This material may be protected by Copyright law (Title 17 U.S. Code)
`
`1
`
`EX2124
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01422
`
`

`

`cross—reactivity of the latter with other N-terminal, C—terminal
`and mid—molecular peptide fragments was <1%. Both antisera
`fully cmss-reaeted with {X— and B-hCGRP and rCGRP. RIA of
`plasma and plasma extracts was carried out in two dilutions in
`duplicate using rCGRP(l—_37) as a standard, as described previ—
`ously (24,27). Sensitivity was 1.0 and 2 fmolr‘tube for the ex—
`traction assay and the direct plasma assay,
`respectively. At
`EDsu’ the intra— and interassay variations were 6% and 9% for
`the extraction assay and 7% and 11% for the plasma assay,
`re—
`speetively.
`
`Ge! Permeation Chromatography
`
`Pooled rat serum (40 ml) was lyophilized and reconstituted
`in 10 ml of 0.2 moh‘l ammonium acetate (pH 5.5), and centri—
`fuged at 4001ng for 20 minutes at 4°C. The supernatant was
`loaded (3 mli'chromatographic run) onto a Scphadcx (3—50 super—
`fine column 1.5 X 85 cm (Pharmaeia) 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 pl of 10 mmolll acetic acid and neutralized
`with 450 yd of 50 mmolt’l TRISJ'HCl containing 0.25% of heat—
`inactivated BSA (assay buffer). Km, values were used for the di-
`rect comparison of the eiution positions of different molecular
`forms of CGRP—Iike immunoreactivity (10). The mean results of
`three experiments are showu in Fig. 5A.
`
`HPLC
`
`C18 Sep-Pak extracts of plasma were redissolved in 1 ml of
`28% acetonitrile in aqueous 0.15% 'I‘FA (va), 450 it] was load—
`ed per chromatograph into a Spherisorb ODS 5 pm, 0.46><25
`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 m1 of rat plasma was extracted similarly
`after adding ml-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.
`l.
`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 ll pmoli‘l, and for the ex-
`tracted plasma the mean value was 35:28 pmoli'l
`(Fig. 2).
`HoWever. when synthetic CGRP(l—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 83:1.3 (n=8 each ex-
`periment) for unlabeled and labeled 'ZSI-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 (,oi0001) (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 (n=98), when ex—
`2
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`CGRP [fmol/tube]
`
`- FIG. 1. Standard curves of I"Ell—CORP obtained with antiserum (CG-39},
`Displacement of
`'zjl—CGRP hy synthetic rCGRP(|—37r') Ellldr'tsr
`[Tyr°]CGRP(28—3?) fragment, using (0) phosphate buffer and (V) blank
`plasma. Displacement of I25I—CGRF’ by rai plasma extracts (V) and nu.
`extracted plasma (0).
`
`pressed as pmolt’g wet weight or pmoli’thyroid gland, also proved
`to be positively correlated with the plasma i-CGRP (r= .93 and
`1': .95, respectively, pdlflfll) (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-
`
`5000 "
`
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`i-CGRI’inrut'plasmatpnmli’l]
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`
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`
`FIG. 2. The correlation of i—CGRP in unextracted (n = 186) (I) and C13
`Sep—Pak extracted (n=108) (V) plasma, and the weight of the rat-9
`(1040.001). Linear regression analysis showed a significant correlation
`between weight of the rat and unextracted plasma (r= .88) and plasma
`following extraction with C18 Sap—Pa]: cartridges (r= .86) (p<0.001).
`
`

`

`CORRELATION OF CORP IN THYROID AND PLASMA
`
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`
`pmol/gwetweight
`
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`Thyroidal i—CGRP (pmoltg wet weight]
`
`100
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`be
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`9-,
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`FIG. 3. The correlation of i—CGRP in CIS Sep-Pak extracted plasma and
`thyroidal content of i—CGRP (11:98). The i—CGRI‘I contents are ex—
`pressed as: (A) prnoli'g wet weight (A) (r—.92) and (B) pmoli'thyroid
`gland (O) (r=.93). Significant positive relationships are shown with
`plasma i—CGRP and thyroidal contents of i-CGRP (pinflfll).
`
`tlition. i-CGRP contents in the thyroid glands were highly corre—
`lated with the weight (age) of the rat (p<0.{)[)l) (r: .88, pmoh’g
`wet weight; r2 .95, pmolr’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 1CGRP(l—3'r').
`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 rp—HPLC and
`the resultant chromatographic fractions were assayed with antise-
`mm CG—39, there were three relatively hydrophilic immunoreac—
`tive peaks (Fig. SB)
`in addition to the major i-CGRP peak
`coclutcd with the synthetic rCGRP( 1—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<0.00|) (Fig. 2).
`'
`
`Weight of the rat. [g]
`
`FIG. 4. The correlation of weight of the rat (g) and thyroidal content of
`i--CGRP: (A) expressed as pmoii'g wet weight (0) and (B) pmolfthyroid
`gland (7). 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 (péllflfll).
`
`Gel permeation chromatography of plasma revealed that only
`third of
`the total
`i—CGRP was coeluting with synthetic
`a
`rCGRP(|—37)
`(Fig. 5A). Furthermore,
`in rp»HPl.C studies of
`plasma extracts. only 45% of the total immunoreactivity coeluted
`with the synthetic CGRPU—BT) (Fig. SB). Therefore, this study
`revealed that the monotnerie 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., solubilized'receptors) or nonspecific serum
`binding protein. The smallest molecular weight form of i—CGRP
`was detected only with antiserum CCi-39 and is likely to repre—
`sent
`the C-tcrminal fragment of CGRP. The difference of total
`i—CGRP detected in the extracted and unextractcd plasma,
`al—
`though unlikely, could partly be due to some interference in the
`unextracted plasma. or dtte 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 (>50,0[)U daltons) as seen with plasma
`3
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`C— cells of the thyroid may undergo hyperplasia and thin Ism-
`contribute to the high incidence of MTC as reported in the ;[.It_
`ing rats (2). Whether this is a true tendency or due to the ‘ iih.
`normal diet“ received by the laboratory rats, thus causing {103”
`hypei'plasia, remains to be resolved. Several authors have also
`reported an age—related increase of plasma immunoreactive [1‘
`levels (both basal and stimulated levels) in the rat (9,13). Fur-
`thermore, CT has been shown to coexist with CGRP it:
`the
`C-cclls 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 im.
`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 perivnscu-
`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 andior clearance of
`CGRP front 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 hyperpiasia
`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
`(andior 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 antisetum used.
`We have previottsly shown that
`these problems can be mini—
`mized by using an assay based on native receptors, which rec-
`ognize only the intact mature CGRP (2]). 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 (1:40.001), but its physiological significance re-
`mains to be elttcidated.
`
`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.
`
`FIG. 5. (A) Gel permeation chromatographic profiles of i—CGRP in rat
`plasma. Chromatographic fractions were measured with RlA using an—
`tiserum CC—Zil'l (O) raised against the intact molecule of CGRP(l—37)_
`and antiserum CG—39 (O)
`raised against a C—terminal
`fragment of
`CGRP(28-—3T). The results were expressed as the percentage of the total
`yield of i-OGRP after chromatography. (A) Dcxtran blue (molwt. 2 X 106,
`V0); (B) trypsinogen (mol. wt 24”000) (C) lactoglobulin (mol. wt IS 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
`am ODS, 0.46X25 cm column) of i—CGRP in rat plasma extracts.
`( .I, )= elution position of synthetic rCGRP(l—3T).
`
`forms present in plasma
`(27). However, some of the moi.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).
`
`J .; Heath. H., Ill. Estimation of circulating monomeric
`1. Body, J.
`caleitonin: Physiological studies in normal and thyroidectomized
`man. J. Clin, Endocrinol. Metab. 571897—903; 1983.
`
`4
`4
`
`2. Boorman, G. A.', van Noord, M. 1.; Hollander, C. F. Naturally oc-
`cttrring tnedullary thyroid carcinoma in the rat. Arch. Pathol. 94:
`35—41; 1972.
`
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`
`

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`5
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`

`PEPTIDES
`AN INTERNATIONAL JOURNAL
`
`Editor-in-Chief
`ABBA .l. 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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`EDITORIAL ADVISORY BOARD
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`I. IZQUIERDO (Porto Alegre. Bras-iii
`5. I. RAPOPOR’I' (Baltimore. MD]
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`8. YEHUIJA (Rental-gait. Local)
`PEPTIDES: AN INTERNATIONAL JOURNAL
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`VA Medical Center
`
`University of New Orteans and Tulane University School of Medicine
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`VOLUME 12 NUMBER 5
`
`,
`
`SEPTEMBER/OCTOBER 1991
`ISSN 0196-9791
`
`PEPTIDES
`
`AN INTERNATIONAL JOURNAL
`
` Published by
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`i a \1
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`Pergamon Press
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