141, lo-16 (1984)
Radioimmunoassay of Nafarelin ([6-(3-(2-Naphthyl)-D-alanine)]-Luteinizing Hormone-Releasing Hormone) in Plasma or Serum C. NERENBERG, J. FOREMAN, N. CHU, M. D. CHAPLIN,
AND S. KUSHINSKY
Department of Analytical and Metabolic Chemistry, Syntex Research, Palo Alto, California 94304 Received October 21, 1983 A procedure which is suitable for the radioimmunoassay (RIA) of nafarelin ([6-(3-(2-naphthyl)rManine)]-luteinizing hormone-releasing hormone) in plasma or serum at concentrations as low as 50 pg/ml is described. Antiserum was prepared by replacing the pyroglutamyl portion of nafarelin with glutaric acid, coupling the product to keyhole limpet hemocyanin, and immunizing rabbits with the resulting conjugate. At a dilution of 1:30,000 the binding was approximately SO?& The antibodies did not cross react with luteinizing hormone-releasing hormone. For RIA, rz51-labeled analyte was used as the tracer and charcoal was used to separate the free and the bound fractions. No purification of samples was required prior to RIA. Accuracy of the method was assessedby adding known quantities of nafarelin to nafarelin-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.050-5.00 r&ml yielded a regression equation of y = 1.01x - 0.066 and a correlation coe&ient of 0.997. At 0.050 rig/ml the CV was 11.3% (interassay). Additional validation was obtained from an in vivo study in which [‘H]nafarelin was administered to monkeys and plasma profiles were determined by RIA, by high-performance liquid chromatography (HPLC), and by an HPLCradiochemical method. The results obtained by RIA agreed well with those obtained by the HPLC methods.
Nafarelin ([6-(3-([email protected]
)]LHRI-I),’ a potent synthetic agonist of LHRH (l), is under development for possible use in several clinical applications, such as for male and female contraception and for the treatment of prostatic and endometrial cancer. In order to provide analytical support for pharmacokinetic and toxicological studies, a radioimmunoassay was needed for the determination of natarelin in plasma or serum. The procedure described in this paper is suitable for the measurement of nafarelin at concentrations as low as 50 pg/ml.
farelin, glutaric acid analog of nafarelin, [desGlyNH:‘] nafarelin, [aza-GlyNH:‘] nafarelin, [6-lo] pentapeptide of nafarelin, [l-5] pentapeptide of nafarelin, [4- IO] heptapeptide of nafarelin, [~Tyr~] nafarelin, and [trimethoxyPhe6] nafarelin. Structures of these analogs are given in Table 1. The sources of the following materials are given in parentheses. Iodine-125 (NEZ-O33H, New England Nuclear, Boston, Mass.); dimethylformamide and sodium borate decahydrate (Mallinckrodt, St. Louis, MO.); boric acid, tris(hydroxymethyl)aminomethane, bovine serum albumin, and EDTA disodium salt (Sigma Chemical Co., St. Louis, MO.); LHRH and keyhole limpet hemocyanin (CaIbiochem-Behring, San Diego, Calif.); chloramine-T (Fisher Scientific Co., Fairlawn, N. J.); I-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (Pierce Chemicals, Rockford, Ill.); sodium
and equipment. The following were synthesized at Syntex: na-
’ Abbreviations used: Nafarelin, [6-(3-(2naphthyl)-D alanine)]-LHRH; LHRH, luteinizing hormone-releasing hormone; RIA, radioimmunoassay; BSA, bovine serum albumin. 0003-2697184 $3.06 Copyright
@ 1984 by Academic F’rcss, Inc. All rigbu of reproduction in any form reserved.
OF NAFARELIN TABLE
Compound L-Pyroglu-L-His-L-TrpL-Ser-L-Tyr-D3[2-naphthyl]Ala-L-~u-L-Arg-L-~o-L~lyNH* (nafarelin) L-Pyroglu-L-His-L-TrgL-Ser-L-Tyr-D-3[2-naph~yl]Ala-L-~u-L-A~-L-Pr~L~lyOH (nafarelin-free acid) Glutaric acid-L-His-L-TrpL-Ser-L-Tyr-D3[2-naphthyl]Ala-L-Leu-L-Arg-L-Pro-L-GlyNH* rPyroglu-L-His-L-Trp-gL-Ser+TyrOH [l-5] pentapeptide of nafarelin) D3[2-Naphthyl]Ala-L-Leu-L-Arg-L-Pro-bGlyNHz ([6-lO]pentapeptide of nafarelin) L-Ser-L-Tyr-D-3[2-naphthyl]Ala-L-Leu-L-A~-L-Pr~L~lyNH* ([4-lO]heptapeptide of nafarelin) L-Pyroglu-L-His-L-Trp-L-Ser-L-Tyr-D-3[2-naphthyl]Ala-L-~u-L-A~-L-~oOH ([desGlyNHz’o]nafarelin) L-Pyroglu-L-His-L-TrpL-Ser-L-Tyr-D3[2-naphthyl]Ala-L-~u-L-A~-L-Pr~L-NH-NHCONH~ ([aza-GlyNHz’o]nafarelin) L-470glu-L-His-L-TrpL-~r-~Tyr-~3[2-naph~yl]Ala-L-~u-L-Arg-L-~~L-GlyNH~ ([bTy?]nafarelin) L-4l0glu-L-His-L-TrL-SeT-L-Tyr-~~methoxy)Phe-L-~u-L-A~-L-~o-L~lyNH* ([trimethoxy-Phe6]nafarelin) LHRH
azide (J. T. Baker Chemical Co., Phillipsburg, N. J.); liquid scintillation fluid, &Blend (WestChem, San Diego, Calif.); Sephadex QAE-A25 (Pharmacia Fine Chemicals, Piscataway, N. J.); Norit A charcoal (Matheson, Coleman and Bell, Norwood, Ohio); Spectrapor 2 membrane dialysis tubing (Spectrum Medical Industries, Los Angeles, Calif.); gelatin (Knox Co., Englewood Cliffs, N. J.); disposable Econo-columns, 1 X 20 cm and 1 X 50 cm (Bio-Rad, Richmond, Calif.). The manufacturers or suppliers of the following equipment are given in parentheses. A Model DPR-6000 refrigerated centrifuge with a swing-out head (IEC/Damon, Needham, Mass.); a Model 3330 liquid scintillation counter (Packard Instruments, Downers Grove, Ill.); a Model 4/600 gamma counter (Micromedic Systems Inc., Horsham, Penn.); a Model FC-80h fraction collector (Gilson Medical Electronics, Inc., Middleton, Wise.); a Model 559 spectrophotometer (Perkin-Elmer, Norwalk, Conn.); a Model 2600 multitube vortex mixer (Scientific Manufacturing Industries, Emeryville, Calif.).
100.00 80.0 31.5 40.001 0.052 0.70 0.46 100.0 1.8 60.1 GO.001
Production of antiserum. The nafarelin hapten-protein conjugate was synthesized by a water-soluble carbodiimide method. Since nafarelin lacks a free carboxyl group as required for this reaction, an analog was synthesized in which the pyroglutamyl residue of nafarelin was replaced by glutaric acid. The coupling reaction was carried out by dissolving 12.0 mg (7.5 pmol) of the glutaric acid analog and 54.7 mg keyhole limpet hemocyanin (Megathura cremdata) in 4.0 ml of water. To this aqueous solution was then added, drop wise, 208 mg (0.5 mmol) of 1-cyclohexyl-3(2-morpholinoethyl)-carbodiimide metho-ptoluenesulfonate that previously had been dissolved in 1.0 ml of water (the carbodiimide dissolves in water more readily if a few drops of dimethylformamide are added). After dropwise addition of the carbodiimide was complete (5-8 min) the reaction was allowed to continue with stirring at room temperature for 3.0 h. Thereafter, the entire reaction mixture was transferred to a dialysis bag and dialyzed exhaustively against saline (9.0 g/liter). After dialysis was complete, the entire volume
in the dialysis bag (7.0 ml) was aliquoted into 0.2-ml aliquots and frozen. Six New Zealand white rabbits were immunized with the hapten-protein conjugate. The immunization emulsion was prepared by dissolving 1.4 mg of the hapten-protein conjugate in 7.0 ml of saline (9.0 g/liter) and emulsifying in 8.0 ml of Freund’s adjuvant. Freund’s complete adjuvant was used for the initial sensitization of the animals; Freund’s incomplete adjuvant was used for subsequent booster shots. Typically, 2.0 ml of this emulsion was injected into each animal at four different sites, one in each hind thigh (inside) and two at lateral sites in the intrascapular area. Each site received a cluster of five injections of 0.1 ml per injection. Booster shots were given at intervals of 3-4 weeks and titers were monitored at the same time. Alter 6-8 months, the antisera were of sufficiently good quality to be used for methods development. Iodine-125 labeling. The labeling reaction was carried out in the vial containing 1 mCi of iodine-125. To the vial were added, in the following order, 50 ~1 of phosphate buffer (0.05 M, pH 7.2), 10 ~1 (1 fig) of nafarelin, and 20 ~l(2 pg) of chloramine-T. The nafarelin and chloramine-T solutions had been prepared previously in water. The reaction was allowed to proceed for 30-40 s and then was quenched by the addition of 500 ~1 of borate buffer (0.1 M, pH 9.2, with 0.1% gelatin). This reaction mixture was transferred immediately to a QAE-A25 Sephadex column (1 X 15 cm) and eluted with the same borate buffer as described above. One-milliliter fractions were collected in 12 X 75-mm test tubes with a fraction collector and 20-~1 aliquots were used for the counting of radioactivity. A column-elution profile was plotted and the fractions corresponding to the center portion of the radioactive peak were pooled (lo-12 ml). An aliquot (1.5-2-O ml) of this pool was rechromatographed on a 1 X 30-cm QAE A-25 Sephadex column using the same borate buffer. Fractions (1 .O ml) were collected in 12 X 75-mm tubes and from the results obtained
by counting 0.1 -ml-fraction aliquots an elution profile was plotted. The fractions corresponding to the center portion of the peak (Fig. la) were pooled (IO- 15 ml) and aliquots of this pool were diluted in RIA buffer for use in the assay. Labeling reactions normally were carried out at intervals of 2-3 weeks. Radioimmunoassay, Standards for the standard curve were prepared from a primary stock solution containing 0.1 mg/ml of nafarelin in 0.1 N HCI. This stock solution was stored at 4OC for periods not exceeding 3-4 weeks. The exact concentration of this primary standard was determined by uv absorption at 275 nm (absorptivity = 9722). The primary standard was diluted in RIA buffer [tris(hydroxymethyl)aminomethane-HCl buffer, 0.1 M, pH 7.2, with 0.5% BSA, 0.001 M EDTA, and 0.00 1% sodium azide] to yield a series of standards containing 2, 5, 7, 10, 20, 30, 50, 70 pg/O.l ml. These standards were stored at 4°C and were discarded after 2-3 weeks. Antiserum was diluted in RIA buffer such that 40-50% binding was achieved. Depending on the characteristics of the antiserum, this dilution ranged from l/30,000 to l/50,000. The diluted antiserum was used for only one assay and then was discarded. The procedure for setting up the assay is as follows: add 0.1 ml of label (3000-4000 cpm), standards, unknowns, antiserum, etc., to appropriate 12 X 75-mm disposable tubes; add RIA buffer to adjust the final volume in each tube to 0.6 ml; cover the tubes with paralilm; vortex briefly to wash down the sides of the tubes; and then incubate the tubes overnight at room temperature. The separation of bound from free radioactivity was carried out using 0.2 ml of a suspension of charcoal in water (5.0 g/liter). After addition of the charcoal the entire rack of tubes was vortexed vigorously for 15 s. The tubes were then placed in an ice bath for 30 min and centrifuged, and the entire supernate was decanted into another tube for gamma counting (1.0 min). Alternatively, an aliquot (0.4 ml) was removed for liquid scintillation
8 4.000 20,000
10 20 30 40 50 60 70 FRACTIONS (ml)
30 40 50 FRACTIONS
60 70 (ml)
FIG. 1. Column elution profiles for nafarelin and ‘*‘I-nafarelin are shown on a I X 30-cm QAE-A25 column. The final purification of ‘*%nafarelin is shown in (a). The separation of tritiated nafarelin (I) and ‘251-nafarelin (II) is shown in (b).
counting (5 to 10 min). All counters were by HPLC (Cl8 PBondapak; mobile phase, 30% equipped with paper punch-tape outputs for acetonitrile in 0.17 M KH2P04). In the direct transmittal of data to computers for autoHPLC assay, quantification was based on uv mated calculation of RIA results by means of absorption at 225 nm. In the HPLC-radioa logistic program (2). Cross-reactivities were chemical assay, the concentration of nafarelin determined in accordance with the method was obtained by dividing the radioactivity described by Abraham (3). found in the appropriate HPLC fraction by Precision and accuracy. Precision and ac- the specific activity of the administered triticuracy were assessed by determining the re- ated nafarelin. covery of different quantities of nafarelin that had been added to plasma. In addition a comRESULTS AND DISCUSSION parative study of RIA, HPLC, and an HPLCradiochemical assay was carried out with The procedure described here for the “‘1 specimens collected serially from monkeys labeling of nafarelin is based on procedures following the administration of tritiated na- reported by Schally et al. (4) and Teuwissen farelin. For both types of HPLC assays, a CL8 et al. (5) for the iodination of LHRH. An reverse-phase extraction column was used for advantage of the procedure described here is preliminary purification prior to separation that the separation involves only a single buffer
FIG. 2. A typical nafarelin standard curve is shown. The antiserum dilution was 1/30,000. Total binding was 59.1%.
and is relatively easy to carry out. The rapid preliminary purification by means of the short column provides a convenient means for the
elimination of unreacted “‘1 and side products that can cause radiochemical degradation of the iodinated derivative. The partially purified derivative is relatively stable (2-3 weeks) and serves as a convenient source of starting material for the second purification step, which separates nonlabeled nafarelin from the iodinated derivative (Fig. 1b). The iodinated derivative obtained after the second purification is suitable for use in the RIA for approximately 10 days. The minimum quantity of m&elm that routinely is distinguishable statistically (P < 0.05) from zero in the RIA is 2.5 pg. A representative standard curve is shown in Fig. 2. Accuracy of the overall procedure was assessed by means of experiments on the recovery of analyte that had been added to plasma. The results of these experiments are summarized in Table 2. The average intraassay coefficient of variation calculated from the results of three separate experiments (n = 6 for
TABLE 2 ACXURACY AND PRECISIONDATA FOR THE CONCENTRATION RANGE 0.050-5.00 @ml)’ Nafamlin measured (&ml)
Nafarelin added Wml) 0.050 0.100 0.200 0.500 1.00
Expt 1 l-3
Expt 1 l-5
Mean b (96 cvy
Meanb (% cvy
Mean b (96 cvy
Group mean (% CV)d
Ratio of measured to added
0.053 (10.0) 0.102 (13.3) 0.191 (5.9) 0.488 (7.0) 0.840
0.045 (14.4) 0.094 (17.6) 0.185 (8.9) 0.538 (5.1) 0.81 (7.5) 1.74 (4.6) 5.27 (6.4)
0.043 (8.4) 0.093 (5.4) 0.173
0.047 (11.3) 0.096 (5.3) 0.183 (5.0) 0.524
1.71 (7.5) 4.97
0.547 (6.9) 0.850
(6.8) 1.70 (4.6) 5.11 (5.0)
0.96 0.92 1.05 0.83
(1.2) 5.12 (2.9)
’ Known amounts of nafarelin were added to blank plasma and assayed by this method. bn = 6. ’ Intraassay. d Interassay.
2 min 5 min 10 min 15 min 20 min 30 min lh 2h 3h 4h 5h 6h
* 15.4 f 9.8 + 4.8 f 6.1 + 5.0 + 2.9 f 2.2 f 0.7 + 0.6 + 0.4 f 0.2 f 0.2
+ SE in 3 monkeys
+ 24.4 + 12.8 f 1.9 k 4.0 +_ 4.9 f 3.5 + 3.5 + I.1 f 0.5 f 0.4 + 0.3 f 0.1
96.5 75.0 41.1 38.5 21.3 25.5 18.1
123.5 88.8 54.3 40.8 33.8 26.8 24.2 6.7 4.5 3.6 2.8 2.1
ASSAYS WITH RIA FOR THE DETERMINATION A SINGLE INTRAVENOUS Doss OF [‘HINAFARELIN
f SE in 6 monkeys
HPLC-RM 101.2 12.2 50.6 37.3 34.0 24.4 16.3 5.8 4.0 2.8 2.1 1.3
OF HPLC AND HPLC-RADIOMETRIC (HPLC-RM) IN PLASMA OF FEMALE RHESUS MONKEYS GIVEN Nafarelin
Z!Y26.1 + 9.7 + 5.6 + 4.6 f 3.1 rt_ 3.9 -+ 1.5 ND ND ND ND ND
HPLC 90.9 75.0 56.9 41.0 33.3 24.0 13.2
z!z 19.2 + 12.5 f 8.0 + 5.4 rt 6.1 -+ 3.4 k 1.9 ND ND ND ND ND
each of seven levels of spiking which ranged from 0.050 to 5.00 @ml) varied from 4.6 to 17.6%. The interassay coefficients of variation calculated from the three mean values ranged from 1.2 to 11.3% over this range of spiking. The following linear regression equation was calculated from the overall mean values: y = 1.01x - 0.066; r = 0.997. Determinations were carried out with no preliminary purification of the nafarelin in plasma or in serum. It was necessary, however, to add an equivalent aliquot of nafarelin-free plasma (either diluted or undiluted as required) to all standard curve tubes in order to minimize differences in the RIA incubation media for standards and unknowns. The data on cross-reactivity are summarized in Table 1. LHRH did not cross-react at all. Of the various synthetic analogs that were tested, only three showed significant crossreactivity. These are the glutaryl analog of nafarelin, nafarelin-free acid, and [azaGlyNH:‘] nafarelin. Of these, only the nafarelin-free acid is a possible metabolite. The extent, if any, to which this cross-reactivity of nafarelin-free acid can perturb measurements
of nafarelin in plasma or serum is not known. Independent evidence of the validity of the RIA was obtained from a comparative study in which plasma from monkeys that had been given tritiated nafarelin by intravenous injection was analyzed by RIA, by HPLC, and by an HPLC-radiochemical method. The results of this comparison are summarized in Table 3. Good agreement was found between the results for RIA and HPLC-radiochemical method (slope = 0.83, r = 0.996) and for RIA and HPLC (slope = 0.95, r = 0.982). The RIA described here has been applied to numerous studies of the bioavailability of nafarelin following administration of the drug by a variety of routes to human subjects and to nonhuman species. These applications have been reported elsewhere (6-8).
ACKNOWLEDGMENTS We are grateful to Brian Rice and Reuben Silva for performing the immunizations, and to Dr. John Nestor for the synthesis of nafuelin and the analogs of nafarelin that were used in these studies.
1. Nestor, J. J., Jr., Ho, T. L., Simpson, R. A., Homer, B. L., Jones, G. H., McRae, G. I., and Vickery, B. H. (1982) J. Med. Chem. 25, 795. 2. Rodbard, D., Hutt, D., Faden, V. B., and Huston, J. C., Jr., (1974) in RIA and Related Procedures in Medicine, pp. 165-192, International Atomic Energy Agency, Vienna. 3. Abraham, G. J. (1969) Clin. EndocrinoL-Metab. 866.
4. Piyachaturawata, P., Pedroza, E., Huang, W. Y., Arimma, and S&ally, A. V. (1980) Life Sci. 26,1309.
ET AL. 5. Teuwissen, B., Fauconnier, J. P., and Thomas, K. (1978) Gynecol. Obstet. Invest. 9, 170. 6. Hertz& M. R., Chaplin, M. D., Nerenbetg, C., Monroe, S. E., and Kushinsky, S. (1983) in Abstracts 30th Annual Meeting, Society for Gynecological Investigation, Abstr. no. 93, March 17-20, Washington, D. C. 7. Vickery, B. H., McRae, G. I., Nerenberg, C., Kushinsky, S. ( 1983) in Abstracts 16th Annual Meeting, Society for the Study of Reproduction, August 1983, Case Western Reserve University, Cleveland. 8. Anik, S. T., McRae, G., Nerenberg, C., Worden, A., Foreman, J., Hwang, J. Y., Kushinsky, S., Jones, R. E., and Vickery, B. (1984) J. Pharm. Sci. 73, 684.