EX2124
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01712
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`1
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`. FIG. 1. Standard curves of 125I—CGRP obtained with antisemm (CG-39},
`Displacement of 125I—CGRP by synthetic rCGRP(l—37) and/or
`[Ty1-°]CGRP(28~37) fragment, using (C) phosphate buffer and (V) blank
`plasma. Displacement of 125I-CGRP by rai plasma extracts (V) and un-
`extracted plasma (O).
`
`pressed as pmol/g wet weight or pmol/thyroid gland, also proved
`to be positively correlated with the plasma i—CGRP (r= .93 and
`r= .95, respectively, p<0.001) (Fig. 3). High circulating levels
`of i-CGRP were detected in rats with a higher concentration of
`i-CGRP in the thyroid gland, especially in the older rats. In ad—
`
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`350
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`50
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`150
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`Weight of the rat
`
`(1;)
`
`FIG. 2. The correlation of i-CGRP in unextracted (n: 186) (O) and C13
`Sep-Pak extracted (n=108) (V) plasma, and the weight of the rats
`(p<0.001). Linear regression analysis showed a significant correlation
`between weight of the rat and unextracted plasma (r= .88) and plasma
`following extraction with C18 Sep-Pak cartridges (r= .86) (p<0.001).
`
`cross-reactivity of the latter with other N—terminal, C-terminal
`and mid—molecular peptide fragments was <1%. Both antisera
`fully cross—reacted with 0t— and B-hCGRP and rCGRP. RIA of
`plasma and plasma extracts was carried out in two dilutions in
`duplicate using rCGRP(1—_37) as a standard, as described previ-
`ously (24,27). Sensitivity was 1.0 and 2 fmol/tube for the ex—
`traction assay and the direct plasma assay,
`respectively. At
`EDSO. the intra— and interassay variations were 6% and 9% for
`the extraction assay and 7% and 11% for the plasma assay, re-
`spectively.
`
`Gel Permeation Chromatography
`
`Pooled rat serum (40 ml) was lyophilized and reconstituted
`in 10 m1 of 0.2 mol/l ammonium acetate (pH 5.5), and centri—
`fuged at 4000><g for 20 minutes at 4°C. The supernatant was
`loaded (3 ml/chromatographic run) onto a Sephadex G-50 super—
`fine column 1.5 X 85 cm (Pharmacia) and eluted at 4°C with the
`same buffer. Corresponding fractions from each chromatograph
`were pooled and lyophilized. Prior to RIA, lyophilized material
`was dissolved in 30 ul of 10 mmol/l acetic acid and neutralized
`with 450 pl of 50 mmol/l TRIS/HCl containing 0.25% of heat-
`inactivated BSA (assay buffer). Kav values were used for the di-
`rect comparison of the elution positions of different molecular
`forms of CGRP-like immunoreactivity (10). The mean results of
`three experiments are shown in Fig. 5A.
`
`HPLC
`
`C18 Sep-Pak extracts of plasma were redissolved in 1 ml of
`28% acetonitrile in aqueous 0.15% TFA (v/v), 450 ul was load—
`ed per chromatograph into a Spherisorb ODS 5 um, 0.46X25
`cm column and eluted with a linear gradient up to 44% acetoni-
`trile in aqueous 0.15% TFA over 80 minutes. Corresponding
`fractions (1 ml) were pooled from each chromatography,
`lyo-
`philized and assayed for i-CGRP as described above (Fig. 5B).
`A further batch of 100 ml of rat plasma was extracted similarly
`after adding 125I-rCGRP (100,000 cpm) to assess recovery dur-
`ing extraction of a large volume of plasma. Following extrac-
`tion with an acid mixture (24,27) and C18 Sep—Pak purification
`(as in the previous experiment), the recovery of the added label
`was 78%.
`
`RESULTS
`
`Displacement curves obtained with C18 Sep-Pak extracted
`and unextracted plasma compared with those of synthetic rCGRP
`are shown in Fig. 1.
`i-CGRP could be detected in all plasma
`samples. When extracted with acid and C18 Sep—Pak cartridges,
`measurable i-CGRP was only 35% of that of the unextracted
`plasma. In rats weighing 200 g, the mean i—CGRP value for un-
`extracted plasma (n=32) was 105 i 11 pmol/l, and for the ex—
`tracted plasma the mean value was 35:2.8 pmol/l
`(Fig. 2).
`However, when synthetic CGRP(1—37) was added to 4 ml of
`plasma, extracted and partly purified with C18 Sep—Pak car-
`tridge, the recoveries were 84: 1.7 and 83 i 1.3 (n: 8 each ex-
`periment) for unlabeled and labeled 125I—CGRP,
`respectively.
`Regression analysis of i-CGRP in 186 of the unextracted and 108
`of the extracted rat plasma samples revealed that the level of
`i—CGRP in plasma was positively correlated (r= .88 and r= .86,
`respectively) with the age of the rat (p<0.001) (Fig. 2). Among
`the oldest rats (weight >450 g, age >52 weeks) both plasma
`i-CGRP levels and the thyroidal contents of i—CGRP were found
`to be markedly higher than other groups (Figs. 2 and 4).
`The i-CGRP levels of thethyroid gland (n298), when ex-
`‘2
`
`2
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`i—CGRPinratthyroidgland
`
`CORRELATION OF CGRP IN THYROID AND PLASMA
`
`1,00
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`Thyroidal i-CGRP (pmol/g wet weight)
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`100
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`20
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`FIG. 3. The correlation of i»CGRP in C18 Sep-Pak extracted plasma and
`thyroidal content of i-CGRP (n=98). The i-CGRP contents are ex-
`pressed as: (A) pmol/g wet weight (A) (r: .92) and (B) pmol/thyroid
`gland (O) (r=.93). Significant positive relationships are shown with
`plasma i-CGRP and thyroidal contents of i-CGRP (p<0.001).
`
`dition, i-CGRP contents in the thyroid glands were highly corre—
`lated with the weight (age) of the rat (p<0.001) (r= .88, pmol/g
`wet weight; r= .95, pmol/thyroid gland, respectively) (Fig. 4).
`The highest concentration of i—CGRP was found in the thyroid
`glands of the oldest (>52 weeks) and the heaviest rats.
`Gel permeation chromatography of rat serum revealed multi-
`ple molecular forms of i—CGRP (Fig. 5A), and only 35—40% of
`the total immunoreactivity was coeluted when checked with the
`synthetic rCGRP(l—37). In addition to the monomeric form of
`CGRP,
`there were three higher mol.wt.
`i-CGRP peaks recog-
`nized by both antisera and one smaller mol.wt. peak recognized
`only by specific antiserum to C-terminal fragment of CGRP
`(CG-39). When plasma extracts were subjected to 1p-HPLC and
`the resultant chromatographic fractions were assayed with antise—
`rum CG-39, there were three relatively hydrophilic immunoreac—
`tive peaks (Fig. 5B)
`in addition to the major
`i-CGRP peak
`coeluted with the synthetic rCGRP(l—37) (45%).
`
`DISCUSSION
`
`Unextracted plasma contains three times higher levels of
`i—CGRP in adult rats (200 g) compared to man (6). The i-CGRP
`levels measured with the extracted rat plasma, however, are
`comparable to the previously reported values in the rat (31) and
`in man (6). The present study shows a significant positive corre—
`lation between i-CGRP in both extracted and unextracted plasma
`with different stages of extra—uterine deVelopment of the rat
`(p<0.001) (Fig. 2).
`’
`
`
`
`pmol/gwetweight 100 —
`
`...
`
`[r:().95)
`
`504
`
`l
`
`H O
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`
`
`
`pmol/thyroidgland
`
`50
`
`150
`
`T
`
`l
`250
`
`l
`
`I
`350
`
`l
`
`450 >500
`
`Weight of the rat (g)
`
`FIG. 4. The correlation of weight of the rat (g) and thyroidal content of
`i--CGRP: (A) expressed as pmol/g wet weight (0) and (B) pmol/thyroid
`gland (V). Linear regressions analysis showed a significant positive re»
`lationship between the content of i—CGRP in the thyroid gland and the
`weight of the rat (p<0.001).
`
`Gel permeation chromatography of plasma revealed that only
`a third of
`the total
`i-CGRP was coeluting with synthetic
`rCGRP(l—37)
`(Fig. 5A). Furthermore,
`in rp—HPLC studies of
`plasma extracts, only 45% of the total immunoreactivity coeluted
`with the synthetic CGRP(l—37) (Fig. 5B). Therefore, this study
`revealed that the monomeric form of CGRP (presumably the ac—
`tive form) in the circulation of the rat is only about 30% of the
`total immunoreactivity.
`The higher mol.wt. forms demonstrated in this study could
`be precursor molecules, molecular aggregates or CGRP bound
`to a specific (e.g., solubilizedreceptors) or nonspecific serum
`binding protein. The smallest molecular weight form of i—CGRP
`was detected only with antiserum CG—39 and is likely to repre—
`sent the C-terminal fragment of CGRP. The difference of total
`i-CGRP detected in the extracted and unextracted plasma,
`al—
`though unlikely, could partly be due to some interference in the
`unextracted plasma, or due to the larger molecular weight forms
`or protein bound CGRP which is not retained by the C18 Sep—
`Pak cartridges (25). As the recovery of added CGRP to plasma
`prior to extraction was >80%, this further suggests that the lat-
`ter
`is
`the case. A similar phenomenon has previously been
`shown with calcitonin (CT) (1,8).
`Multiple mol.wt. forms of i-CGRP are present in the extracts
`of the thyroid glands and spinal cord, but these do not contain
`the highest mol.wt. peak (>50,000 daltons) as seen with plasma
`3
`
`3
`
`

`

`1146
`
`NO
`
`H O
`
`
`
`0/0oftotalrecovery
`
`0.4 .4
`
`o to
`
`
`
`i<rCGRP(pmol/fraction)
`
`
`
`ACN %
`
`J._;
`
`fraction number
`
`FIG. 5. (A) Gel permeation chromatographic profiles of i-CGRP in rat
`plasma. Chromatographic fractions were measured with RIA using an-
`tiserum CC—2/l (O) raised against the intact molecule of CGRP(1—37),
`and antiserum CG-39 (O)
`raised against a C—terminal
`fragment of
`CGRP(28—37). The results were expressed as the percentage of the total
`yield of i-CGRP after chromatography. (A) Dextran blue (mol.wt. 2 X 106,
`V0); (B) trypsinogen (mol.wt. 24,000); (C) lactoglobulin (mol.wt. 18,400);
`(D) horse heart cytochrome c (mol.wt. 12,400); (E) aprotinin (mol.wt.
`6500); (F) synthetic rat CGRP (mol.wt. 3900),
`(G) human calcitonin
`(mol.wt. 3200) and (H) NaCl (salt volume, V,). (B) rp-HPLC profile (5
`pm ODS, 0.46X25 cm column) of i—CGRP in rat plasma extracts.
`( i )=eluti0n position of synthetic rCGRP(l—37).
`
`in plasma
`forms present
`(27). However, some of the mol.wt.
`are likely to have originated from these tissues (22,27). These
`mol.wt. forms in plasma, as well as in the thyroid gland, do not
`dramatically change during ageing except for the increase of
`i-CGRP peak corresponding to mol.wt. 12,500. This confirms
`that the contribution of i-CGRP from the thyroid gland to the
`circulation,
`indeed,
`increases with age. We have also recently
`demonstrated the presence of multiple mol.wt. forms of i—CGRP
`in plasma of both healthy volunteers and in patients with
`MTC (22).
`It has been shown that a major portion of plasma CGRP de-
`rives from the perivascular nerves (30), and an increase of
`plasma i-CGRP and thyroidal CGRP was found in the old rats
`(31). The present study shows that there is a positive relation—
`ship of plasma i-CGRP with thyroidal CGRP which even extends
`throughout
`the extra-uterine development of the rat
`(Fig. 4).
`
`WiM.ziLA\A.«'xa..x;;,~_ix
`
`C-cells of the thyroid may undergo hyperplasia and lllls ”My
`contribute to the high incidence of MTC as reported in the gigs:
`ing rats (2). Whether this is a true tendency or due to the “:‘h
`normal diet" received by the laboratory rats, thus causing C-celi
`hyperplasia, remains to be resolved. Several authors have am,
`reported an age-related increase of plasma immunoreactive Cf
`levels (both basal and stimulated levels) in the rat (9,13). Fur»
`thermore, CT has been shown to coexist with CGRP in {11¢
`C-cells of the thyroid (7) and to cosecrete from cultured MTC
`C—cells (15). In man,
`i—CGRP in plasma also increases with ad-
`vancing age (personal observations). Therefore,
`it is likely that
`not only the proportion of CGRP, but also the total quantity lib~
`erated from the thyroid to the circulation, may also increase
`with age.
`CGRP is a potent vasodilator, but the physiological signifi-
`cance of circulating CGRP or the increased levels of i—CGRp
`associated with age (both in plasma and in the thyroid gland) is
`uncertain. Although a relative deficiency of CGRP has been inn-
`plicated as a causative factor in Raynaud’s phenomenon in man
`(4,17), its excess so far has not been correlated with disease ex-
`cept for MTC. A wide distribution of i-CGRP and its receptors
`in the cardiovascular system (26) and i—CGRP in the perivascu~
`l'ar nerves (14) suggests that
`it
`is likely to have a role in the
`control of local blood flow, through neural release of CGRP.
`It
`is tempting to postulate that the rigidity of the vasculature or the
`down-regulation of vascular receptors for CGRP with age may
`act as a stimulus to further secretion of CGRP, thereby increas-
`ing the plasma levels. Other possibilities of an increase of CGRP
`with age are an increase in the percentage of CGRP bound to
`serum proteins (e. g.,
`increase in serum CGRP binding protein)
`or a decrease of the rate of metabolism and/or clearance of
`CGRP from the circulation, and increased synthesis and release
`of CGRP from other cells. Similarly,
`in spite of the raised
`plasma immunoreactive CT in patients with C-cell hyperplasia
`and in MTC, patients do not present with biological effects of
`excess CT, such as hypocalcemia or hypophosphatemia. This
`phenomenon is most likely due to the down-regulations of both
`renal and bone receptors for CT, as CT extracted from plasma
`(and/or MTC tissues) is biologically active (12,32).
`This study, in addition to demonstrating the immunochemical
`heterogeneity of CGRP in normal rat plasma, also shows that
`the monomeric form of CGRP is only about a third of the total
`circulating i-CGRP. Furthermore, the total i—CGRP measured by
`RIA depends on the recognition of these multiple immunochem-
`ical forms (specificity), and their affinities to the antiserum used.
`We have previously shown that
`these problems can be mini~
`mized by using an assay based on native receptors, which we
`ognize only the intact mature CGRP (21). This assay has been
`successfully applied for the estimation of CGRP in a number of
`physiological and pharmacological studies (28),
`including the
`demonstration of a diurnal variation of circulating CGRP in man
`(23). The relationship of both thyroidal and plasma i—CGRP with
`age is striking (p<0.001), but its physiological significance re—
`mains to be elucidated.
`
`ACKNOWLEDGEMENTS
`
`This study was partly supported by a grant from the British Heart
`Foundation.
`I thank Dr. R. D. Gunasekera for helpful criticism of this
`manuscript and for the secretarial assistance of Mrs. Wendy Grant.
`
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`'and thyroidectomized
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`
`4
`
`2. Boorman, G. A.; van Noord, M. J .; Hollander, C. F. Naturally 00'
`curring medullary thyroid carcinoma in the rat. Arch. Pathol. 94:
`35—41; 1972.
`
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`4
`
`

`

`CORRELATION OF CGRP IN THYROID AND PLASMA
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`
`
`
`5
`
`

`

`PEPTIDES
`AN INTERNATIONAL JOURNAL
`
`Edit0r=in-Chief
`ABBA J. KASTIN
`VA Medical Center
`
`University of New Orleans and Tulane University School of Medicine
`1601 Perdido Street, New Orleans, LA 70146
`
`All manuscripts should be sent to the Editor-in—Chief for review and processing.
`EDITORIAL ADVISORY BOARD
`
`R. QUIRION (Quebec, Canada)
`H. IMURA (Kyoto, Japan)
`A. ARIMURA (New Orleans, LA)
`S. I. RAPOPORT (Baltimore, MD)
`I. IZQUIERDO (Porto Alegre, Brasil)
`A. BALASUBRAMANIAM (Cincinnati, OH)
`P. L. RAYFORD (Little Rock, AR)
`R. T. JENSEN (Bethesda, MD)
`B. E. BECKWITH (Grand Forks, ND)
`F. RIOUX (Quebec, Canada)
`G. F. KOOB (La Jolla, CA)
`J. P. H. BURBACH (Utrecht, The Netherl.)
`0. P. RORSTAD (Calgary, Canada)
`R. M. KOSTRZEWA (Johnson City, TN)
`J. CHRISTOPHE (Brussels, Belgium)
`S. I. SAID (Chicago, IL)
`J. M. LIPTON (Dallas, H)
`J. M. CONLON (Omaha, NE)
`C. A. SANDMAN (Orange, CA)
`S. M. MCCANN (Dallas, TX)
`D. H. COY (New Orleans, LA)
`A. V. SCHALLY (New Orleans, LA)
`P. W. MANTYH (Minneapolis, MN)
`T. P. DAVIS (Tucson, AZ)
`J. M. STEWART (Denver, CO)
`P. MELCHIORRI (Roma, Italy)
`D. M. DORSA (Seattle, WA)
`F. STRAND (New York, NY)
`R. P. MILLAR (Cape Town, South Africa)
`R. H. EHRENSING (New Orleans, LA)
`H. VAUDRY (Mont-Saint-Aignan, Francs»;
`T. W. MOODY (Washington, DC)
`R. EKMAN (Lund, Sweden)
`J. M. WALKER (Providence, RI)
`J. E. MORLEY (St. Louis, MO)
`E. G. ERDOS (Chicago, IL)
`J. WALSH (L05 Angeles, CA)
`E. E. MULLER (Milan, Italy)
`J. FAHRENKRUG (Copenhagen, Denmark)
`M. J. WAYNER (San Antonio, 730
`G. A. OLSON (New Orleans, LA)
`G. FEHM-WOLFSDORF (Kiel, Germany)
`R. S. YALOW (Bronx, NY)
`R. D. OLSON (New Orleans, LA)
`K. FUXE (Stockholm, Sweden)
`
`
`G. PELLETIER (Quebec, Canada)T, HOKFELT (Stockholm, Sweden) S. YEHUDA (Ramat-gan, Israel)
`PEPTIDES: AN INTERNATIONAL JOURNAL
`
`‘
`
`Editorial Offices:
`Abba J. Kastin
`VA Medical Center
`
`University of New Orleans and Tulane University School of Medicine
`1601 Perdido Street, New Orleans, LA 70146
`
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`VOLUME 12 NUMBER 5
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`SEPTEMBER/OCTOBER 1991
`ESSN 0196-9781
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`PEPTI (ES
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`AN INTERNATIONAL JOURNAL
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`Publishedby
`Pergamon Press
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`NEW YORK/OXEQRDJSEOUL/TOKYO 7 ‘
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