Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 1 of 123 PageID #: 610
`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 1 of 123 PagelD #: 610
`
`
`
`
`
`EXHIBIT 1
`EXHIBIT 1
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 2 of 123 PageID #: 611
`
`As filed with the Securities and Exchange Commission on April 8, 2002
`
`SECURITIES AND EXCHANGE COMMISSION
`WASHINGTON, D.C. 20549
`
`FORM 20-F
`
`REGISTRATION STATEMENT PURSUANT TO SEGION 12(b) OR (g)
`OF THE SECURITIES EXCHANGE AG OF 1934
`OR
`ANNUAL REPORT PURSUANT TO SEGION 13 OR 15(d)
`OF THE SECURITIES EXCHANGE AG OF 1934
`For the fiscal year ended December 31, 2001
`OR
`TRANSITION REPORT PURSUANT TO SEGION 13 OR 15(d)
`OF THE SECURITIES EXCHANGE AQ OF 1934
`to
`For the transition period from
`
`Commission file number: 1-10378
`
`0
`
`0
`
`Ave ntis
`
`(Exad name of Registrant as specified in its charter)
`Republic of France
`Not applicable
`(Translation of Registrant's name into English)
`aurisdidion of incorporation or organization)
`67917 Strasbourg cedex 9
`France
`(Address of principal executive offices)
`
`Securities registered or to be registered pursuant to Section 12(b) of the Act:
`
`Title of each class:
`
`Name of each exchange on which registered
`
`American Depositary Shares, each representing one Ordinary Share
`nominal value € 3.82 per share ................................... .
`Ordinary Shares, nominal value € 3.82 per share* .................... .
`Guarantee of 81/s% Cumulative Preference Shares of Aventis Overseas Ltd ... .
`
`New York Stock Exchange
`New York Stock Exchange
`New York Stock Exchange
`
`Securities registered or to be registered pursuant to Section 12(g) of the Act:
`
`American Depositary Shares, each representing one quarter of a Participating Share Series A par value € 70.89 per
`share.**
`
`Securities for which there is a reporting obligation pursuant to Section 15(d) of the Act: None
`
`(*)
`
`Listed not for trading or quotation purposes, but only in connection with the registration of the American Depositary Shares pursuant to the
`requirements of the Securities and Exchange Commission.
`
`(**) The American Depositary Shares representing Participating Shares Series A were removed from listing and registration on the New York Stock
`Exchange effective july 31, 1995.
`
`Indicate the number of outstanding shares of each of the issuer's classes of capital or common stock as of the
`close of the period covered by the annual report:
`
`Ordinary Shares, nominal value € 3.82 per Share: 795,621,603
`
`Indicate by check mark whether the registrant (1) has filed all reports required to be filed by Section 13 or 15(d) of
`the Securities Exchange Act of 1934 during the preceding 12 months (or for such shorter period for which the
`registrant was required to file such reports), and (2) has been subject to such filing requirements for the past 90 days.
`No 0
`Yes lZl
`Indicate by check mark which financial statement item the registrant has elected to follow.
`Item 17 0
`
`Item 18 lZl
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 3 of 123 PageID #: 612
`
`Diabetes
`
`Lontus (insulin glargine) is the first true once-daily long-acting basal insulin for treatment of type 1 and
`type 2 diabetes. After one daily injection before bedtime, Lantus is released into the body steadily and
`continuously with little variation in the amount of insulin in the body and provides patients with 24-hour
`basal glucose (blood sugar) control. First launched in Germany in june 2000, Lantus was launched in the
`United States in May 2001.
`
`Amory/ (glimepiride) is a once-daily sulfonylurea for the oral treatment of type 2 diabetes, which
`accounts for more than 90% of the people diagnosed with diabetes worldwide, as an adjunct to diet and
`exercise. Amory/ reduces the body's blood glucose {blood sugar) level primarily by helping the body produce
`more insulin. Amaryl is the first oral diabetes drug in its class to receive three indications: either as a
`monotherapy or in combination with insulin or metformin, another oral diabetes treatment. First launched
`in 1996, Amary/ is now available in more than 50 countries worldwide.
`
`lnsumon (human insulin) is a biosynthetic insulin identical to that produced by the human body and is
`used for treatment of type 1 and type 2 diabetes. It was launched in Germany in 1999, followed by other
`European countries. /nsuman is currently available in more than 13 countries, led by Germany and Austria.
`Aventis does not sell this product in the United States.
`
`Arthritis/Osteoporosis
`
`Adonel (risedronate sodium) is a novel bisphosphonate approved for treatment and prevention of
`osteoporosis, a disease that causes bones to become weaker, in postmenopausal women and for the
`treatment of corticosteroid-induced osteoporosis. It is also approved for treatment of Paget's disease, which
`leads to excessive formation of abnormal bone that is weak and at risk of fracturing. Actonel is the only
`bisphosphonate that has shown rapid clinical vertebral fracture reduction in as early as six months and has
`shown sustained fracture reduction over up to five years. Actonel is being co-developed and co-marketed in
`partnership with Procter & Gamble Pharmaceuticals. It was approved and launched in Sweden in 1999,
`received U.S. and EU approval in 2000 and is currently approved in more than 65 countries worldwide.
`
`Arovo (leflunomide) is a novel disease-modifying anti-rheumatic drug ("DMARD") for first-line treatment
`of rheumatoid arthritis, a debilitating disease that leads to inflammation in the lining of the joints. It is the
`first drug to be indicated to reduce the signs and symptoms of rheumatoid arthritis and to retard structural
`damage, such as erosions and joint-space narrowing, as evidenced by X-ray. Arava offers once-daily dosing
`and can be used in both early and late stages of the disease. This brand was launched in the United States
`in 1998 and is available in 37 countries, including the EU and areas of Latin America.
`
`Anti-lnfectives
`
`Ketek (telithromycin) is the world's first ketolide antibiotic to be marketed and has been developed
`specifically to treat community-acquired respiratory tract infections, including those caused by bacteria
`resistant to commonly used antibiotics. The unique mode of action of Ketek results from dual-binding sites
`for bacteria as opposed to single-binding sites for currently available antibiotics. The pharmacokinetic profile
`of this drug allows for a convenient, once-daily regimen, short-course treatment option for most indications
`that is expected to encourage better patient adherence to treatment.
`
`Ketek received EU marketing approval in july 2001 for the treatment of community-acquired respiratory
`tract infections, including those caused by bacteria resistant to commonly used antibiotics. The first market
`launch was Germany in October 2001, followed by other EU countries. Ketek has received an approvable
`letter from the U.S. Food and Drug Administration {"FDA") for the treatment of community-acquired
`pneumonia {"CAP") as well as for acute bacterial exacerbation of chronic bronchitis ("ABECB") and acute
`bacterial sinusitis ("ABS"), although non-approvable for tonsillitis/pharyngitis without additional data. Aventis
`is working closely with the FDA to gain approval for Ketek.
`
`23
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 4 of 123 PageID #: 613
`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 4 of 123 PagelD #: 613
`
`
`
`
`
`EXHIBIT 2
`EXHIBIT 2
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 5 of 123 PageID #: 614
`1111 m 11111111111111111111111111111111111~ 111
`US005656722A
`5,656,722
`[111 Patent Number:
`[451 Date of Patent:
`Aug. 12, 1997
`
`United States Patent [19]
`Dorschug
`
`-, 8 341
`[54] A21
`- MODIFIED INSULIN
`DERIVATIVES HAVING AN ALTERED
`ACTION PROFILE
`
`[75]
`
`Inventor: Michael Dorschug. Bochum. Germany
`
`[73] Assignee: Hoechst AktiengeseDschaft. Frankfurt
`am Main, Germany
`
`[21] AppL No.: 304,593
`
`[22] Filed:
`
`Sep. 12, 1994
`
`Related U.S. AppHcation Data
`
`[63]
`
`Continuation of Ser. No. 46,481, Apr. 9, 1993, abandoned,
`which is a continuation ofSer. No. 929,510,Aug. 19, 1992,
`abandoned, which is a continuation of Ser. No. 431,844,
`Nov. 6, 1989, abandoned.
`
`[30]
`
`Foreign AppHcation Priority Data
`
`Nov. 8, 1988
`[DE] Germany .......................... 38 37 825.6
`Int. CL 6 ..................................................... A61K 38/28
`[51]
`[52] U.S. Ct .............................................. 530/303; 530/304
`[58] Field of Search ..................................... 530/303, 304;
`514/3, 12
`
`[56]
`
`References Cited
`
`U.S. PATENf DOCUMENTS
`
`4,701,440 10/1987 Grau ............................................ 51413
`
`FOREIGN PATENT DOCUMENTS
`
`62066/86
`75916/87
`13976/88
`0046979
`0133285
`0214826
`0254516
`
`5/1986 Auslllll.ia .
`1/1988 Auslllll.ia .
`9/1988 Auslllll.ia .
`8/1981 European Pat. Off ..
`211985 European Pat. Off ................ 495/351
`3/1987 European Pat. Off ..
`6/1987 European Pat. Off ..
`
`OTHER PUBUCATIONS
`
`Schome et al. Diabetes vol. 27 (1978) 8-15.
`Geigee, Chem-Zeitung vol. 100 No. 3 pp. 54-56 (Jan.
`1976).
`Thompson et al Int'l J. Pept. Prot. Res. vol. 23 (1984)
`394-401.
`Schwartz et al PNAS voL 84 pp. 6408--6411 (Sep. 1987).
`Volund Diabetic Medicine 8. 839, 1991.
`Neubauer. "The Immunogenicity of Different lnsulins in
`Several Animal Species". Diabetes, vol. 27. No. 1 (1977).
`"Galenics Of Insulin. The Physico-chemical and Pharma(cid:173)
`ceutical Aspects of Insulin and Insulin Preparations". J.
`BRANGE: Springer-Verlag Berlin Heidelberg 1987. pp.
`35-36.
`Markus sen et al., Soluble. prolonged-acting insulin deriva(cid:173)
`tives. II. Degree of protraction and crystallizability of insu(cid:173)
`lins substituted in positions A17, B8, B13, B27 and B30,
`Protein Engineering 1(3):215-223 (1987).
`
`Markussen et al., Soluble, prolonged-acting insulin deriva(cid:173)
`tives, ill. Degree of protraction, crystallizability and chemi(cid:173)
`cal stability of insulins substituted in positions A21. Bl3,
`B23. B27, and B30, Protein Engineering 2(2):157-166
`(1988).
`Zinman, Bernard, The Physiologic Replacement of Insulin.
`Medical Intelligence, vol. 32, No.6. pp. 363-370 (1989).
`Sundby, F., Separation and Characterization of Acid-in(cid:173)
`duced Insulin Transformation Products by Paper Electro(cid:173)
`phoresis in 7 M Urea, The Journal of Biological Chemistry,
`vol. 237, No. 11, pp. 3406-3411 (1962).
`Burgermeister, W., et al., The Isolation of Insulin from the
`Pancreas, Reprint from the Handbook of Experimental Phar(cid:173)
`macology, pp. 715-727 (1975) Insulin Humanum. European
`Pharmacopeia 838 ( 1993 ).
`Primary Examiner-David Lukton
`Anome>' Agent, or Firm-Finnegan, Henderson, Farabow,
`Garrett & Dunner, L.L.P.
`[57]
`ABSTRACT
`
`New insulin derivatives, the use thereof, and a pharmaceu(cid:173)
`tical composition containing them
`
`Insulin derivatives having an isoelectric point between 5 and
`8.5, or physiologically tolerated salts thereof, of the Formula
`n
`
`Al
`
`S
`
`S
`
`A21
`
`(ll)
`
`H-Gly__j_rhain~ R2
`s
`s
`I
`I
`s
`I
`
`s
`
`BlO
`B2
`R ' -Val-B-chain- X
`
`B29
`R30-Rll
`
`in which:
`R 1 at position B1 denotes H or H-Phe;
`R 2 at position A21 denotes a genetically encodable L-amino
`acid selected from the group consisting of Gly, Ala. Val,
`Leu, lle. Pro. Phe, Trp, Met, Ser, Thr, Cys, Tyr. Asp, and
`Glu;
`R30 represents the residue of a neutral genetically encodable
`L-amino acid selected from the group consisting of Ala,
`Thr. and Ser;
`R 31 represents 1, 2, or 3 neutral or basic alpha amino acids,
`wherein at least one of the alpha amino acids is selected
`from the group consisting of Arg, Lys. Hyl. Om, Cit, and
`His;
`X represents His at position B10; and
`the sequences Al to A20 and Bl to B29 in Formula ll
`correspond to a mammalian insulin;
`excluding those insulin derivatives in which simultaneously:
`R 1 at position Bl denotes Phe; and
`R 3 is one alpha amino acid having a terminal carboxyl
`group.
`
`15 Claims, No Drawings
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 6 of 123 PageID #: 615
`
`5,656,722
`
`1
`A21
`-, B341
`- MODIFIED INSULIN
`DERIVATIVES HAVING AN ALTERED
`ACTION PROFILE
`
`This application is a continuation, of application Ser. No. 5
`08/046,481 filed Apr. 9, 1993, abandoned, which is a con(cid:173)
`tinuation of application Ser. No. 07/929,510, filed Aug. 19,
`1992, abandoned, which is a continuation of application Ser.
`No. 07/431,844, filed Nov. 6, 1989, now abandoned.
`
`2
`The abovementioned depot principle resulting from basic
`modification of the insulin has also been further utilized by
`the provision and corresponding use of other insulin deriva(cid:173)
`tives with basic modifications, mainly within the A and B
`chains; cf. for example EP-A 0,194,864 and EP-A 0,254.
`516.
`In the insulin derivatives specified in EP-A 0.194,864, a
`basic amino acid is incorporated in the B27 position and/or
`10 a neutral amino acid is located at positions A4, A17, B 13
`and/or B21; in addition, the C-terminal carboxyl group of
`the B chain is blocked by an amide or ester residue.
`The insulin derivatives specified in EP-A 0,254,516 are
`15 very similar to those specified in the abovementioned EP-A;
`however, in this case, with the aim of increasing the stability
`of the relevant pharmaceutical compositions at the weakly
`acid pH values, the amino acid Ash in position A21 can also
`be replaced by other amino acids which are more stable in
`20 acid medium, such as. for example, Asp. As is known. Ash
`(=asparagine) differs from Asp (=aspartic acid) by the block(cid:173)
`ing of one of the two carboxyl groups by the amide group:
`
`BACKGROUND OF THE INVENTION
`
`As is known, insulin and insulin derivatives are required
`in considerable quantities for the treatment of the disease
`diabetes mellitus, and some of them are also produced on an
`industrial scale. Despite the considerable number of insulin
`compositions and modifications with different action pro(cid:173)
`files which are already in existence, there is still a need,
`because of the variety of organisms with their inter- and
`intraindividual variations, for other insulin products which
`in turn have other properties and action characteristics.
`Insulin derivatives with a delayed action are described, for
`example, in EP-B 132,769 and EP-B 132,770. These are
`specifically derivatives with a basic modification in position
`B31 of the insulin B chain. of the following formula I:
`
`M
`H-Gly:.__j_i-chain~Asn-OH
`
`S
`
`~1
`
`S
`
`s
`
`s
`
`I
`I
`
`B10
`B2
`Rl-Val-B-chain-His
`
`s
`
`s
`
`I
`I B29
`
`R30-R31
`
`(I)
`
`25
`
`30
`
`35
`
`COOH
`I
`H2N-C-H
`I
`CH2
`I
`CONH2
`asparagine
`
`COOH
`I
`HzN-C-H
`I
`CHz
`I
`COOH
`aspartic acid
`
`Rapid-acting insulin derivatives are said to result from yet
`another modification of the insulin molecule in the A and B
`chain, in particular by replacing the amino acid His, which
`is responsible for the formation of a complex with zinc-and
`thus for a certain delaying action, in the B10 position by
`other appropriate amino acids; cf. EP-A 0,214,826.
`All the insulin derivatives specified in the 3 lastmentioned
`publications are mainly modified within the A and B chains;
`they are prepared by genetic engineering routes.
`
`SUMMARY OF THE INVENTION
`
`in which R 1 denotes H or H-Phe, R30 represents the residue
`of a neutral, genetically encodable L-amino acid, and R31
`represents a physiologically acceptable organic group which
`is basic in nature and has up to 50 carbon atoms, in whose 40
`structure 0 to 3 a-amino acids are involved and whose
`terminal carboxyl group which is present where appropriate
`can be free, in the form of an ester functionality, an amide
`functionality, a lactone or reduced to CH20H.
`Characteristic of these insulin derivatives is an isoelectric
`point between 5.8 and 8.5 (measured by isoelectric
`focusing). The fact that the isoelectric point is shifted from
`the isoelectric point of unmodified natural insulin or proin(cid:173)
`sulin (at pH=5.4) into the neutral range derives from the
`additional positive charge(s) located on the surface of the so
`molecule as a result of the basic modification. This makes
`these insulin derivatives with a basic modification less
`soluble in the neutral range than, say, natural insulin or
`proinsulin, which are normally dissolved in the neutral
`
`45
`
`In the attempt to increase the stability in acid medium of
`the insulin derivatives with a basic modification on the
`C-terminal end of the B chain as specified in the European
`Patents EP-B 0,132,769 and EP-B 0,132.770 mentioned in
`55 the introduction. and, where appropriate, also to alter the
`range.
`f
`d
`thi b
`The delaying or depot action of the insulin derivatives
`f;
`~ctichion. proedfil'e theradeo , it has now been obun ~at. sAo ~1~
`with a basic modification, of the formula L derives from
`ding to
`1s a
`ev m an vantageous manner y rep.acmg sn
`th ·
`·
`· t. A
`1 bility at th
`· 1 tri
`err sparmg sou
`th
`tai
`b
`ticall
`'d
`hi h
`e ISoe ec c pom
`ccor
`dahl
`·
`Y 0
`Y enco
`.er gene
`e llllll;no act s w ~ co~ 81~
`the two abovementioned publications, the redissolution of
`the insulin derivatives under physiological conditions is 60 no amtde grou~ and. where appr~te. b~ replacmg His
`achieved by elimination of the additional basic groups,
`by other genetically encodable amtno ac1ds.
`which is brought about, depending on the derivative, by
`trypsin or trypsin-like and/or carboxypeptidase B or carbox(cid:173)
`ypeptidase B-like and/or esterase activity. The eliminated
`groups are in each case either purely physiological metabo- 65
`lites or else easily metabolized physiologically acceptable
`substances.
`
`DESCR1PfiON OF THE PREFERRED
`EMBODIMENfS
`
`Hence the invention relates to insulin derivatives of the
`formula n
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 7 of 123 PageID #: 616
`
`5,656,722
`
`3
`
`AI
`S
`S
`A21
`H-Gly___l_rhain~ Rl
`
`(TI)
`
`s
`
`I
`R'-!~-!_cbain-~10
`
`s
`
`s
`
`s
`
`I
`I B:29 RlO-Rll
`
`4
`suitable basic groups for this being, for example. the above(cid:173)
`mentioned basic groups-in the case where no a-amino
`acids are involved in the structure of R31
`• Of course, these
`basic ester or amide groups can also block the carboxyl
`5 group of basic a-amino acids. Also possible and suitable for
`blocking the carboxyl group of the basic a-amino acids
`are--if the blocking is desired-neutral ester or amide
`groups such as, for example, (C1-CJ-alkoxy, (C3-C6)(cid:173)
`cycloalkyloxy, NH2, (C1-C6)-alkylamino or di-(C1-CJ-
`10 alkylamino.
`Of course, the terminal carboxyl group can be in the fonn
`of a lactone only if the terminal amino acid is a
`hydroxyamino acid.
`Moreover, the terminal carboxyl group can also be
`reduced to CH20H.
`R 31 is preferably composed of 1, 2 or 3 of the abovemen-
`tioned basic naturally occurring amino acids; R31 is particu(cid:173)
`larly preferably Arg-OH or Arg-Arg-OH.
`Suitable genetically encodable L-amino acids-for
`X-are the same amino acids as for R2
`, but the genetically
`20 encodable L-amino acids which contain an amide group(cid:173)
`which are Ash and Gin-are also possible in this case; the
`latter-Asn and Gln-are in fact preferred in this case. If
`Asn or Gin is located in position BlO, the amide group is at
`least stable in weakly acid medium (in contrast toAsn or Gin
`25 in position A21). The sequences (Al-A20) and (Bl-B9,
`B 11-B29) are preferably the sequences of human, porcine or
`bovine insulin, especially the sequences of human insulin.
`Examples of insulin derivatives of the formula n are:
`
`15
`
`in which:
`R 1 denotes H or H-Phe,
`R2 denotes a genetically encodable L-amino acid which
`contains no amide group,
`R 30 represents the residue of a neutral genetically encod(cid:173)
`able L-amino acid,
`R31 represents a physiologically acceptable organic group
`which is basic in nature and has up to 50 carbon atoms, in
`whose structure 0 to 3 a-amino acids are involved and
`whose terminal carboxyl group which is present where
`appropriate can be free, in the fonn of an ester functionality,
`an amide functionality. a lactone or reduced to CH20H. and
`X represents a genetically encodable L-amino acid, hav(cid:173)
`ing an isoelectric point between 5 and 8.5. and the physi(cid:173)
`ologically tolerated salts thereof.
`The new insulin derivatives and the physiologically tol(cid:173)
`erated salts thereof are stable at the weakly acid pH values
`of appropriate pharmaceutical compositions even for
`extended periods and have--especially when His810 has
`also been replaced by other amino acids-an altered 30
`(shorter) action profile compared with the known(cid:173)
`unaltered-insulin derivatives with a basic modification of
`the formula I indicated in the introduction.
`R 1 in formula n is preferably H-Phe.
`Genetically encodable L-amino acids containing no 35
`amide group-for R2 -are Gly, Ala, Ser, Thr, Val, Leu, Tie,
`Asp, Glu. Cys, Met, Arg, Lys, His, Tyr, Phe, Trp. Pro;
`Gly, Ala, Ser, Thr, Asp and Glu are preferred, especially
`Asp.
`Neutral genetically encodable L-amino acids-for R30
`-
`are Gly, Ala, Ser, Thr. Val, Leu, Tie, Ash, Gln, Cys, Met, Tyr,
`Phe and Pro; Ala. 'lbr and Set are preferred.
`R31 is a physiologically acceptable organic group which
`is basic in nature and has up to 50 carbon atoms and in
`whose structure 0-3 a-amino acids are involved. When no 45
`a-amino acids are involved in the structure of R 31
`, examples
`of suitable basic groups for this residue are the following:
`amino-(C2-C6)-alkoxy, (C1-C4)-alkylamino-(C2-C6)(cid:173)
`alkoxy, di-(C1-C4 )-alkylamino-(C2-C6)-alkoxy. tri-(C 1-CJ
`ammonio-(C2-C6)-alkoxy, amino-(C2-C6)-alkylamino, so
`[ ( C 1-C4 )-alkyl-amino ]- (C2-C6)-alkylamino, di-(C 1-C4 )(cid:173)
`alkylamino-(C2-C6)-alkylamino or (tri-(C 1-C4 )(cid:173)
`alkylamino]-(C2-C6)-alkylamino, especially -0-[CH2 ]
`-NR2 and -0-[CH2]-N3~3• -NH-(CH2]p-NR2 or
`-NH-[CH2]P-"'R3, in which pis 2 to 6. and R is identical 55
`or different and represents hydrogen or (C1-C4)-alkyl.
`When up to 3 a-amino acids are involved in the structure
`of R 31
`• these are primarily neutral or basic naturally
`occurring L-amino acids and/or the D-amino acids corre(cid:173)
`sponding thereto. Neutral naturally occurring amino acids 60
`are, in particular, Gly. Ala, Ser. Thr. Val. Leu. Tie. Ash. Gin,
`Cys, Met. Tyr, Phe, Pro and Hyp. Basic naturally occurring
`amino acids are, in particular, Arg. Lys. Hyl, Om, Cit and
`His. If only neutral a-amino acids are involved. the terminal
`carboxyl group thereof cannot be free--in order for R31 to 65
`be basic in nature; on the contrary. the carboxyl group must
`in this case be amidated or esterified with a basic group,
`
`40
`
`AspA21-Human iosu!in-Arg"-'1-0H
`Glu-""1-Human insulin-Arg'131-0H
`Gly""1-Human insulin-Arg'131-0H
`Ser"2'-Human iDsulin-Arg"-'1-0H
`'I'hr""1-Human insulin-Arg"-'1-0H
`Ala""'-Human insulin-Arg"-'1-0H
`AspA21-Human insulin-Arg"-'1-Arg"-'2-0H
`Glu-""1-Human insulin-Arg"-'1-Ati'32-0H
`GiyA>'-Human insulin-Arg"31-Arg"32-0H
`&rA2' -Human insulin-Arg"-'1-Arg"-'2-0H
`'I'hr""1-Human insulin-Arg"-''-Arg"-'2-0H
`AlaA21-Human iosu!in-Arg"-'1-Arg"-'2-0H
`AspA2'-AJm.Blo_HUIIlliD iosu!in-Arg"3'-0H
`Glu""1-AJm810-Human insulin-Arg"-'1-0H
`Gly""1-Asn1010-Human insulin-Arg"-'1-0H
`Ser"21--Asn810 -Human insulin-Arg"-'1-0H
`'I'hr""'-Asr/'10-Human insulin-Arg"-'1-0H
`AlaA21-Asrl'10-Human insulin-Arg"31-0H
`AspA21_AJma'o-Human insuJin-Arg"-''-Arg"-'2-0H
`Gllf'21--Asn810-Human ill8ulin-AziU'-Ati'32-0H
`Gly""1-Alm810-Human insulin-Arg"-''-Arg"32-0H
`Ser"'l'-AJm.BlO.Human iosu!in-Arg"-''-Arg"-''-OH
`~ 1-Asrl" 0-Human insulin-Arg"-'1-Arg"-'2-0H
`AlaA21--AsnB10.Humao insulin-Arg"-''-Arg"-'2-0H
`
`The insulin derivatives of the formula n are prepared
`mainly by a genetic manipulation by means of site-directed
`mutagenesis using standard methods.
`For this purpose, a gene structure coding for the desired
`insulin derivative of the fonnula n is constructed and its
`expression is brought about in a host cell-preferably in a
`bacterium such as E. coli or a yeast. in particular Saccha(cid:173)
`romyces cerevisiae-and-if the gene structure codes for a
`fusion protein~e insulinderivative of the formula n is
`liberated from the fusion protein; analogous methods are
`described, for example, in EP-A 0,211,299. EP-A 0,227 ,938.
`EP-A 0,229,998, EP-A 0,286,956 and German Patent Appli(cid:173)
`cation P 38 21 159.9 dated Jun. 23, 1988 (HOE 88/F 158).
`After cell disruption, the fusion protein portion is elimi(cid:173)
`nated either chemically using cyanogen halide-cf. EP-A
`0.180,920 or enzymatically using lysostaphin-ct. DE-A
`3,739,347.
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 8 of 123 PageID #: 617
`
`5,656,722
`
`6
`A) Preparation By Genetic Manipulation
`
`EXAMPLE 1
`Construction of a plasmid for the preparation of Gly
`(A21)-human insulin Arg (B31-0H)
`The plasmid pSW3 has been described in German Patent
`Application P 38 21 159.9 (HOE 88/F 158). The plasmid
`DNA is reacted with the restriction enzymes Pvull and Sail
`and subsequently treated with bovine alkaline phosphatase.
`10 The two resulting fragments are separated by gel
`electrophoresis, and the large fragment is isolated. This
`fragment is linked in a T4 DNA ligase reaction with the
`following synthetic DNA sequence:
`
`5
`The insulin precursor is then subjected to oxidative suUi(cid:173)
`tolysis by the method described. for example, by R. C.
`Marshall and A. S. Inglis in "Practical Protein
`Chemistry-A Handbook" (edited by A. Darbre) 1986,
`pages 49-53. and subsequently renatured in the presence of 5
`a thiol with the formation of the correct disulfide bridges, for
`example by the method described by G. H. Dixon and A. C.
`Wardlow in Nature (1960), pages 721-724.
`The C peptide is removed by cleavage with trypsin-for
`example by the method of Kemmler et al., J. B. C. (1971),
`pages 6786-6791, and the insulin derivative of the formula
`II is purified by known techniques such as
`chromatography-d., for example, EP-A-0,305,760-and
`crystallization.
`The insulin derivatives of the formula II with R 2=Asp and
`X=His are expediently prepared by hydrolysis of the known 15
`insulin derivatives which have a basic modification and the
`formula I in aqueous acidic medium (because only the amide
`group of the asparagine in position A21 must be hydrolyzed
`in this case), preferably at pH values between about 2 and
`about 4, in particular of about 2.5, and at temperatures of 20
`about 0° to about 40° C., preferably at room temperature.
`The insulin derivatives of the formula II, according to the
`invention, and/or the physiologically tolerated salts thereof
`(such as, for example. the alkali metal or ammonium salts)
`are mainly used as active substances for a pharmaceutical 25
`composition for the treatment of diabetes mellitus.
`The pharmaceutical composition is preferably a solution
`or suspension for injection; it contains at least one insulin
`derivative of the formula II and/or at least one of the
`physiologically tolerated salts thereof in dissolved, amor- 30
`phous and/or crystalline-preferably in dissolved-form.
`The composition preferably has a pH between about 2.5
`and 8.5, in particular between about 4.0 and 8.5, and
`contains a suitable tonicity agent, a suitable preservative
`and, where appropriate. a suitable buffer, as well preferably 35
`a certain zinc ion concentration, all, of course, in sterile
`aqueous solution. All the ingredients of the composition
`apart from the active substance form the composition
`vehicle.
`Examples of suitable tonicity agents are glycerol, glucose, 40
`mannitol. NACl. and calcium or magnesium compounds
`such as CaC~. MgCl2 etc.
`The choice of the tonicity agent and/or preservative
`influences the solubility of the insulin derivative or the
`physiologically tolerated salt thereof at the weakly acid pH 45
`values.
`Examples of suitable preservatives are phenol, m-cresol,
`benzyl alcohol and/or p-hydroxybenzoic esters.
`Examples of bu1fer substances which can be used, in
`particular for adjusting a pH between about 4.0 and 8.5, are so
`sodium acetate, sodium citrate, sodium phosphate etc.
`Otherwise. also suitable for adjusting the pH are physiologi(cid:173)
`cally acceptable dilute acids (typically HCl) or alkalis
`(typically NaOH).
`When the composition contains zinc a content of 1 Jlg to ss
`2 mg. in particular from 5 Jlg to 200 Jlg, of zinc/ml is
`preferred.
`In order to vary the action profile of the composition
`according to the invention it is also possible to admix
`unmodified insulin. preferably bovine. porcine or human
`insulin. in particular human insulin.
`Preferred concentrations of active substance are those
`corresponding to about 1-1500, also preferably about
`5-1000, and in particular about 40-400, international units/
`ml.
`The invention is now explained in detail by the examples
`which follow.
`
`5'- CTO OAA AAC TAC TOT GOT TOA TAO
`OAC CTT TTOATOACACCAACT ATC AOCT-5'
`
`Competent E. coli W3110 cells are transformed with the
`ligation mixture. The transformation mixture is plated out on
`NAplates which contain 20 Jlg of Ap (=Ampicillin)lml and
`incubated at 37° C. overnight An overnight culture is
`obtained from single colonies, and plasmid DNA is obtained
`from this. This DNA is characterized by means of restriction
`analysis and DNA sequence analysis. Correct plasmids
`which encode the modified A chain are called piK100.
`Expression is carried out in analogy to Example 3 of the
`abovementioned German Patent Application P 38 21 159.9.
`The modified mono-Arg-insulin is likewise prepared in
`analogy to the preparation of the unmodified mono-Arg(cid:173)
`insulin described in this German Patent Application.
`EXAMPLE2
`Construction of a plasmid for the preparation of Ser
`(A21)- human insulin (Arg B31-0H)
`The construction corresponds to the route described in the
`above example. The synthetic DNA sequence is, however.
`modified as follows:
`
`S'- CTG OAA AAC TAC TOT TCA TOA TAO
`OAC CTT TTO ATG ACA Arrf ACT ATC AOCT- 5'
`
`The plasmid piKllO which has an additional BspHI
`recognition sequence is obtained.
`EXAMPLE3
`Construction of a plasmid for the preparation of Gly
`(A21)- Asn(BlO)-human insulin Arg(B31-0H)
`DNA from the plasmid piK100 is cleaved with the
`restriction enzymes Hpal and Draiii and treated with bovine
`alkaline phosphatase. The two resulting fragments are sepa(cid:173)
`rated by gel electrophoresis, and the larger of the two
`fragments is isolated. The fragment is ligated with the
`synthetic DNA sequence
`
`S'- AAC CAA CAC TTO TOT GOT TCT AAC TTO
`TTO OTT OTO AAC ACA CCA AOA TTO
`- 5'
`
`and competent E. coli W3110 cells are transformed with the
`ligation mixture. Further characterization of the resulting
`60 plasmid piK101 is carried out as described in Example 1.
`EXAMPLE4
`Construction of a plasmid for the preparation of Ser
`(A21)- Asn(B10)-human insulin
`The construction corresponds to the cloning described in
`Example 3, but starting from DNA from the plasmid
`piKllO. The newly constructed plasmid is called piKlll.
`
`65
`
`

`

`Case 1:14-cv-00113-RGA-MPT Document 21-1 Filed 04/10/14 Page 9 of 123 PageID #: 618
`
`5,656,722
`
`7
`EXAMPLES
`
`Construction of an expression plasmid for monkey pro-
`insulin
`
`8
`and 5 ml of a 1% strength zinc chloride solution per liter
`with a protein concentration of 5 gll~t H 6.0. The yield is
`390 mg of As-A21_human insulin-
`1_ .. .....B32_
`ru~;
`I'
`C) Preparation of an Injection Solution
`Monkey proinsulin differs from human proinsulin merely 5
`The insulin derivative from B is dissolved at a concen-
`tration of 1.4 mg1m1 in a sterile vehicle solution of the
`by replacement of a single amino acid in the C peptide
`(B37-Pro in place of Leu in this position of human
`following composition (per ml):
`18 mg of glycerol, 10 mg of benzyl alcohol, 80 Jlg of
`proinsulin).
`Zn2
`+, pH 4.0.
`The plasmid pSW3 is opened with Hpal and San and the
`D) Action Profile of an As~21-Human Insulin-Arg"31-
`remainingplasmidDNAis isolated. The Dram-San monkey 10
`proinsulin fragment is isolated from the plasmid pK50 ~32-0H Composition in dogs by comparison with human
`insulin-Arg"3 1-~2-0H and basal H insulin Hoechst<R>=
`described in EP-A0.229,998. The two fragments are linked
`to the synthetic DNA fragment
`an NPH (neutral protamine Hagedorn) composition contain(cid:173)
`ing about 10 Jlg of Zn2+.
`
`5'- AAC CAO CAC CTO TGC GOT TCT CAC CTA
`- 5'
`TTO OTC OTO OAC ACO CCA AOA GI'O
`
`15
`
`Blood glucose as a % of the
`initial level in hours U!l
`
`1 h 2h 3h 5h 7h
`Product
`w -------------