2, NH2), and 6.8-7.9 ppm (m, 4, aromatic). Anal.--Calc. for C27H2sN20K: C, 70.42; H, 6.13; N, 6.08. Found: C, 70.28; H, 6.19; N, 6.06. Naltrexone-3-Acetylsalicylate(31, Naltrexone-3-Benzoate (4), and NaltrexoneJ-Pivalate (5); (See Scheme I)-Naltrexone base (3.4 g, 0.01 mol) was typically dissolved in methylene chloride (50 mL) and triethylamine (8mL, 0.055 mol) a t 0 to 5 "C in an ice bath. A solution of the corresponding acid chloride (0.012 mol) was added in a dropwise manner with stirring. After addition was complete, the ice bath was removed and the mixture was stirred a t ambient temperature for 5 h. The methylene chloride solution was washed with 10% aqueous sodium carbonate and then with water, dried over anhydrous sodium sulfate, filtered, and reduced to a small volume by evaporation under reduced pressure. The ester was precipitated by adding excess petroleum ether. After filtering, the precipitate was air dried to provide the desired compound. Compound 3 was obtained as a white solid, mp 174-176 "C; 'H NMR (Me2SO-d6):6 0.13-2.28 (m, 10, aliphatic), 2.3 ( 8 , 3, acetylmethyl), 2.3-3.23 (m, 8,aliphatic), 5.0 ( 8 , 1, C-5), 5.16 ( 8 , 1, OH), and 6.76-8.13 ppm (m, 6, aromatic). Anal.-Calc. for C.J-129N0,*1/4 H20: C, 68.56; H, 5.85; N, 2.76. Found: C, 68.53; H, 5.89; N, 2.75. Compound 4 was obtained as a white solid, mp 174-177 "C; 'H NMR (Me2SO-d6):6 0.133.23 (m, 18, aliphatic), 4.93 ( 8 , 1, C-5h5.17 (8, 1, OH), and 6.76-8.13 ppm (m, 7, aromatic). Anal.-Calc. for C27H27NOK:C, 72.8; H, 6.1; N, 3.14. Found: C, 72.68; H, 6.18; N, 3.12. Compound 5 was obtained as a white solid, mp 161-163 "C; 'H NMR (Me2SO-d6):6 0.13-0.93 (m,5, cyclopropyl protons), 1.3 ( 8 , 9, C-(CH&,), 1.33-3.2 (m, 13, aliphatic), 4.93 ( 8 , 1, C-5), 5.17 ( 8 , 1, OH), and 6.66-6.83 ppm (m, 2, aromatic). AnaZ.--Calc. for CZbH31N06: C, 70.56; H, 7.34; N, 3.29. Found: C, 70.48; H, 7.36; N, 3.28. Analytical-Prodrug Hydrolysis in Plasm-The hydrolysis rates of 2, 3, 4, and 5 to naltrexone were determined in both dog and human plasma. Human plasma was obtained from a local blood bank and was kept frozen between the time of receipt and initiation of the experiments. The dog plasma was obtained from beagle dogs, anticoagulated with heparin, and kept frozen until the start of the experiment. Prodrug hydrolysis was performed in 3 mL of plasma at 37 "C with a prodrug concentration of 35 At various times of incubation, a 200-pL aliquot of plasma was taken and added to 200 pL of acetonitrile, which was then centrifuged. The clear supernatant was injected directly onto the HPLC column. The components of the HPLC system were a solvent delivery pump (Du Pont Instruments); an autosampler (Waters, WISP 710B); a reversed phase, 25 cm x 4.6 mm i.d., stainless steel column prepacked with 6-pm C-8 particles (Zorbax C-8, Du Pont) attached to a guard column with C-18packing; a variable wavelength spectrophotometric detector (DuPont Instruments, model 8800) set at A = 276 nm; and a recording integrator (Hewlett-Packard, model 3392A). The mobile phase contained 0.1 M acetate buffer (pH 3.8):acetonitri1e:tetrahydrofuran:sodiumheptane sulfonate (600 mL:170 mL:190 mL:lg), and the flow rate was 1.1 mumin. Retention times for the prodrugs were: 2 = 7.6, 3 = 6.5, 4 = 8.0, 5 = 7.9, and naltrexone salicylate = 8.9 min. Biwuailability-Naltrexone hydrochloride (1)was administered iv or orally to three female beagle dogs, and the prodrugs were administered orally in a crowver experiment. For iv dosing, 1 was dissolved in water, and 2.9 pmol/kg (0.5 m u g ) was injected via the cephalic vein. Compound 1 was administered orally a8 an aqueous solution (11.9 pmolkg; 1 m u g ) . Compounds 2 , 3 , 4 , and 5 were also administered as solutions of the prodrugs in 0.05 M HCI (11.9 WoUkg; 1 mukg). All oral doses were followed by the oral administration of 50 mL of water. Blood (5 mL) was collected by jugular venipuncture into evacuated tubes containing sodium EDTA as the anticoagulant. Plasma was separated and stored frozen. Animals were fasted overnight prior to each experiment. Plasma naltrexone concentrations were determined by HPLC after solvent extraction using a method similar to those previously de-
a.
scribed by Meyer et aL5 and Garrett and El-Koussi.12 Nalbuphine hydrochloride (Du Pont Pharmaceuticals) was added to plasma as an internal standard prior to extraction. Plasma aliquots (0.5 mL) were buffered with 0.2 mL of carbonate buffer (1.0 M, pH 9) and extracted twice into 4-mL volumes of to1uene:ethyl acetate:isopropyl alcohol (70:29:1). Back extractions into 0.2 mL of 0.3 M phosphoric acid were then performed. The phosphoric acid solution was injected onto the HPLC column. A 25 cm x 4.6 mm i.d., reversed-phase, stainless steel column prepacked with 6 - p n C-8 particles (Zorbax, C-8 Du Pont) was used. The mobile phase was 11-12% acetonitrile and 0.2% tetrahydrofuran in 0.055 M phosphate buffer at pH 3-4. Electrochemical detection (Bioanalytical Systems, LCB4) a t an oxidative potential of +0.98 V was used. Typical retention times for naltrexone and nalbuphine were 8 and 11 min, respectively. Calibration curves were made each day that analyses were performed. The prodrugs were tested for stability during the extraction procedure and were found to be stable. Only the plasma concentration of naltrexone and not that of the prodrugs were measured. Oral naltrexone bioavailability (F)was evaluated using the areaunder-the-plasma-concentrationversus time curve (AUC), as shown in eq 1.
(1) The AUC was calculated for each dog using the trapezoidal method with residual area calculated by dividing C (plasma naltrexone concentration) at the time of the last sample gy K (the elimination rate constant). Individual AUC, and AUCi, values were used.
Results and Discussion The hydrolysis half-lives of the prodrugs in vitro in dog and human plasma are shown in Table I. All prodrugs exhibited apparent first-order kinetics. The hydrolysis half-lives for ( t l / 2 ) for 3 and 4 were similar in both dog and human plasma. Such was not the case for 2 and 5 ; the t 1 / 2 in human plasma was longer than that in the dog. The hydrolysis rate profile of 3 in human plasma is shown in Figure 1. Compound 3 was found to rapidly hydrolyze to naltrexone salicylate ( t l l z was -0.25 min), and subsequently naltrexone salicylate hydrolyzed to naltrexone with a t1,2 of 30 min. In dog plasma, the t 1 / 2 for the first step was 5 min and that of the second step was similar to that determined for human plasma (35 m i d . Bioavailability data are shown in Table I1 and Figure 2 for the various prodrugs. Naltrexone bioavailability after oral administration of 1 was very low (1.1 0.1%),indicating extensive first-pass metabolism. Naltrexone bioavailability after oral administration of 2 and 3 was 45 and 28 times higher than that of oral naltrexone, respectively. The enhancement in naltrexone bioavailability after the administration of 4 and 5 was very much smaller. There is no
*
Table CHydrolysls Half-Llves of the Anthranllate (2), 3Acetyisallcylate (3), Benzoate (4), and the Plvalate (5) Esters of Naltrexone (1) in Human and Dog Plasma Prodrug
Hydrolysis Half-Life, h Human Plasma ~~
2
3 4 5
~
-480.004' 0.5' 2.0 7.5
Dog Plasma
--24' 0.086 0.6' 2.2 2.0
'Disappearance was followed for only 24 h. 'Compound 3 to naitrexone salicylate. 'Naltrexone salicylate to 1. Journal of Pharmaceutical Sciences / 357 Vol. 76, No. 5, May 1987
apparent correlation between the in vitro hydrolysis half-life in dog plasma and oral bioavailability. In this case, the rate of hydrolysis in plasma could not be used as a criteria for predicting in vivo characteristics of the prodrugs. For example, even if a derivative fails to generate the parent compound in plasma in vitro within a reasonable time, there is still a high probability that it may be cleaved in other organs that are rich in hydrolytic enzymes. In conclusion, the anthranilate and acetylsalicylate prodrugs were useful for enhancing the oral bioavailability of naltrexone in dogs.
1000
t I a
;
I-
100
0
03
a:E 3 a
3
10
9
z 4
4
n
'0°1
1 HOURS
Figure 2-Plasma naltrexone concentrations (mean + SEM) in dogs administered 1 iv (V); (2.9 pmollkg) and equimolar doses (1 1.9pmol/ kg) of 1 (O),2 (m), 3 (A),4 (A), and 5 (0) oral/y.
References and Notes
TIME (MINI
Figure 1-Hydrolysis of 3 in human plasma. Key: (0) disappearance of 3; (3)appearance and disappearance of naltrexone salicylate; (A) appearance of 1 .
Table II-Oral Naltrexone Bloevallablllty ( F ) In Dog8 attor Admlnlstratlon of Naltrexone ( l ) , and the Anthranllate (2), 3Acetylsallcylate (3), Benzoate (4), and Plvalate (5) Esters F, Yo
Compound Administered
Dose'
1 2
1.1 2 49.2 2 31.0 ? 2.7 2 8.0 ?
3 4 5
'Results are expressed as mean
* SEM (n = 3).
358 / Journal of Pharmaceutical Sciences Vol. 76, No. 5, May 1987
0.1 9.3 7.0 1.3
0.6
1. Fact Sheet on Naltrexone, National Clearinghouse for Drug Abuse Information, National Institute on Drug Abuse, Rockville, MD, Series 38 (1-7), December, 1978. 2. Resnick, R.; Volavka, J.; Freedman, A. M.; Thomas. M. A m . J. Psychiat. 1974,131,646-650. 3. Brahen, L.; Capone, T.; Wiechert, V.; Desiderio, D. Arch. Gen. Pqchiat. 1977,34, 1181-1184. 4. Wall, M. E.; Brine, D. R.; Perez-Reyes, M. Drug. Metab. Disp. isni.9.369-m. 5. Meyer, 'M. C.; Straughn, A. B.; Lo,M.-W.; Schary, W. L.; Whitney, C. C. J . Clin. Psychiatr. 1984,45, 15-19. 6. Konan, M. J.; Verebey, K.; Mule, S. J. Res. Commun. Chem. Pathol. Pharmacol. 1977, 18, 29-34. 7. Cone, E. J.; Gorodetzky, C. W.; Yeh, S. Y. Drug Metab. Dispos. 1974.2. 506-512. 8. Hornblad, Y.; Ripe, E.; Magnusson, P. 0.; Tegner, K. Eur. J . Clin. Pharmacol. 1976, 10, 9-18. 9. Bodor, N.; Sloan, K. B.; Higuchi, T.; Saaahar, K. J . Med. Chem. 1977,20, 1435-1445. 10. Vickers, S.; Duncan, C. A.; White, S. D.; Breault, G. 0.;Royds, R. B.: DeScheDDer. P. J.: TemDero. - . K. F. D r w Metab. Disms. 197n,8,640-6kii. ' 11. Vickers, S.; Duncan, C. A.; Ramjit, H. G.; Dobrinska, M. R.; Dollery, C. T.; Gomez, H. J.; Leidy, H. L.; Vincek, W. C. Drug Metab. Dispos. 1984, 12, 242-246. 12. Garrett, E. R.; El-Koussi, A.E.-D.A.J. Pharm. Sci. 1985,74,5056. 13. Aungst, B. J.; Myers, M. J.; Shefler, E.; Shami, E. G. Znt. J . Pharmuceut., in press. 14. Martin, W. R.; Sandquist, V. L. Arch. Gen. Psychiatr. 1974,30, 31-33. I
.
Abstract 3 In an effort to improve the oral bioavailability of naltrexone [ 17-(cyclopropylmethyl)-4,5a-epoxy-3,14-dihydroxymorphinan-
6-one;l],a number of prodrug esters on the 3-hydroxyl group were prepared: the anthranilate (2), acetylsalicylate (3), benzoate (4), and pivalate (5). The oral bioavailability of these prodrugs was determined in dogs. Compounds 2 and 3 exhibited the greatest enhancement of naltrexone bioavailability (45and 28 times greater than 1, respectively). No correlation was found between the rates of plasma hydrolysis and bioavailability. Naltrexone-3-acetylsalicylate hydrolyzed in human and dog plasma with a fast deacetylation step to naltrexone salicylate followed by a slower hydrolysis step to naltrexone.
tal animal in the study of the central nervous system consequences of narcotic antagonists.14 This article describes the evaluation of four such prodrugs in dogs.
Experimental Section Materials-Naltrexone hydrochloride [17-(cyclopropylmethyl)4,5a-epoxy-3,14-dihydroxymorphinan-6-one; 11 and nalbuphine hydrochloride [17-(cyclobutylmethyI)-4,5a-epoxymorphinan-3,6a,l4-
trio11 were obtained from Du Pont Pharmaceuticals. Isatoic anhydride and 4-dimethylaminopyridine were obtained from Fluka -~~~ .. .. .~ __ Chemical Corporation. NJV-Dimethylformamide and petroleum ether were obtained from J. T. Baker Chemical Company. Triethylamine, acetic acid, and phosphoric acid were purchased fmm E. M. Naltrexone hydrochloride Il7-(cyclopropylmethyl)-4,5a- Science. Methylene chloride, acetonitrile (HPLC grade), tetrahydmfuran (HPLC grade), sodium acetate, isopropyl alcohol, and toluene epoxy-3,14-dihydroxymorphinan-6-one; Trexan; Du Pont were obtained from Fisher Scientific Company. d,-Deuterated diPharmaceuticals] is currently used for the treatment of methyl sulfoxide was purchased from Columbia Organic Chemical opioid addiction. It is nonaddicting’ and has minimal side Company. Trimethylacetyl chloride, acetylsalicyloyl chloride, and effects.2.:’ Naltrexone has been shown to be well absorbed benzoyl chloride were purchased from Aldrich Chemical Company. Sodium heptanesulfonate was obtained from Sigma Chemical Comfrom the gastrointestinal tract.4 However, it undergoes extensive first-pass metabolism when given orally, being rapidpany. Melting points were determined on a FisherJohns melting point ly cleared by the gut and/or liver. This results in a relatively apparatus and are uncorrected. Proton NMR spectra were recorded low systemic bioavailability for t h e drug. Systemic bioavailwith a Bruker, WP2OOSY NMR spectrometer (IBM). ability of a single oral dose to healthy volunteers was Synthesis-”ltrexone-3-Anthranilate (2); (See Scheme I)-A estimated to be 5-6% in one study,6 and 20-22% after acute mixture of naltrexone (3.4 g, 0.01 mol), isatoic anhydride (2 g, 0.012 or chronic dosing i n another study.6 The major naltrexone mol), and 4-dimethylaminopyridine (1.2 g, 0.01 mol) in NJV-dimethmetabolites in humans are naltrexol and conjugated naltrexylformamide (25 mL) was heated at 80 “C for 4 h. After cooling, 50 one and naltrexol. It is not known whether first-pass metabomL of water was added and the mixture was stirred at ambient lism is selective for reduction to naltrexol or conjugation. temperature for 0.5 h. The precipitate was collected by filtration, Plasma levels in humans of conjugated naltrexone were higher than naltrexol levels after a single oral 50-mg dose,‘ 1 Rx H but urinary recovery of naltrexol was higher t h a n that of conjugated n a l t r e ~ o n e . ~ Through the use of prodrugs, i t is sometimes possible to reduce the extent of first-pass metabolism and improve the t R : oral bioavailability of a drug. This approach has been successfully used for phenols and catechols, such as terbutaline8 and d dopa.^ The bioavailability of methyldopa in rats and humans was enhanced when administered as the pivaloyloxyethyl ester.Lo,llThere are very few published reports discussing the use of prodrug derivitization of phenolic opioids for enhancing their oral bioavailability. A number of ester derivatives on the 3-phenolic hydroxyl group of naltrexone were examined for their ability to overcome the extensive first-pass metabolism of the parent drug. In dogs administered iv naltrexone, naltrexol was not a metabolic product.12 Specifically, the goal of this study was to block conjugation during absorption. The dog is a valid animal model for initially testing the hypothesis of using prodrugs to block naltrexone conjugation because conjugation is the major metabolic route in this species. Hydrolysis 5 a: rates of prodrugs in dog plasma were expected to be similar to CH, rates in human plasma, based on previous data obtained with nalbuphine p r o d r u g ~ ,in ~ ~contrast to those in rats and monkeys. In addition, the dog is a commonly used experimenScheme CNalfrexone (1) and nalfrexone prodrugs (2-5).
!-Q
356 / Journal of Pharmaceutical Sciences Vol. 76, No. 5, May 1987
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0022-3549/87/05OO-O356$0 l.OO/O 1987,American Pharmaceutical Association
washed with water, and air dried. The product was dissolved in methylene chloride and treated with charcoal to remove the tan color. The methylene chloride solution was evaporated and the resulting product was a white solid recrystallized from ethyl acetate, mp 179-181 "C, 75% yield; 'H NMR (Me2SO-d,): 60.13-3.23 (m, 18, aliphatic), 4.93 ( 8 , 1, C-5), 5.17 ( 8 , 1, OH), 6.53-6.66 (m, 2, aromatic), 6.73 (8, 2, NH2), and 6.8-7.9 ppm (m, 4, aromatic). Anal.--Calc. for C27H2sN20K: C, 70.42; H, 6.13; N, 6.08. Found: C, 70.28; H, 6.19; N, 6.06. Naltrexone-3-Acetylsalicylate(31, Naltrexone-3-Benzoate (4), and NaltrexoneJ-Pivalate (5); (See Scheme I)-Naltrexone base (3.4 g, 0.01 mol) was typically dissolved in methylene chloride (50 mL) and triethylamine (8mL, 0.055 mol) a t 0 to 5 "C in an ice bath. A solution of the corresponding acid chloride (0.012 mol) was added in a dropwise manner with stirring. After addition was complete, the ice bath was removed and the mixture was stirred a t ambient temperature for 5 h. The methylene chloride solution was washed with 10% aqueous sodium carbonate and then with water, dried over anhydrous sodium sulfate, filtered, and reduced to a small volume by evaporation under reduced pressure. The ester was precipitated by adding excess petroleum ether. After filtering, the precipitate was air dried to provide the desired compound. Compound 3 was obtained as a white solid, mp 174-176 "C; 'H NMR (Me2SO-d6):6 0.13-2.28 (m, 10, aliphatic), 2.3 ( 8 , 3, acetylmethyl), 2.3-3.23 (m, 8,aliphatic), 5.0 ( 8 , 1, C-5), 5.16 ( 8 , 1, OH), and 6.76-8.13 ppm (m, 6, aromatic). Anal.-Calc. for C.J-129N0,*1/4 H20: C, 68.56; H, 5.85; N, 2.76. Found: C, 68.53; H, 5.89; N, 2.75. Compound 4 was obtained as a white solid, mp 174-177 "C; 'H NMR (Me2SO-d6):6 0.133.23 (m, 18, aliphatic), 4.93 ( 8 , 1, C-5h5.17 (8, 1, OH), and 6.76-8.13 ppm (m, 7, aromatic). Anal.-Calc. for C27H27NOK:C, 72.8; H, 6.1; N, 3.14. Found: C, 72.68; H, 6.18; N, 3.12. Compound 5 was obtained as a white solid, mp 161-163 "C; 'H NMR (Me2SO-d6):6 0.13-0.93 (m,5, cyclopropyl protons), 1.3 ( 8 , 9, C-(CH&,), 1.33-3.2 (m, 13, aliphatic), 4.93 ( 8 , 1, C-5), 5.17 ( 8 , 1, OH), and 6.66-6.83 ppm (m, 2, aromatic). AnaZ.--Calc. for CZbH31N06: C, 70.56; H, 7.34; N, 3.29. Found: C, 70.48; H, 7.36; N, 3.28. Analytical-Prodrug Hydrolysis in Plasm-The hydrolysis rates of 2, 3, 4, and 5 to naltrexone were determined in both dog and human plasma. Human plasma was obtained from a local blood bank and was kept frozen between the time of receipt and initiation of the experiments. The dog plasma was obtained from beagle dogs, anticoagulated with heparin, and kept frozen until the start of the experiment. Prodrug hydrolysis was performed in 3 mL of plasma at 37 "C with a prodrug concentration of 35 At various times of incubation, a 200-pL aliquot of plasma was taken and added to 200 pL of acetonitrile, which was then centrifuged. The clear supernatant was injected directly onto the HPLC column. The components of the HPLC system were a solvent delivery pump (Du Pont Instruments); an autosampler (Waters, WISP 710B); a reversed phase, 25 cm x 4.6 mm i.d., stainless steel column prepacked with 6-pm C-8 particles (Zorbax C-8, Du Pont) attached to a guard column with C-18packing; a variable wavelength spectrophotometric detector (DuPont Instruments, model 8800) set at A = 276 nm; and a recording integrator (Hewlett-Packard, model 3392A). The mobile phase contained 0.1 M acetate buffer (pH 3.8):acetonitri1e:tetrahydrofuran:sodiumheptane sulfonate (600 mL:170 mL:190 mL:lg), and the flow rate was 1.1 mumin. Retention times for the prodrugs were: 2 = 7.6, 3 = 6.5, 4 = 8.0, 5 = 7.9, and naltrexone salicylate = 8.9 min. Biwuailability-Naltrexone hydrochloride (1)was administered iv or orally to three female beagle dogs, and the prodrugs were administered orally in a crowver experiment. For iv dosing, 1 was dissolved in water, and 2.9 pmol/kg (0.5 m u g ) was injected via the cephalic vein. Compound 1 was administered orally a8 an aqueous solution (11.9 pmolkg; 1 m u g ) . Compounds 2 , 3 , 4 , and 5 were also administered as solutions of the prodrugs in 0.05 M HCI (11.9 WoUkg; 1 mukg). All oral doses were followed by the oral administration of 50 mL of water. Blood (5 mL) was collected by jugular venipuncture into evacuated tubes containing sodium EDTA as the anticoagulant. Plasma was separated and stored frozen. Animals were fasted overnight prior to each experiment. Plasma naltrexone concentrations were determined by HPLC after solvent extraction using a method similar to those previously de-
a.
scribed by Meyer et aL5 and Garrett and El-Koussi.12 Nalbuphine hydrochloride (Du Pont Pharmaceuticals) was added to plasma as an internal standard prior to extraction. Plasma aliquots (0.5 mL) were buffered with 0.2 mL of carbonate buffer (1.0 M, pH 9) and extracted twice into 4-mL volumes of to1uene:ethyl acetate:isopropyl alcohol (70:29:1). Back extractions into 0.2 mL of 0.3 M phosphoric acid were then performed. The phosphoric acid solution was injected onto the HPLC column. A 25 cm x 4.6 mm i.d., reversed-phase, stainless steel column prepacked with 6 - p n C-8 particles (Zorbax, C-8 Du Pont) was used. The mobile phase was 11-12% acetonitrile and 0.2% tetrahydrofuran in 0.055 M phosphate buffer at pH 3-4. Electrochemical detection (Bioanalytical Systems, LCB4) a t an oxidative potential of +0.98 V was used. Typical retention times for naltrexone and nalbuphine were 8 and 11 min, respectively. Calibration curves were made each day that analyses were performed. The prodrugs were tested for stability during the extraction procedure and were found to be stable. Only the plasma concentration of naltrexone and not that of the prodrugs were measured. Oral naltrexone bioavailability (F)was evaluated using the areaunder-the-plasma-concentrationversus time curve (AUC), as shown in eq 1.
(1) The AUC was calculated for each dog using the trapezoidal method with residual area calculated by dividing C (plasma naltrexone concentration) at the time of the last sample gy K (the elimination rate constant). Individual AUC, and AUCi, values were used.
Results and Discussion The hydrolysis half-lives of the prodrugs in vitro in dog and human plasma are shown in Table I. All prodrugs exhibited apparent first-order kinetics. The hydrolysis half-lives for ( t l / 2 ) for 3 and 4 were similar in both dog and human plasma. Such was not the case for 2 and 5 ; the t 1 / 2 in human plasma was longer than that in the dog. The hydrolysis rate profile of 3 in human plasma is shown in Figure 1. Compound 3 was found to rapidly hydrolyze to naltrexone salicylate ( t l l z was -0.25 min), and subsequently naltrexone salicylate hydrolyzed to naltrexone with a t1,2 of 30 min. In dog plasma, the t 1 / 2 for the first step was 5 min and that of the second step was similar to that determined for human plasma (35 m i d . Bioavailability data are shown in Table I1 and Figure 2 for the various prodrugs. Naltrexone bioavailability after oral administration of 1 was very low (1.1 0.1%),indicating extensive first-pass metabolism. Naltrexone bioavailability after oral administration of 2 and 3 was 45 and 28 times higher than that of oral naltrexone, respectively. The enhancement in naltrexone bioavailability after the administration of 4 and 5 was very much smaller. There is no
*
Table CHydrolysls Half-Llves of the Anthranllate (2), 3Acetyisallcylate (3), Benzoate (4), and the Plvalate (5) Esters of Naltrexone (1) in Human and Dog Plasma Prodrug
Hydrolysis Half-Life, h Human Plasma ~~
2
3 4 5
~
-480.004' 0.5' 2.0 7.5
Dog Plasma
--24' 0.086 0.6' 2.2 2.0
'Disappearance was followed for only 24 h. 'Compound 3 to naitrexone salicylate. 'Naltrexone salicylate to 1. Journal of Pharmaceutical Sciences / 357 Vol. 76, No. 5, May 1987
apparent correlation between the in vitro hydrolysis half-life in dog plasma and oral bioavailability. In this case, the rate of hydrolysis in plasma could not be used as a criteria for predicting in vivo characteristics of the prodrugs. For example, even if a derivative fails to generate the parent compound in plasma in vitro within a reasonable time, there is still a high probability that it may be cleaved in other organs that are rich in hydrolytic enzymes. In conclusion, the anthranilate and acetylsalicylate prodrugs were useful for enhancing the oral bioavailability of naltrexone in dogs.
1000
t I a
;
I-
100
0
03
a:E 3 a
3
10
9
z 4
4
n
'0°1
1 HOURS
Figure 2-Plasma naltrexone concentrations (mean + SEM) in dogs administered 1 iv (V); (2.9 pmollkg) and equimolar doses (1 1.9pmol/ kg) of 1 (O),2 (m), 3 (A),4 (A), and 5 (0) oral/y.
References and Notes
TIME (MINI
Figure 1-Hydrolysis of 3 in human plasma. Key: (0) disappearance of 3; (3)appearance and disappearance of naltrexone salicylate; (A) appearance of 1 .
Table II-Oral Naltrexone Bloevallablllty ( F ) In Dog8 attor Admlnlstratlon of Naltrexone ( l ) , and the Anthranllate (2), 3Acetylsallcylate (3), Benzoate (4), and Plvalate (5) Esters F, Yo
Compound Administered
Dose'
1 2
1.1 2 49.2 2 31.0 ? 2.7 2 8.0 ?
3 4 5
'Results are expressed as mean
* SEM (n = 3).
358 / Journal of Pharmaceutical Sciences Vol. 76, No. 5, May 1987
0.1 9.3 7.0 1.3
0.6
1. Fact Sheet on Naltrexone, National Clearinghouse for Drug Abuse Information, National Institute on Drug Abuse, Rockville, MD, Series 38 (1-7), December, 1978. 2. Resnick, R.; Volavka, J.; Freedman, A. M.; Thomas. M. A m . J. Psychiat. 1974,131,646-650. 3. Brahen, L.; Capone, T.; Wiechert, V.; Desiderio, D. Arch. Gen. Pqchiat. 1977,34, 1181-1184. 4. Wall, M. E.; Brine, D. R.; Perez-Reyes, M. Drug. Metab. Disp. isni.9.369-m. 5. Meyer, 'M. C.; Straughn, A. B.; Lo,M.-W.; Schary, W. L.; Whitney, C. C. J . Clin. Psychiatr. 1984,45, 15-19. 6. Konan, M. J.; Verebey, K.; Mule, S. J. Res. Commun. Chem. Pathol. Pharmacol. 1977, 18, 29-34. 7. Cone, E. J.; Gorodetzky, C. W.; Yeh, S. Y. Drug Metab. Dispos. 1974.2. 506-512. 8. Hornblad, Y.; Ripe, E.; Magnusson, P. 0.; Tegner, K. Eur. J . Clin. Pharmacol. 1976, 10, 9-18. 9. Bodor, N.; Sloan, K. B.; Higuchi, T.; Saaahar, K. J . Med. Chem. 1977,20, 1435-1445. 10. Vickers, S.; Duncan, C. A.; White, S. D.; Breault, G. 0.;Royds, R. B.: DeScheDDer. P. J.: TemDero. - . K. F. D r w Metab. Disms. 197n,8,640-6kii. ' 11. Vickers, S.; Duncan, C. A.; Ramjit, H. G.; Dobrinska, M. R.; Dollery, C. T.; Gomez, H. J.; Leidy, H. L.; Vincek, W. C. Drug Metab. Dispos. 1984, 12, 242-246. 12. Garrett, E. R.; El-Koussi, A.E.-D.A.J. Pharm. Sci. 1985,74,5056. 13. Aungst, B. J.; Myers, M. J.; Shefler, E.; Shami, E. G. Znt. J . Pharmuceut., in press. 14. Martin, W. R.; Sandquist, V. L. Arch. Gen. Psychiatr. 1974,30, 31-33. I