APOTEX ET AL. - EXHIBIT 1065
`Apotex Inc. et al. v. Novartis AG
`IPR2017-00854
`
`

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`dence For Two Distinct Conformations”, Aids 7:639—46
`(1993). Abstract only.
`Linsley, P.S., et al., “Effects Of Anti—gp120 Monoclonal
`Antibodies On CD4 Receptor Binding By The Env Protein
`Of Human Immunodeficiency Virus Type 1”, J. 0f Virology
`62:3695—3702 (1988). Abstract only.
`Rini, J .M., et al., “Crystal Structure Of A Human Immuno-
`deficiency Virus Type 1 Neutralizing Antibody, 50.1, In
`Complex With Its V3 Loop Peptide Antigen”, Proceedings
`Of The Nat’l. Academy ofSciences Of The United States Of
`America 90:6325—9 (1993). Abstract only.
`Subramanyam, W.G., et al., “Mechamism Of HIV—1 Tat
`Induced Inhibition Of Antigen—Specific T Cell Responsive-
`ness”,J. OfImmunol. 150:2544—2553 (1993),Abstract only.
`Dang, N.H., et al., “Cell Surface Modulation Of CD26 By
`Anti—1F7 Monoclonal Antibody: Analysis Of Surface
`Expression And Human T Cell Activation”, J. 0f Immanol.
`145:3963—3971 (1990). Abstract only.
`De Caestecker, M.P ., et al., “The Detection Of Intercyto-
`plasmic Interleukin 1 (Alpha) Expression In Human Mono-
`cytes Using Two Colour Immunoflourescence Flow Cytom-
`etry”, J. Immunol. Methods 154:11—20 (1992). Abstract
`only.
`
`Fauci, AS, “The Human Immunodeficiency Virus: Infec-
`tivity And Mechanisms Of Pathogenesis”, Science
`239:617—722 (1988). Abstract only.
`
`Kinder, D ., et al., “Analogues of Carbamyl Aspartate as
`Inhibitors .
`.
`. ”J. Med. Chem, (1990), 33:819—823.
`
`Snow, R., et al., “Studies on Proline Boronic Acid Dipeptide
`Inhibityors of Dipeptidyl
`.
`.
`. ”J. Med. Chem, (1990),
`116:10860—10869.
`
`Wijdenes et al., “Monoclonal Antibodies (mAb) against
`gp130 Imitating Cytokines Which Use the gp130 for Signal
`Transduction”, (Jul. 1995), p. 303.
`
`Blumenstein et al., “Synthetic Non—Peptide Inhibitors of
`HIV Protease,” vol. 163, No. 2 (1989), pp. 980—987.
`
`Luftig et al., “Update on Viral Pathogenesis,” ASM News
`(1990) vol. 56, No. 7, pp. 366—368.
`
`Jiang et al., “Inhibition of Human Immunodeficiency Virus
`Type 1 Infection in a T—Cell Line (CEM) by New Dipep-
`tidyl—Peptides IV (CD26) Inhibitors,” Res. Virol. (1997),
`vol. 148, pp. 255—266.
`
`Coutts et al., “Structure—Activity Relationships of Boronic
`Acid Inhibitors of Dipeptidyl Peptidase IV, 1. Variation of
`the P2 Position of XZZ—boroPro Dipeptides,” J. Med. Chem.
`(1996), vol. 39, pp. 2087—2094.
`
`Ostresh et al., “Generation of Use of Nonsupport—Bound
`Peptide
`and Peptidomimetic Combinatorial Libraries,”
`Methods in Enzymology, (1996) vol. 267, pp. 230—234.
`
`Bristol, L, et al., “Inhibition of CD26 Enzyme Activity with
`Pro—boropro
`Stimulates Rat Granulocyte/Macrophage
`Colony Formation and Thymocyte Proliferation in Vitro,”
`Blood, vol. 85, No. 12 (1995), pp. 3602—3609.
`
`Ansorge S., et al., “CD26/Dipeptidyl Peptidase IV in Lym-
`phocyte Growth Regulation,” Advances in Medicine and
`Biology, (1997), vol. 421, pp. 127—40.
`
`Reinhold, Dirk, et al., “Inhibitors of Dipeptidyl PepdaseIV
`(DP IV, CD26) Induces Secretion of Transforming Growth
`Factor—[31 .
`.
`. ”, Immunology Letters, 58(1997), pp. 29—35.
`
`* cited by examiner
`
`

`

`US. Patent
`
`Aug. 3, 2004
`
`Sheet 1 0f 6
`
`US 6,770,628 B2
`
`Figure 1
`
`
`
`Figure 2
`
`Duration 0f PT—‘IOO Treatment
`
`1400
`
`1200
`
`E
`{5 100°
`3
`g 30°
`
`- 96- . saline
`- - o - -PT-1eo 0.1ug oral
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`-— -A-— PT-1oo 5ug oral
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`600
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`7
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`
`DAYS AFTER CYCLOPHOSPHAMIDE TREATMENT
`
`

`

`US. Patent
`
`Aug. 3, 2004
`
`Sheet 2 0f 6
`
`US 6,770,628 B2
`
`Figurc 3
`Duration Of PT-1OO Treatment
`:22:
`
`1200
`
`000
`
`1
`
`-- -x- - sallne
`
`--o—1 ug PT~100 5.0.
`— 1e.— 5 ug PT—100 3.0.
`
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`DAYS AFTER CYCLOPHOSPHAMIDE TREATMENT
`
`Figure 4
`
`Duration of PT-100 Treatment
`
`[2:222:21
`
`1 800
`
`1600
`
`1400
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`200
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`CELLNUMBER(x10‘lml)
`
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`
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` .. .5. .. PT-100 2ug. oral
`
`+6438}: 8.6.
`
`
`
`

`

`US. Patent
`
`Aug. 3, 2004
`
`Sheet 3 0f 6
`
`US 6,770,628 B2
`
`Figure 5
`
`Duration Of Treatment
`
`Em
`
`- ->€-
`
`- Saline
`
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`DAYS AFTER CYCLOPHOSPHAMIDE TREATMENT
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`CELLNUMBER(x1o‘IML)
`
`1°00
`900
`
`800
`
`700
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`
`Figure 6
`
`[:1
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`Duration of Treatment
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`- 9(- - saline
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`—B—-—-1 day
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`— 9—. 3 days
`53“
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`1200
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`1000
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`
`DAYS AFFER CYCLOPHOSPHAMIDE TREATMENT
`
`

`

`US. Patent
`
`Aug. 3, 2004
`
`Sheet 4 0f 6
`
`US 6,770,628 B2
`
`o0ooo00oo0420861.1.1
`
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`Figure 7
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`DAYS AFTER CYCLOPHOSPHAMIDE TREATMENT
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`Figure 8
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`
`
`
`
`
`

`

`US. Patent
`
`Aug. 3, 2004
`
`Sheet 5 0f 6
`
`US 6,770,628 B2
`
`a
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`
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`

`

`US. Patent
`
`Aug. 3, 2004
`
`Sheet 6 0f 6
`
`US 6,770,628 B2
`
`Figure 10
`
`human G—CSF
`
`4500
`
`4000
`
`3500
`
`3000
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`pg/mlhumanG—CSF
`
`

`

`US 6,770,628 B2
`
`2
`poietic cells. The teaching also suggests that cytokines be
`administered in conjunction with the inhibitors to increase
`the production of hematopoietic cells in a subject.
`
`SUMMARY OF THE INVENTION
`
`The invention is based upon a variety of surprising and
`unexpected findings. It has been discovered, unexpectedly,
`that the agents useful according to the invention stimulate
`growth factor production by stromal cells. It also has been
`discovered, unexpectedly, that the agents useful according to
`the invention stimulate proliferation of primitive hemato-
`poietic progenitor cells, but do not stimulate directly the
`differentiation or proliferation of committed progenitor
`cells. It further has been discovered, unexpectedly, that the
`agents useful according to the invention can be administered
`at doses much lower than would have been expected accord-
`ing to the teachings of the prior art. Another unexpected
`finding is that the agents according to the invention can
`accelerate the time it takes to achieve hematopoietic cell
`recovery after treatment with an hematopoietic cell inhibitor.
`Another unexpected finding is that the agents useful accord-
`ing to the invention can at relatively low doses, restore
`normal levels of neutrophils at least as fast as the most
`successful commercially available product used worldwide
`for this purpose, except that the agents useful according to
`the invention can be used orally, whereas the commercially
`available product (which represents more than a billion
`dollar market) must be injected. These unexpected results
`have important therapeutic and experimental research impli-
`cations.
`
`According to one aspect of the invention, a method is
`provided for treating a subject to stimulate hematopoiesis in
`the subject. The invention involves administering to a sub-
`ject
`in need of such treatment an amount of an agent
`effective to increase the number of hematopoietic cells or
`mature blood cells in the subject, wherein the amount is less
`than 1 mg/kg body weight per day and wherein the agent is
`a compound of Formula I.
`
`The agents useful according to the invention are com-
`pounds of Formula I:
`
`Formula I
`
`1
`HEMATOPOIETIC STIMULATION
`
`RELATED APPLICATIONS
`
`This application is a continuation application of prior US.
`patent application Ser. No. 09/304,199, filed May 3, 1999,
`entitled Hematopoietic Stimulation, now issued as US. Pat.
`No. 6,300,314 B1 the entire contents of which are incorpo-
`rated herein by reference and which claims priority under 35
`U.S.C. § 119(e) to US. provisional application serial No.
`60/084,128 filed May 4, 1998.
`BACKGROUND OF THE INVENTION
`
`The present invention relates to methods and products for
`producing increased numbers of hematopoietic cells, of
`restoring to preselected normal levels numbers of hemato-
`poietic cells, to therapies for treating deficiencies in hemato-
`poietic cells and to in vitro methodologies for culturing
`hematopoietic cells.
`PT-100 is a dipeptide consisting of valine-prolineboronic
`acid (ValboroPro) designed to interact with the cell surface
`receptor CD26. CD26, a type II transmembrane protein is
`expressed on the cell surface of a number of cell types,
`including lymphocytes (Marguet, D. et al., Advances in
`Neuroimmzmol. 3:209—215 (1993)), hematopoietic cells
`(Vivier, I. et al.,J. Immunol. 147:447—454 (1991); Bristol, et
`al.,J. Immunol. 149:367 (1992)) thymocytes (Dang, N. H. et
`al., J. Immunol. 147:2825—2832 (1991), Tanaka, T. et al., J.
`Immunol. 149:481—486 (1992), Darmoul, D. et al., J Biol.
`Chem. 267:4824—4833 (1992)),
`intestinal brush border
`membrane and endothelial cells. Cell surface associated
`
`CD26 is a sialoglycoprotein, with most of its mass on the
`outside of the cell.
`
`CD26 has been best characterized on peripheral T cells
`where it functions as a potent costimulatory signal for T cell
`activation. Its surface expression is upregulated upon T cell
`activation (Dong, R. P. et al., Cell 9:153—162 (1996),
`Torimoto, Y. et al.,J. Immunol 147:2514 (1991), Mittrucker,
`H-W. et al., Eur.JImmzm. 25:295—297 (1995), Hafler, D. A.
`et al,J. Immunol. 142:2590—2596 (1989), Dang, N. H. et al.,
`J. Immunol 144:409 (1990)). CD26 has also been identified
`in rodents as an important regulatory surface receptor in
`hematopoiesis and lymphoid development (Vivier, I. et al.,
`J. Immunol. 147:447—454 (1991)). The primary structure of
`CD26 is highly conserved between species (Ogata, S. et al.,
`J. Biol. Chem. 264:3596—3601 (1998)). In humans CD26
`seems to be involved in the regulation of thymocyte
`activation, differentiation and maturation (Dang, N. H. et al.,
`J. Immunol. 147:2825—2832 (1991); Kameoka, J. et al.,
`Blood 85:1132—1137 (1995)). We have evidence that CD26
`is expressed within the human and murine hematopoietic
`systems.
`CD26 is an ectoenzyme with activity identical to that of
`Dipeptidyl Peptidase IV (DPP-IV), a serine type exopepti-
`dase with high substrate specificity. It cleaves N-terminal
`dipeptides from proteins if the penultimate amino acid is
`proline, or in some cases alaninie (Fleischer, B. Immunol.
`Today 152180 (1994)). PT-100 is a potent
`inhibitor of
`DPP-IV activity.
`The prior art PCT published application WO94/03055
`teaches methods of producing increased numbers of hemato-
`poietic cells by administering inhibitors of DPP-IV. The
`teaching of this published application, however,
`is that
`dosages of at least 1 mg/kg body weight are necessary to
`achieve such hematopoietic cell increases. This published
`application also teaches that inhibitors are administered to
`mammals which have an established deficiency of hemato-
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`
`
`A— N
`I
`H2C\
`
`111
`
`O
`H
`II
`I
`C—C Al— N
`I
`I
`/CH2
`H2C\
`E12
`
`
`
`X1
`H
`/
`I
`C— B
`I
`\X
`/CH2
`I{:12
`
`2
`
`wherein m is an integer between 0 and 10, inclusive; A and
`A1 are L-amino acid residues (for glycine there is no such
`distinction) such that the A in each repeating bracketed unit
`can be a different amino acid residue; the C bonded to B is
`in the L-configuration; the bonds between A and N, A1 and
`C, and between A1 and N are peptide bonds; and each X, and
`X2 is, independently, a hydroxyl group or a group capable of
`being hydrolysed to a hydroxyl group in aqueous solution at
`physiological pH. By “the C bonded to B is in the
`L-configuration” is meant that the absolute configuration of
`the C is like that of an L-amino acid.
`
`

`

`US 6,770,628 B2
`
`Thus, the
`
`group has the same relationship to the C as the —COOH
`group of an L-amino acid has to its (X carbon. In some
`embodiments, Aand A1 are independently proline or alanine
`residues;
`In is 0; X1 and X2 are hydroxyl groups;
`the
`inhibitor is L-Ala-L-boroPro; and the inhibitor is L-Pro-L-
`boroPro.
`In one important aspect of the invention, the subject has
`an abnormally low level of hematopoietic cells or mature
`blood cells and the agent is administered in an amount
`effective to restore levels of a hematopoietic cell-type or
`mature blood cell-type to a preselected normal or protective
`level. The agent preferably is administered to the subject in
`at least 2 doses in an 18 hours period. The invention has
`particularly important applications in the restoration of
`normal or protective levels of neutrophils, erythrocytes and
`platelets. The most preferred agent is ValBoroPro.
`According to another aspect of the invention, a method is
`provided for shortening or eliminating the time that a subject
`has an abnormally low level of hematopoietic or mature
`blood cells resulting from treatment with a hematopoietic
`cell inhibitor. An agent is administered to a subject in need
`of such treatment in an amount effective to increase the
`
`number of hematopoietic cells or mature blood cells in the
`subject, wherein the administration of the agent begins prior
`to or substantially simultaneous with administration of the
`hematopoietic cell inhibitor. The agents and the preferred
`agent are as described above. In one important embodiment,
`the hematopoietic cell inhibitor causes an abnormally low
`level of hematopoietic cells or mature blood cells in the
`subject and the agent is administered in an amount effective
`to restore levels of a hematopoietic cell type to a preselected
`normal or protective level. Preferably, the agent is admin-
`istered to the subject in at least 2 doses in an 18 hour period.
`In important embodiments, the agent is used to restore in the
`subject normal or protective levels of neutrophils, erythro-
`cytes or platelets. The preferred effective amount of age