356 Kohno et al.

© 1998 Wiley-Liss, Inc.

Journal of Clinical Laboratory Analysis 12:356–362 (1998)

Determination of the Bioavailability of Intranasal Elcatonin inHumans: Development of a Sandwich Transfer Enzyme

Immunoassay for ElcatoninTakeyuki Kohno, 1* Noriaki Murasugi, 1 Hiroki Sakurai, 1 Kazuhito Watabe, 1

Hiromichi Nakamuta, 2 Masao Koida, 2 Yohko Sugie, 3,4 Toshiyo Ogouchi, 5

Tadashi Inoue, 5 Masao Yanaka, 5 Masakatsu Nomura, 6 and Akira Yanagawa 6

1Department of Environmental Health Sciences, Faculty of Pharmaceutical Sciences,Setsunan University, Osaka, Japan

2Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan3Laboratory of Diagnostics, Research Institute for Production Development, Kyoto, Japan

4Department of Clinical Research, Pharmaplot Co., Ltd., Shiga, Japan5Fujiyakuhin Co., Ltd., Saitama, Japan

6Department of Pharmacotherapeutics, St. Marianna University School of Medicine, Kanagawa, Japan

Grant sponsor: Ministry of Education, Science, Sports and Culture of Japan,Grant-in-Aid for Scientific Research; Grant numbers: 05857262, 06557136,and 09470514.*Correspondence to: Takeyuki Kohno, Ph.D., Associate Professor of De-partment of Environmental Health Sciences, Faculty of PharmaceuticalSciences, Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan.Received 26 January 1998; Accepted 23 July 1998

A sandwich transfer enzyme immunoas-say for elcatonin (ECT) and its usability forthe pharmacokinetic study are described.The anti-salmon calcitonin (SCT) antibodywas used for the present assay. The assayprocedure consisted of the reaction of ECTwith 2,4-dinitrophenylbiotinyl anti-SCT IgGand anti-SCT Fab′-β-D-galactosidase conju-gate, trapping onto (anti-2,4-dinitrophenyl bo-vine serum albumin) IgG-coated polystyreneballs, eluting with εN-2,4-dinitrophenyl-L-lysine and transferring to streptavidin-coatedpolystyrene balls and fluorometric detectionof β-D-galactosidase activity. The practicaldetection limit of ECT was 0.15 pg (44amol)/50 µl of sample and 3 pg/ml as theconcentration. The application of this method

has enabled us to directly estimate thebioavailability of ECT dosed intranasaly ata therapeutic level (100 IU, 17 µg) for itsanti-osteoporotic effect as compared to anintramuscular dose (40 IU, 6.7 µg). The phar-macokinetic parameters of the intranasalECT (n = 6) thus estimated were as follows:the area under the serum concentration-timecurve (AUC) = 2,570 ± 1,650 (SD) pg × min/ml, and the maximal concentration (Cmax)= 60 ± 25 (SD) pg/ml with the maximaltime (Tmax) = 17.5 ± 6.9 (SD) min, when theAUC for the intramuscular ECT (n = 9) = 9,460± 5,870 (SD) pg × min/ml and the Cmax =165 ± 79 (SD) pg/ml with the Tmax = 16.1 ±4.2 (SD) min. J. Clin. Lab. Anal. 12:356–362,1998. © 1998 Wiley-Liss, Inc.

Key words: pharmacokinetics; osteoporosis; osteomalacia; thyroid; hormone

INTRODUCTION

Calcitonins are a family of natural inhibitors of the mam-malian osteoclast (1–3) and have been successfully prescribedas the therapeutic for osteoporotic diseases caused by patho-logically increased osteoclastic activity (4). Elcatonin (ECT)is an eel calcitonin analogue that has a dicarbon bridge in-stead of the natural disulfide bridge, rendering the moleculemore stable biologically than eel calcitonin (4,5). In spite ofthe established therapeutic effectiveness of ECT, however,there is no knowledge about its pharmacokinetic propertiesdue to the lack of a sensitive assay method.

We recently have described a highly sensitive enzyme im-munoassay for salmon calcitonin (SCT) (6–8). The applica-tion of the assay method had enabled us to directly estimatethe bioavailability of SCT dosed subcutaneously (6) and in-

tranasally (7,8) at the therapeutic levels in rats (6,7) andhumans (8).

The aims of this study are: 1) to develop a sensitive en-zyme immunoassay technique for ECT, and 2) to test the ap-plicability for the pharmacokinetic study of intranasal ECTto be dosed to humans.

Bioavailability of Elcatonin 357

MATERIALS AND METHODS

Calcitonins

Elcatonin (ECT, Lot No. EC-6), which was synthesized bythe method of Sako et. al. (Japanese patent application num-ber 03325035), was obtained from Bio-Chiba Co., Ltd., To-kyo, Japan. The biological activity (6,000 IU/mg) and thepurity (≥ 99 %) were estimated by the methods of Nogata etal. (9) and Kimura et al. (10), respectively. Salmon calcitonin(SCT, SMC 20-051, Lot No. 91049; 4,220 IU/mg (EuropeanPharmacopoeia method)) was a gift from Sandoz Pharma-ceuticals, Ltd. (Basel, Switzerland). Human calcitonin (LotNo. 111H49501, purity ≥99%), synthesized by the method ofSieber et al. (11), was obtained from Sigma Chemical Co. (StLouis, MO).

2,4-Dinitrophenyl Bovine Serum Albumin

Thiol groups were introduced into bovine serum albuminmolecules using N-succinimidyl-S-acetylthioacetate (12) andwere reacted with maleimide groups introduced into eN-2,4-dinitrophenyl-L-lysine molecules using N-succinimidyl-6-maleimidohexanoate (13). The amounts of bovine serumalbumin, 2,4-dinitrophenyl groups and 2,4-dinitrophenyl bo-vine serum albumin were calculated from the absorbance at280 and 360 nm (14). The average number of 2,4-dinitrophenyl groups introduced per bovine serum albuminmolecule was 6.0 (13).

Antisera

Albino rabbits (New Zealand White, b.w., 2.5–3.0 Kg) werefirst intracutaneously injected with SCT (100 µg) and 2,4-dinitrophenyl bovine serum albumin (100 µg) in 1.5 ml ofFreund’s complete adjuvant (Nakalai Tesque, Inc., Kyoto,Japan), and thereafter twice boosted with SCT (50 µg) and2,4-dinitrophenyl bovine serum albumin (50 µg) in 1.5 ml ofFreund’s incomplete adjuvant (Nakalai Tesque, Inc.) at 3-weekintervals. Blood was collected two weeks after the last injec-tion, and the antisera were stored at –80°C.

IgG and Its Fragments

IgG was prepared from serum by fractionation with Na2SO4

followed by passage through a column of diethylaminoethyl-cellulose (15). F(ab′)2 was prepared by digestion of IgG withpepsin from porcine gastric mucosa (15), and Fab′ was pre-pared by reduction of F(ab′)2 with 2-mercaptoethylamine (15).The amounts of IgG and its fragments were calculated fromthe absorbance at 280 nm (15).

2,4-Dinitrophenyl Biotinyl Anti-SCT IgG

Rabbit anti-SCT IgG (23 mg) in 0.1 mol/l sodium phos-phate buffer, pH 7.0 (2.4 ml) was incubated with 33 mmol/lN-succinimidyl-S-acetylthioacetate (Research Organics Inc.,

Cleveland, OH) in N,N-dimethylformamide (0.24 ml) at 30°Cfor 30 min. The reaction mixture was incubated with 0.12 mlof 0.1mol/l EDTA, pH 7.0 and 0.31 ml of 1 mol/l hydroxy-lamine · HCl, pH 7.0 at 30°C for 15 min. After incubation,the reaction mixture was subjected to gel filtration on a col-umn (1 × 30 cm) of Sephadex G-25 (Pharmacia LKB Bio-technology, Uppsala, Sweden) using 0.1 mol/l sodiumphosphate buffer, pH 6.0, containing 5 mmol/l EDTA. Theaverage number of thiol groups introduced per IgG moleculewas 13 (15). The mercaptoacetylated IgG (22 mg) in 0.1 mol/l sodium phosphate buffer, pH6.0, containing 5 mmol/l EDTA(5.9 ml) was incubated with the maleimide-biocytin solution(8) (1.5 ml) at 30°C for 30 min. The average number of biotinresidues introduced per the mercaptoacetylated IgG moleculewas 5.1, which was calculated from the decrease in the num-ber of thiol groups (15). The mercaptoacetylated biotinyl IgGsolution (0.68 ml) was incubated with the maleimide-eN-2,4-dinitrophenyl-L-lysine solution (13) (0.11 ml) at 30°C for 30min. After incubation, the reaction mixture was subjected togel filtration on a column (1 × 30 cm) of Sephadex G-25 us-ing 10 mmol/l sodium phosphate buffer, pH 7.0, containing0.1 mol/l NaCl, 1 mmol/l MgCl2, 1 g/lNaN3, and 0.1 g/l bo-vine serum albumin (fraction V, Intergen Co., Purchase, NY).The average number of 2,4-dinitrophenyl groups introducedper IgG molecule was 6.3, which was calculated from theabsorbance at 280 and 360 nm (14,15). The amount of the2,4-dinitrophenylbiotinyl IgG was calculated from the absor-bance at 280 and 360 nm (14,15).

SCT-BovineThyroglobulin Conjugate

SCT was reduced with dithiothreitol and conjugated tobovine thyroglobulin using N-succinimidyl-6-maleimido-hexanoate as described previously (6). The conjugate wasquantified by using the commercial protein assay kit (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories, Richmond, CA)using bovine thyroglobulin as a standard.

Protein-Coupled Sepharose 4B

SCT-bovine thyroglobulin conjugate and 2,4-dinitrophenylbovine serum albumin were coupled to CNBr-activatedSepharose 4B according to the instructions of Pharmacia LKBBiotechnology (5,14).

Affinity Purification of Antibodies

Anti-SCT F(ab′)2 and 2,4-dinitrophenyl biotinyl anti-SCTIgG were affinity-purified using columns of SCT-bovine thy-roglobulin conjugate-coupled Sepharose 4B (6). Anti-2,4-dinitrophenyl bovine serum albumin IgG was affinity-purifiedusing a column of 2,4-dinitrophenyl bovine serum albumin-coupled Sepharose 4B (14). The specific antibodies wereeluted from the corresponding columns with 3.2 mmol/l HCl,pH 2.5, and neutralized by the addition of 1 mol/l sodiumphosphate buffer, pH 7.0 (16).

358 Kohno et al.

Affinity-Purified Anti-SCT Fab ¢-b-D-GalactosidaseConjugate

Affinity-purified rabbit anti-SCT Fab′ was conjugated to β-D-galactosidase from E. coli (enzyme label for enzyme immu-noassay, Boehringer Mannheim GmbH, Mannheim, Germany)using o-phenylenedimaleimide (15). The amount of conjugatewas calculated from β-D-galactosidase activity (17).

Biotinyl Nonspecific Rabbit IgG

Thiol groups were introduced into nonspecific rabbit IgGusing N-succinimidyl-S-acetylthioacetate as described aboveand were reacted with the maleimide-biocytin solution (8).The average number of biotin residues introduced per IgGmolecule was 13, which was calculated from the decrease inthiol groups (15).

Protein-Coated Polystyrene Balls

Polystyrene balls (Immuno Chemical, Inc., Okayama, Ja-pan) were coated with affinity-purified rabbit (anti-2,4-dinitrophenyl bovine serum albumin) IgG (0.1 g/l), andbiotinyl nonspecific rabbit IgG (0.1 g/l) by physical adsorp-tion (18). Streptavidin-coated polystyrene balls were preparedby incubating biotinyl nonspecific rabbit IgG coated-poly-styrene balls with streptavidin (0.1 g/l, GIBCO BRL Life-Technologies, Inc., Gaithersburg, MD) (19).

Human Serum

Blood samples were withdrawn into glass tubes (Veno-ject VT-SA, Termo Corp., Tokyo, Japan), and centrifugedto separate the serum. Serum samples were obtained fromeight healthy subjects (four males and four females, aged22–40 years).

Calcitonin Standards

ECT and human calcitonin were dissolved in 33 mmol/lsodium acetate buffer, pH 4.1, containing 0.1 mol/l NaCl at aconcentration of 1 mg/ml and diluted with a pooled serumfrom the eight healthy subjects and used as standards. In someexperiments (recovery test), ECT was diluted with 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl,1mmol/lMgCl2, 1 g/l NaN3, and 1 g/l bovine serum albumin.

Sandwich Transfer Enzyme Immunoassay for ECT

An assay sample (ECT or human calcitonin standards orserum samples, 0.05ml) were incubated with 2,4-dinitrophenylbiotinyl affinity-purified anti-SCT IgG (50 fmol) and affin-ity-purified anti-SCT Fab′-β-D-galactosidase conjugate (20fmol) in 10 mmol/l sodium phosphate buffer, pH 7.0, con-taining 0.55 mol/l NaCl, 1 mmol/l MgCl2, 1.5 mmol/l 2-mercaptoethylamine, 1 g/l NaN3, and 1 g/l bovine serumalbumin (0.1 ml) at 20°C for 2 hr. The reaction mixture was

incubated with two colored polystyrene balls coated with af-finity-purified (anti-2,4-dinitrophenyl bovine serum albumin)IgG at 20°C overnight. After incubation, the colored polysty-rene balls were washed twice by addition and aspiration of 2ml of 10 mmol/l sodium phosphate buffer, pH 7.0, contain-ing 0.1 mol/l NaCl, 1mmol/l MgCl2, 1 g/l NaN3, and 0.1 g/lbovine serum albumin. The washed polystyrene balls wereincubated with 1mmol/l eN-2,4-dinitrophenyl-L-lysine ⋅ HCl(Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) in 10 mmol/lsodium phosphate buffer, pH 7.0, containing 0.1mol/l NaCl,1 mmol/l MgCl2, 1 g/l NaN3, and 1 g/l bovine serum albumin(0.15ml) and two white polystyrene balls coated withstreptavidin at 20°C for 1 hr. The colored polystyrene ballswere removed, and the incubation was continued at 20°C for2 hr. The white polystyrene balls were washed as above, andβ-D-galactosidase activity bound to the white polystyrene ballswas assayed by fluorometry (20) as follows. The white ballswere preincubated with 10 mmol/l sodium phosphate buffer,pH 7.0, containing 0.1 mol/l NaCl, 1 mmol/l MgCl2, 1 g/lNaN3, and 0.1 g/l bovine serum albumin (0.1 ml) at 30°C for5 min. The enzyme reaction was initiated by addition of 0.3mmol/l 4-methylumbelliferyl-β-D-galactopyranoside (0.05ml) (Boehringer Mannheim GmbH) and incubated at 30°Cfor 150 min. The enzyme reaction was then terminated by theaddition of 0.1 mol/l glycine-NaOH buffer, pH 10.3 (2.5 ml)and the fluorescence intensity was measured using aspectrofluorophotometer (F-3010, Hitachi,Tokyo, Japan) at360 nm for excitation and 450 nm for emission. The scales of0 and 100 were adjusted by using 0.1 mol/l glycine-NaOHbuffer, pH 10.3, and 10–8 mol/l 4-methylumbelliferone(Nakalai Tesque, Inc.) in the same buffer, respectively. Thefluorescence intensity was measured relative to the 4-methyl-umbelliferone solution (20).

Measurement of Bioavailability

The participants were fifteen healthy postmenopausalwomen (aged 48–64 years, menopausal age ≥ 3 years, es-tradiol < 25 pg/ml, FSH ≥ 50 mU/ml, ± 20% of ideal bodyweight). None of them had previously been treated withcalcitonin and bisphosphonate. They had taken no sex hor-mones and anti-osteoporotic drugs within 4 mon. Nonehad a history of allergic reactions and bronchial asthma.Six of them were administered ECT (100 IU (17 µg), LotNo. I001QR100, NDE-1003, Fujiyakuhin Co., Ltd.,Saitama, Japan) intranasally.

Intranasal ECT formulation was prepared by mixingECT solution with calcium carbonate powder and freeze-drying. They loaded the capsule containing the ECT for-mulation on the novel device Jetlizer® (UNISIA JECS, Co.,Gunnma, Japan) and puffed it by themselves five timesinto both nasal cavities. The detailed procedures for theformulation and the device will be published elsewhere.Nine of them were administered ECT (40 IU (6.7µg), Lot

Bioavailability of Elcatonin 359

No. I119QK, Elbestal® Inj., Fujiyakuhin Co., Ltd.) intra-muscularly. Before and after (5, 10, 15, 20, 30, 45, 60, 90,120, and 180 min) administration, blood samples werewithdrawn, and centrifuged to separate the serum. Serumsamples were kept at –80°C until assayed. The ECT con-centration was measured in duplicate by the sandwichtransfer enzyme immunoassay as described above, and wascalculated by curve-fitting using the ECT serum standards.The area under the serum concentration-time curve (AUC)was calculated with a personal computer using a linear trap-ezoidal equation (21,22). Informed consent was obtainedfrom each participant, and the protocol was approved by theInstitutional Review Board of Medical Corp. Seikoukai, NewMedical Research System Clinic, Tokyo, Japan.

RESULTS

Sandwich Transfer Enzyme Immunoassay forElcatonin

Concept of the assay procedure

The assay procedure was designed to proceed as follows:1) formation of immune complex consisting of elcatonin(ECT), 2,4-dinitrophenyl biotinyl anti-salmon calcitonin(SCT) IgG and anti-SCT Fab′-β-D-galactosidase conjugate,2) trapping of the immune complex onto polystyrene ballscoated with (anti-2,4-dinitrophenyl group) IgG, 3) elimina-tion of unreacted Fab′-β-D-galactosidase conjugate by spe-cific elution of the immune complex from the polystyreneballs with eN-2,4-dinitrophenyl-L-lysine, 4) the final trap-ping of the immune complex onto streptavidin-coated poly-styrene balls, and 5) measurement of the β-D-galactosidaseactivity bound to the streptavidin-coated polystyrene ballsby fluorometry. The whole procedure was completed withintwo days.

In the present assay, anti-SCT antibody was used to mea-sure ECT. The reason for this will be described in the Discus-sion section.

Sensitivity and Specificity

ECT was diluted with a pooled serum from eight healthysubjects as described in the Materials and Methods section,and subjected to the present assay. The assay was able to de-tect 0.15 pg (44 amol)/50 µl of a given sample, that is theminimum concentration for detection being 3 pg/ml (Fig. 1).The detection limit was taken to be the minimal concentra-tion obtained 2 SD (n = 8) above the serum background. Thestandard curve was linear up to a concentration of 1,000 pg/ml (Fig. 1). The assay is specific for ECT and practically nointerference occurred by human calcitonin up to10 ng/ml (Fig.1). In addition, the background ECT concentration in the eightsera (0.33 ± 0.99 pg/ml, ranged from –1.1 to 1.7 pg/ml) (Table1, left column) was lower than the detection limit (3 pg/ml),

indicating that normal human serum appears to contain nosubstance detected as ECT.

Assay Variation

When examined at three different serum levels in the rangeof 10–1,000 pg/ml for within-assay and between-assay varia-tion, the CVs were 3.8–8.5% (n = 8) and 3.9-8.6 % (n = 8),respectively (Table 2). The samples using the assay varia-tion were prepared by mixing ECT with the pooled serum.

The ECT serum standards (0, 10, 100, and 1,000 pg/ml)

Fig. 1. Dose-response curves for calcitonin by sandwich transfer enzymeimmunoassay. Circles and triangles indicate the curves for elcatonin (ECT)and human calcitonin, respectively, in the presence of a pooled serum fromeight healthy subjects. Each value indicates the mean of 4 determinations(ECT 0–10 pg/ml), 3 determinations (ECT 30–3,000 pg/ml) and 2 determi-nations (human calcitonin), and small horizontal bars with vertical bars in-dicate ± SD.

TABLE 1. The Background in the Presence of HumanSerum and the Recovery Rate of Elcatonin Mixed WithHuman Serum

Recovery rate of elcatoninmixed with seruma (%)Elcatonin concentration

Serum Background (pg/ml)

no. (pg/ml as elcatonin) 20 200

1 1.1 95 872 –0.8 110 963 1.1 90 824 0.3 95 895 –1.1 100 966 –0.3 100 867 1.7 85 868 0.6 90 98

aRecovery rate was calculated by curve-fitting of a standard curve in theabsence of serum.

360 Kohno et al.

were subjected to the present assay eight times; the CVs forfluorescence intensity for specifically bound β-D-galactosi-dase activity were 12.0–14.3% (Fig. 2). The between-assayvariation and ECT standards variation were run over a pe-riod of 26 days.

Recovery

ECT was mixed with eight serum samples at concen-trations of 20 and 200 pg/ml and assayed. The recoveryrates of ECT were 96 ± 7.8 (SD)%(ranged 85 to 110%,CV = 8.1%) and 90 ± 5.9 (SD)% (ranged 82 to 98%, CV =6.6%) (Table 1, middle and right columns), respectively,which were calculated by curve-fitting of a standard curvein the absence of serum.

Stability of ECT in Serum Matrix

First, ECT was mixed with the pooled serum at concentra-tions of 100 and 1,000 pg/ml, incubated at 20°C or 37°C for0–2 hr, and subjected to the present assay. The recovery ratesof ECT in the serum matrix were satisfactory until 2-hr incu-bation at 20°C and 1-hr incubation at 37°C (Fig. 3a).

Second, ECT was mixed with the pooled serum at concen-

trations of 20, 200, and 1,000 pg/ml, stored at –80°C for 0–12 mo, and assayed. The recovery rates of ECT were satis-factory until 12 mo storage (Fig. 3b).

Third, ECT was mixed with the pooled serum at concen-trations of 100 and 1,000 pg/ml, and subjected to freezing(–80°C) and thawing (room temperature) three times. The

TABLE 2. Within-Assay and Between-Assay Imprecision

Elcatonin level Elcatonin level Coefficientadded to serum Number of detected mean ± SD of variation

Assay (pg/ml) determinations (pg/ml) (%)

Within-assay 10 8 10.6 ± 0.9 8.5100 8 110 ± 7.0 6.4

1000 8 1095 ± 42 3.8Between-assay 20 8 19.7 ± 1.7 8.6

200 8 197 ± 17 8.62000 8 963 ± 38 3.9

Fig. 2. Intermediate precision of standard curve for elcatonin.The elcatoninstandards (0, 10, 100, and 1,000 pg/ml) were assayed by the present assayeight times, and the variation coefficients for fluorescence intensity for spe-cifically bound β-D-galactosidase activity were calculated.

Fig. 3. Stability of elcatonin (ECT) in serum matrix. (a) ECT was mixedwith a pooled serum at concentrations of 100 pg/ml (circles) and 1,000 pg/ml (triangles), incubated at 20°C (open symbols) or 37°C (closed symbols),and assayed. (b) ECT was mixed with a pooled serum at concentrations of20 (circles), 200 (triangles), and 1,000 (squares) pg/ml, stored at –80°C, andassayed. (c) ECT was mixed with a pooled serum at concentrations of 100pg/ml (circles) and 1,000 pg/ml (triangles), subjected to freezing-thawing,and assayed.

Bioavailability of Elcatonin 361

thawed samples were assayed. The recovery rates were satis-factory until three freeze-thaw cycles (Fig. 3c).

Bioavailability of Intranasal ECT

Figure 4 shows the appearance-disappearance patternof intranasal ECT (100 IU, 17 µg) and intramuscular ECT(40 IU, 6.7 µg) in humans. The areas under the serum con-centration-time curve (AUCs) were calculated to be 2,570± 1,650 (SD) and 9,460 ± 5,870 (SD) pg × min/ml, re-spectively (Table 3). The comparative AUC (bioavail-ability) of intranasal ECT as compared with intramuscularECT was 11%. The maximal concentration (Cmax) of in-tranasal ECT was calculated to be 60 ± 25 pg/ml with themaximal time (Tmax) of 17.5 ± 6.9 min and the Cmax ofintramuscular ECT was calculated to be 165 ± 79 pg/mlwith the Tmax of 16.1 ± 4.2 min (Table 3).

DISCUSSION

This is the first study in which the bioavailability of intra-nasal elcatonin (ECT) in humans has been directly measured.The detection limit of elcatonin by the present assay was 3pg/ml and was at least 20-fold lower than other immunoas-say methods reported so far (23,24).

The reasons that anti-salmon calcitonin (SCT) antibodywas used in the present assay were as follows. First, theamino acid sequence of N-terminal portion (residues 1–7), forming a ring structure, is common to mammals andfish, so that the antigenicity of the portion was expectedto be low. In fact, we had failed to prepare antibody againstthe ring structure using conventional and SPF rabbits.

Second, anti-ECT antibodies were prepared by immuniz-ing rabbits with ECT-ovalbumin (OVA) and with ECT, andthen were affinity-purified and labeled to develop assaymethods. However, the detection limit was too high toexamine pharmacokinetic properties of intranasal ECT.The ECT-OVA was prepared by introducing thiol groupsinto ECT using NH2 reactive cross-linking reagent. Andthe specific antibodies were purified by using Sepharose4B, which had linked with NH2 groups on ECT. Accord-ingly, the modification of amino groups of ECT (N-termi-nal, Lys11, and Lys18-ECT) might cause damage toantigenicity. The above result also suggested that the anti-genicity of C-terminal portion (residues 26–31) includingone of ECT specific region is not satisfactory. Thus, it wasconsidered that antibodies against the central portion ofECT might be essential to develop the highly sensitivemethod. The central portion of ECT (residues 8–25) isidentical with SCT; therefore, anti-SCT antibodies whichrecognize the central portion can be used to measure ECT.The antibody used in the present assay recognized the cen-tral portion. This was determined by using the EPITOPESCANNING KIT (Chiron Mimotopes Pty Ltd., ClaytonVictoria, Australia).

In terms of specificity, no degradation product of ECT andserum protein cross-reacted with the antibody has been foundin serum samples of volunteers injected intramuscularly withECT. This was examined by HPLC using a column ofCOSMOSIL 5C 18-P.

The detection limit of ECT by the present assay (3 pg/ml)is closer to the minimum effective concentration (approxi-mately 1 pM) of calcitonin that inhibits pit formation by os-teoclasts in vitro (25,26). Therefore, the assay developedherein can provide us with a reliable and sensitive methodwith which to estimate the clinical bioavailability of ECTapplied in a therapeutic dose range to humans.

ACKNOWLEDGMENTS

We are grateful to Sandoz Pharmaceuticals, Ltd. (Basel, Swit-zerland and Tokyo, Japan) for providing SCT and also to Dr. S.Yamashita, Department of Pharmacy, Faculty of Pharmaceuti-cal Sciences, Setsunan University, for his valuable suggestionson the evaluation of pharmacokinetic studies.

TABLE 3. Pharmacokinetic Parameters After ElcatoninAdministration

AUCa Cmaxb Tmaxc

Dose mean ± SD mean ± SD mean ± SD(IU) (pg·min/ml) (pg/ml) (min)

Intranasal 100 2570 ± 1650 60 ± 25 17.5 ± 6.9Intramuscular 40 9460 ± 5870 165 ± 79 16.1 ± 4.2

aArea under the serum concentration-time curve.bMaximal concentration.cMaximal time.

Fig. 4. Concentration of elcatonin (ECT) after an intranasal administra-tion of ECT (100 IU (17 µg)/subject, n = 6, circles) and intramuscular ad-ministration of ECT (40 IU (6.7 µg)/subject, n = 9, triangles) to human.Each value indicates the mean, and small horizontal bars with vertical barsindicate ± SD.

362 Kohno et al.

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