ANnFOLATE DRUGS IN
`ANTIFOLATE DRUGS IN
`CANCER THERAPY
`
`Edited by
`Edited by
`ANN L. JACKMAN
`The Cancer Research Campaign Centre
`The Cancer Research Campaign Centre
`for Cancer Therapeutics,
`for Cancer Therapeutics,
`The Institute of Cancer Research,
`The Institute of Cancer Research,
`Sutton, Surrey, UK
`sutton, Surrey, UK
`
`!NT
`~NT
`
`I
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`
`HUMANA P~ss
`HUMANA PRESS
`TOTOWA, N~W
`TOTOWA, NEW JERSEY
`
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`

`
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`I
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`
`Sandoz Inc. IPR2016-00318
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1122-0002
`Sandoz v. Eli Lilly, Exhibit 1122-0002
`
`

`
`8
`8
`
`Preclinical Pharmacology Studies
`Preclinical Pharmacology Studies
`and the Clinical Development
`and the Clinical Development
`of a Novel Multitargeted
`of a Novel Multitargeted
`Antifolate, MTA (LY231514)
`Antifolate, MTA (LY231514)
`
`Chuan Shih and Donald E. Thornton
`Chuan Shih and Donald E. Thornton
`
`CONTENTS
`CONTENTS
`
`INTRODUCTION
`INTRODUCTION
`PRECLINICAL PHARMACOLOGY STUDIES OF MT A
`PRECLINICAL PHARMACOLOGY STUDIES OF MTA
`CLINICAL STUDIES OF MT A
`CLINICAL STUDIES OF MTA
`CONCLUSION AND PERSPECTIVE
`CONCLUSION AND PERSPECTIVE
`
`1. INTRODUCTION
`1. INTRODUCTION
`Since the ~arly 1950s, extensive resear.ch effoRs have been devoted to the discovery
`Since the early 1950s, extensive researGh efforts have been devoted to the discovery
`and development of antifolate antimetabolites as chemotherapeutic agents for the man-
`and development of antifolate antimetabolites as chemotherapeutic agents for the man(cid:173)
`agement of neoplastic diseases. However, it was only in the last 10-15 yr, because of the
`agement of neoplastic diseases. However, it was only in the last 10-15 yr, because of the
`rapid advances of medicinal chemistry, X-ray protein crystallography, molecular biol-
`rapid advances of medicinal chemistry, X-ray protein crystallography, molecular biol(cid:173)
`ogy, pharmacology, and clinical medicine, that a significant number of new generation
`ogy, pharmacology, and clinical medicine, that a significant number of new generation
`antifolates were brought forward for clinical development. Several folate-based an-
`antifolates were brought forward for clinical development. Several folate-based an(cid:173)
`timetabolites are currently being investigated in clinical trials. These include lometrexol
`timetabolites are currently being investigated in clinical trials. These include lometrexol
`(6R-5,10-dideazatetrahydrofolic acid)(1-3), LY309887 (4), and AG2034 (5), which are
`(6R-5,1O-dideazatetrahydrofolic acid) (1-3), LY309887 (4), and AG2034 (5), which are
`potent and selective inhibitors of glycinamide ribonucleotide formyltransferase
`potent and selective inhibitors of glycinamide ribonucleotide formyltransferase
`(GARb-T), an enzyme in the purine de novo biosynthetic pathway; trimetrexate (6), eda-
`(GARFT), an enzyme in the purine de novo biosynthetic pathway; trimetrexate (6), eda(cid:173)
`trexate (7,8), and PT523 (9) which act on dihydrofotate reductase (DHFR); raltitrexed
`trexate (7,8), and PT523 (9) which act on dihydrofolate reductase (DHFR); raltitrexed
`(10,11), AG337 (12), BW1843U89 (13), and ZD933 (14) which specifically target the
`(10,11), AG337 (12), BW1843U89 (13), and ZD933 (14) which specifically target the
`enzyme thymidylate synthase (TS) involved in pyrimidine biosynthesis.
`enzyme thymidylate synthase (TS) involved in pyrimidine biosynthesis.
`N- [4- [2- (2-amino-3,4-dihydro-4-oxo-7H-pyrrolo [2,3-d]pyrimidin-5 -yl)ethyl] -ben-
`N -[ 4-[2-(2-amino-3,4-dihydro-4-oxo-7H -pyrrolo[2,3-d]pyrimidin-5 -yl)ethyl] -ben(cid:173)
`zoyt]-c-glutamic acid, LY231514, is a structurally novel antifolate that possesses a
`zoyl]-L-glutamic acid, LY231514, is a structurally novel anti folate that possesses a
`unique 6-5 fused pyrrolo[2,3-d]pyrimidine nucleus instead ofthe more common 6-6
`unique 6-5 fused pyrrolo[2,3-d]pyrimidine nucleus instead of the more common 6-6
`fused pteridine or quinazoline ring structure. LY231514 was discovered through struc-
`fused pteridine or quinazoline ring structure. L Y231514 was discovered through struc-
`
`From: Anticancer Drug Development Guide: Antifolate Drugs in Cancer Therapy .
`From: Anticancer Drug Developinent Guide: Antifolate Drugs in Cancer Therapy
`Edited by: A.L. Jackman © Humana Press Inc., Totowa, NJ
`Edited by: A.L. Jackman © Humana Press Inc., Totowa, NJ
`
`183
`183
`
`Sandoz Inc. IPR2016-00318
`Sandoz Inc. IPR2016-00318
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`
`

`
`184
`184
`
`Shih and Thornton
`Shih and Thornton
`
`~ ~ 6 _ r?-)~~H
`HN~ .. X..t"'-./~ H
`H2NA N
`
`o ~ooe
`
`3H
`eOOH
`
`N
`H
`
`Lometrexol
`Lometrexol
`(6R-DDATHF)
`(6R-DDATHF)
`
`_COOH
`
`30H
`
`MTA (LY231514)
`MTA (LY231514)
`Fig. 1. The structures oflometrexol (6R-5,1O-dideazatetrahydrofolic acid, DDATHF) and MTA (N(cid:173)
`Fig. 1. The structures of lometrexol (6R-5, l 0-dideazatetrahydrofolic acid, DDATHF) and MTA (N- .
`[4-[2-(2-amino-3 ,4-dih ydro-4-oxo-7H -pyrrol 0 [2,3-d] pyrimidin -5 -Y I )eth y I]-benzoy 1]-L-glutamic
`[4-[2-(2-amin~-3~4-dihydr~-4-~x~-7H-pyrr~~~[2,3-d]pyrirnidin-5-y~)ethy~]-benz~y~~-L-g~utamic
`acid).
`acid).
`
`ture activity relationship (SAR) studies of the novel antipurine antifolate lometrexol se(cid:173)
`ture activity relationship (SAR) studies of the novel antipurine antifolate lometrexol se-
`ries, by eliminating the C5 methylene of lometrexol and converting the sp3 center at C6
`ries, by eliminating the C5 methylene of lometrexol and converting the sp3 center at C6
`to sp2 geometry (Fig. 1) (15,16). These modifications give rise to a very potent cytotoxic
`to sp2 geometry (Fig. 1) (15,16). These modifications give rise to a very potent cytotoxic
`agent (IC50 = 15 nM) against human CCRF-CEM leukemia cells in culture. However,
`agent (IC5o = 15 nag) against human CCRF-CEM leukemia cells in culture. However,
`the end-product reversal pattern of this new pyrrolopyrimidine-based antifolate was
`the end-product reversal pattern of this new pyrrolopyrimidine-based antifolate was
`completely different to the GARFT inhibitor lometrexcil. The purine precusor hypoxan(cid:173)
`completely different to the GARFT inhibitor lometrex01. The purine precusor hypoxan-
`thine (100 f.LM) or aminoimidazole carboxamide (AICA) (300 !JM) was incapable of
`thine (100 IxM) or aminoimidazole carboxamide (AICA) (300 IxM) was incapable of
`protecting the cells from the cytotoxicity of L Y231514. In contrast, thymidine (5 f.LM)
`protecting the cells from the cytotoxicity of LY231514. In contrast, thymidine (5 btM)
`was able to provide partial protection to the cells up to lOX IC50 concentrations of
`was able to provide partial protection to the cells up to 10X IC5o concentrations of
`L Y231514. The replacement of the tetrahydropyridine ring of lometrexol with a pyrrole
`LY231514. The replacement of the tetrahydropyridine ring of lometrexol with a pyrrole
`moiety caused a major loss of activity in the inhibition of purine biosynthesis and shifted
`moiety caused a major loss of activity in the inhibition of purine biosynthesis and shifted
`the major site of action of LY231514 to the inhibition of pyrimidine biosynthesis
`the major site of action of LY231514 to the inhibition of pyrimidine biosynthesis
`(thymidylate cycle). As a "classical" antifolate, LY231514 was found to be one of the
`(thymidylate cycle). As a "classical" antifolate, LY231514 was found to be one of the
`best known substrates for mammalian folylpolyglutamate synthetase (FPGS) (17) and it
`best known substrates for mammalian folylpolyglutamate synthetase (FPGS) (17) and it
`is believed that polyglutamation and the polyglutamated metabolites of L Y231514 play
`is believed that polyglutamation and the polyglutamated metabolites of LY231514 play
`profound roles in determining both the selectivity and antitumor activity of this novel
`profound roles in determining both the selectivity and antitumor activity of this novel
`agent. Recent studies have shown that the polyglutamates ofLY231514, (e.g., the triglu(cid:173)
`agent. Recent studies have shown that the polyglutamates of LY231514, (e.g., the triglu-
`tamate glu3 and the pentaglutamate glu5) potently inhibit several key enzymes of the fo(cid:173)
`tamate glu3 and the pentaglutamate glus) potently inhibit several key enzymei of the fo-
`late metabolism, including TS, DHFR, GARFT, and aminoimidazole carboxamide
`late metabolism, including TS, DHFR, GARFT, and aminoimidazole carboxamide
`ribonucleotide formyltransferase (AICARFT) (18). As a result of this activity against
`ribonucleotide formyltransferase (AICARHF) (18). As a result of this activity against
`several enzymes, L Y231514 has become known as MT A, multi targeted antifolate.
`several enzymes, LY231514 has become known as MTA, multitargeted antifolate.
`The phase I clinical evaluation of MT A began in late 1992. Objective tumor responses
`The phase I clinical evaluation of MTA began in late 1992. Objective tumor responses
`were observed in patients with colorectal cancer and pancreatic cancer, some of whom
`were observed in patients with colorectal cancer and pancreatic cancer, some of whom
`had failed treatment with other TS inhibitors such as 5FU and raltitrexed ( 19-21). Phase·
`had failed treatment with other TS inhibitors such as 5FU and raltitrexed (19-21). Phase-
`II studies have shown activity in a range of solid tumors, including colorectal, breast and
`II studies have shown activity in a range of solid tumors, including colorectal, breast and
`nonsmall-celliung cancers (22-27). The purpose of this chapter is to comprehensively
`nonsmall-cell lung cancers (22-27). The purpose of this chapter is to comprehensively
`review the unique biochemical and pharmacological modes of action, and the recent
`review the unique biochemical and pharmacological modes of action, and the recent
`phase I and II clinical findings of this novel multi targeted antifolate, MTA ..
`phase I and II clinical findings of this novel multitargeted antifolate, MTA.
`
`Sandoz Inc. IPR2016-00318
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1122-0004
`Sandoz v. Eli Lilly, Exhibit 1122-0004
`
`CI
`
`M
`M
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`M
`M
`
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`+
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`po
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`to I
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`
`

`
`:m
`
`Chapter 8/ MTA (LY231514)
`Chapter 8 / MTA (LY231514)
`
`185
`185
`
`Table 1
`Table 1
`Inhibitory Activity of MTA, Methotrexate and Their Polyglutamates Against rhTS,
`Inhibitory Activity of MTA, Methotrexate and Their Polyglutamates Against rhTS,
`rhDHFR, rmGARFT, and rhAICARFT (Ki [mean ± SE, nM])
`rhDHFR, rmGARFT, and rhAICARFT (Ki [mean + SE, nM])
`
`Compound
`Compound
`
`MTA
`MTA
`MTA-glu3
`MTA-glu3
`MTA-glus
`MTA-glu5
`MTX
`MTX
`MTX-glus
`MTX-glu5
`
`rhTS
`rhTS
`
`109 ± 9
`109 -- 9
`1.6 ± 0.1
`1.6 + 0.1
`1.3 ± 0.3
`1.3 +- 0.3
`13,000
`13,000
`47
`47
`
`rhDHFR
`rhDHFR
`
`7.0 ± 1.9
`7.0 -+ 1.9
`7.1 ± 1.6
`7.! __ 1.6
`7.2 ± 0.4
`7.2 _+ 0.4
`0.004
`0.004
`0.004
`0.004
`
`rmGARFT
`rmGARFT
`
`rhAICARFT
`rhAICARFT
`
`9300 ± 690
`9300 -+ 690
`380 ± 92
`380 -+ 92
`65 ± 16
`65 -- 16
`80,000
`80,000
`2500
`2500
`
`3580
`3580
`480
`480
`265
`265
`143,000
`143,000
`56
`56
`
`2. PRECLINICAL PHARMACOLOGY STUDIES OF MTA
`2. PRECLINICAL PHARMACOLOGY STUDIES OF MTA
`
`2.1. Folate Enzyme Inhibition Studies
`2.1. Folate Enzyme Inhibition Studies
`The inhibition of recombinant human (rh)TS, rhDHFR, recombinant mouse
`The inhibition of recombinant human (rh)TS, rhDHFR, recombinant mouse
`(rm)GARFT, and rhAICARFT by MTA and its polyglutamates (glu3 and glus) (18) is
`(rm)GARFT, and rhAICARFT by MTA and its polyglutmnates (glu3 and glus) (18) is
`summarized in Table 1. The parent monoglutamate MT A inhibited rhTS with a Ki of 109
`summarized in Table 1. The parent monoglutamate MTA inhibited rhTS with a Ki of 109
`± 9 nM. It has been well documented that mammalian TS shows a strong preference for
`+- 9 nM. It has been well documented that mammalian TS shows a strong preference for
`polyglutamated folate substrates. The longer chain 'Y-glutamyl derivatives of MTA had
`polyglutamated folate substrates. The longer chain y-glutamyl derivatives of MTA had
`significantly enhanced affinity toward rhTS. The addition of two extra 'Y-glutamyl
`significantly enhanced affinity toward rhTS. The addition of two extra y-glutamyl
`residues (glu3) to MT A resulted in 68-fold reduction of the Ki value (Ki = 1.6 nM). Fur(cid:173)
`residues (glu3) to MTA resulted in 68-fold reduction of the Ki value (Ki -- 1.6 nM). Fur-
`ther extension of the glutamate tail (MTA-glus) only slightly increased the affinity to(cid:173)
`ther extension of the glutamate tail (MTA-glus) only slightly increased the affinity to-
`ward rhTS (Ki = 1.3 nM). MTA was also found to be a very potent inhibitor of human
`ward rhTS (Ki = 1.3 nM). MTA was also found to be a veyy.potent inhibitor of human
`DHFR (Ki = 7.0 nM). In contrast to rhTS, attachment of additional 'Y-glutamyl residues
`DHFR (Ki = 7.0 nM). In contrast to rhTS, attachment of additional y-glutamyl residues
`to MTA had little effect on the inhibition ofDHFR; MTA-glu3 and MTA-glus exhibited
`to MTA had little effect on the inhibition of DHFR; MTA-glu3 and MTA-glu5 exhibited
`identical Ki values against rhDHFR, 7.1 nM. Tight-binding analysis showed that MTA(cid:173)
`identical Ki values against rhDHFR, 7.1 nM. Tight-binding analysis showed that MTA-
`glun inhibited both TS and DHFR competitively. When MTA was tested against the en(cid:173)
`glu,~ inhibited both TS and DHFR competitively. When MTA was tested against the en-
`zymes along the purine de novo biosynthetic pathway, it only demonstrated moderate
`zymes along the purine de novo biosynthetic pathway, it only demonstrated moderate
`inhibition toward rmGARFT (Ki = 9.3 j-LM). The triglutamate and pentaglutamate of
`inhibition toward rmGARFT (Ki = 9.3 b~M). The triglutamate and pentaglutamate of
`MTA had significantly enhanced inhibitory activity against GARFT, with Ki values of
`MTA had significantly enhanced inhibitory activity against GARFT, with Ki values of
`380 nM (24-fold) and 65 nM (l44-fold), respectively. The pentaglutamate ofMTA also
`380 nM (24-fold) and 65 nM (144-fold), respectively. The pentaglutamate of MTA also
`inhibited human AICARFT with a Ki of 265 nM. Kinetic analysis confirmed the com(cid:173)
`inhibited human AICARFT with a Ki .of 265 nM. Kinetic analysis confirmed the com-
`petitive inhibition pattern of MT A polyglutamates against both GARFT and AICARFT.
`petitive inhibition pattern of MTA polyglutamates against both GARFT and AICARFT.
`Finally, MTA and its polyglutamates were competitive inhibitors of both the dehydro(cid:173)
`Finally, MTA and its polyglutamates were competitive inhibitors of both the dehydro-
`genase and synthetase domains of Cl tetrahydrofolate synthase. The Ki values for the
`genase and synthetase domains of C1 tetrahydrofolate synthase. The Ki 7¢alues for the
`mono-, tri- and pentaglutamyl derivatives of MTA were 9.9, 3.9, and 4.7 j-LM, respec(cid:173)
`mono-, tri- and pentaglutamyl derivatives of MTA were 9.9, 3.9, and 4.7 ~M, respec-
`tively, for dehydrogenase and 329,25,4 and 1.6j-LM for synthetase. MTA was a relatively
`tively, for dehydrogenase and 329,25,4 and 1.6 b~M for synthetase. MTA was a relatively
`Iess potent inhibitor of Cl tetrahydrofolate synthase than other enzyme targets such as
`less potent inhibitor of C1 tetrahydrofolate synthase than other enzyme ~argets such as
`TS, DHFR, and GARFT. However, cell-culture experiments have suggested that the in(cid:173)
`TS, DHFR, and GARFT. However, cell-culture experiments have suggested that the in-
`tracellular drug concentration of MT A can reach levels of 50 J.1M (RM Schultz, unpub(cid:173)
`tracellular drug concentration of MTA can reach levels of 50 ~ (RM Schultz, unpub-
`lished observation), and at these concentrations the activity of Cl tetrahydrofolate
`lished observation), and at these concentrations the activity of C1 tetrahydrofolate
`synthase can also be greatly suppressed by MT A polyglutamates. The important role of
`synthase can also be greatly suppressed by MTA polyglutamates. The importantrole of
`TS in serving as a rate-limiting enzyme in folate metabolism, as well as the relative or(cid:173)
`TS in serving as a rate-limiting enzyme in folate metabolism, as well as the relative or-
`der of inhibitory potency toward TS by MTA-glun indicate that TS is a major site of ac(cid:173)
`der of inhibitory potency toward TS by MTA-glun indicate that TS is a major site of ac-
`tion for MT A. Inhibition of DHFR and other enzymes in the de novo purine biosynthetic
`tion for MTA. Inhibition of DHFR and other enzymes in the de novo purine biosynthetic
`pathway may also contribute significantly to the overall antiproliferative effect of MT A
`pathway may also contribute significantly to the overall antiproliferativ~ effect of MTA
`
`c
`
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`:6
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`[1-
`Jf
`1)
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`)(cid:173)
`Ie
`st
`
`~s
`m
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`ld
`ly
`Ilt
`
`Sandoz Inc. IPR2016-00318
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1122-0005
`Sandoz v. Eli Lilly, Exhibit 1122-0005
`
`

`
`186
`186
`
`Shih and Thornton
`Shih and Thor.nton
`
`Multitargeted
`Antifolate
`(LY231514)
`
`~ 5-FU, TomudeX®
`dUMP
`dTMP --. --. DNA
`
`5,10- 2-T~HF
`( "
`~1Jt- NADPH
`\ . ~I ~' I DHF~ Methotrexate
`PRPP",
`GAR~ THF
`fGAR
`"-. ~ AMP, GMP
`
`NADP+
`
`DNA,RNA
`
`10-CHO-THF
`
`L Y309887
`
`TS: thymidylate synthase (5-FU, Tomudex)
`T$: thymidylate synthase (5-FU, Tomudex)
`DHFR: dihydroiolate reductase (Methotrexate)
`DHFR: dihydrofolate reductase (Methotrexate)
`GARFT: glycinamide ribonucleotide iormyltransierase
`GARFT: glycinamide dbonucleotide formyltransferase
`
`Fig. 2. Inhibition of multiple folate enzymes (TS, DHFR, and GARFT) by MTA and its polygluta(cid:173)
`Fig. 2. Inhibition of multiple folate enzymes (TS, DHFR, and GARFT) by MTA and its polygluta-
`mated metabolites.
`mated metabolites.
`
`(Fig. 2). This unique mode of action was further supported by additional cell-based stud(cid:173)
`(Fig. 2). This unique mode of action was further supported by additional cell-based stud-
`ies (vide infra).
`ies (vide infra).
`As a reference for comparison, the polyglutamates of methotrexate (MTX) also in(cid:173)
`As a reference for comparison, the polyglutamates of methotrexate (MTX) also in-
`hibit multiple folate-dependent enzymes. Chabner et al (28) reported that the pentaglu-
`hibit multiple folate-dependent enzymes. Chabner et al (28) reported that the pentaglu(cid:173)
`tam ate ofMTX (MTX-glus) demonstrated a significant increase in affinity toward rhTS
`tamate of MTX (MTX-glu5) demonstrated a significant !ncrease in affinity toward rhTS
`(Kj = 47 nM) and AICARFT (Kj = 56 nM) when compared with the parent monogluta(cid:173)
`(Ki = 47 nM) and AICARFT (Ki = 56 nM) when compared with the parent monogluta-
`mate. However, the affinity of MTX and its polyglutamates for DHFR (Ki = 4 pM) was
`mate. However, the affinity of MTX and its polyglutamates for DHFR (Kj = 4 pM) was
`several orders of magnitude (> 12,000-fold) higher than its affinity for TS and
`several orders of magnitude (>12,000-fold) higher than its affinity for TS and
`AICARFT, suggesting that the primary intracellular target of MTX may still be DHFR.
`AICARFT, suggesting that the primary intracellular target of MTX may still be DHFR.
`
`2.2. Cell-Based End-Product Reversal Studies
`2.2. Cell-Based End-Product Reversal Studies
`MT A is very cytotoxic against CCRF-CEM leukemia cells in culture. This potent an(cid:173)
`MTA is very cytotoxic against CCRF-CEM leukemia cells in culture. This potent an-
`tiproliferative effect of MTA can be prevented by leucovorin, whereas only partial pro(cid:173)
`tiproliferative effect of MTA can be prevented by leucovorin, whereas only partial pro-
`tection was observed with thymidine. In the presence of 5 IxM thymidine, the ICs0 of
`tection was observed with thymidine. In the presence of 5 J.LM thymidine, the ICso of
`MTA increased only 6-10-fold and this was significantly less than that of a pure TS in(cid:173)
`MTA increased only 6-10-fold and this was significantly less than that of a pure TS in-
`hibitor such as raltitrexed. This reversal pattern of MTA was further characterized in var-
`hibitor such as raltitrexed. This reversal pattern of MTA was further characterized in var(cid:173)
`ious human tumor cell lines such as GC3/C1 colon carcinoma and HCT-8 ileocecal
`ious human tumor cell lines such as GC3/C1 colon carcinoma and HCT-8 ileocecal
`carcinoma (Table 2). It was observed that 5 J.LM thymidine fully protected the cells from
`carcinoma (Table 2). It was observed that 5 bUI4 thymidine fully protected the cells from
`cytotoxicity with raltitrexed, whereas similar treatment with thymidine only increased
`cytotoxicity with raltitrexed, whereas similar treatment with thymidine only increased
`the ICso of MT A by 18.7-fold (GC3/C 1), and by 15-fold (HCT -8). Hypoxanthine (100
`the ICs0 of MTA by 18.7-fold (GC3/C1), and by 15-fold (HCT-8). Hypoxanthine (100
`J.LM) alone did not markedly influence the cytotoxicity of MTA. Similarly, AICA (300
`b~M) alone did not markedly influence the cytotoxicity of MTA. Similarly, AICA (300
`J.LM) did not modulate cytotoxicity. However, the combination of thymidine plus hy(cid:173)
`txM) did not modulate cytotoxicity. However, the combination of thymidine plus hy-
`poxanthine completely reversed the cytotoxicity of MT A in all cell lines (ICsos > 20
`poxanthine completely reversed the cytotoxicity of MTA in all cell lines (ICs0s > 20
`J.LM). The reversal pattern of MTA was also significantly different from that of MTX.
`b~M). The reversal pattern of MTA was also significantly different from that of MTX.
`Neither thymidine nor hypoxanthine could protect the cells from the cytotoxic actions-of
`Neither thymidine nor hypoxanthine could protect the cells from the cytotoxic actions of
`MTX at all drug concentrations. The unusual reversal pattern observed for MTA sug(cid:173)
`MTX at all drug concentrations. The unusual reversal pattern observed for MTA sug-
`gests that in addition to TS, other important inhibitory sites may exist for this agent. The
`gests that in addition to TS, other important inhibitor5, sites may exist for this agent. The
`higher degree of protection by thymidine at low drug concentrations indicates that TS is
`higher degree of protection by thymidine at low drug concentrations indicates that TS is
`a major target for MT A. Addition of hypoxanthine together with thymidine fully re-
`a major target for MTA. Addition of hypoxanthine together with thymidine fully re-
`
`Sandoz Inc. IPR2016-00318
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1122-0006
`Sandoz v. Eli Lilly, Exhibit 1122-0006
`
`

`
`fhornton
`Fhornton
`
`Chapter 8/ MTA (LY231514)
`Chapter 8 / MTA (LY231514)
`
`187
`187
`
`Table 2
`Table 2
`End-Product Reversal Studies with MTAa (IC5o (uM))
`End-Product Reversal Studies with MTAa (IC5o (na¢))
`dThdb
`dThdb
`(5 jJM)
`(5 tzM)
`
`Hypoxanthine C
`Hypoxanthinec
`(lOa jJM)
`(100 tzM)
`
`dThd and Hypoxanthine
`dThd and Hypoxanthine
`
`MTA
`MTA
`alone
`alone
`
`25
`25
`34
`34
`220
`220
`
`13S
`138
`637
`637
`3104
`3104
`
`32
`32
`34
`34
`1077
`1077
`
`>40,000
`>40,000
`>40,000
`>40,000
`>40,000
`>40,000
`
`Cell line
`Cell line
`
`CCRF-CEM
`CCRF-CEM
`GC3/Cl
`GC3/C1
`HeT
`HCT
`
`aCytotoxicity detennined by MIT analysis after 72 h exposure to drug, SE of triplicate detenninations
`aCytotoxicity determined by MTI" analysis after 72 h exposure to drug, SE of triplicate determinations
`did not exceed 10% of mean.
`did not exceed 10% of mean.
`bWith the addition of 5 fLM of thymidine.
`bWith the addition of 5 IxM of thymidine.
`CWith the addition of 100 ,LM of hypoxanthine.
`cWith the addition of 100 IxM of hypoxanthine.
`
`Table 3
`Table 3
`Substrate Activity of MTA and Other Antifolates for Mouse and Hog Liver FPGS
`Substrate Activity of MTA and Other Antifolates for Mouse and Hog Liver FPGS ,
`reI. Vma/Kmc
`rel. V,,~/K,nc
`
`Km(,uMya
`Km ( tzM)a
`
`reI. V ma}
`rel. Vma.rb
`
`Compound
`Compound
`
`Mouse Liver FPGS
`Mouse Liver FPGS
`Lometrexol
`Lometrexol
`MTA
`MTA
`Methotrexate
`Methotrexate
`
`Hog Liver FPGS
`Hog Liver FPGS
`Lometrexol
`Lometrexol
`MTA
`MTA
`Methotrexate
`Methotrexate
`
`9.3 ± 1.6
`9.3 ± 1.6
`O.SO ± 0.11
`0.80 ± 0.11
`166.0 ± 14
`166.0 ± 14
`
`16.4 ± 1.0
`16.4 _ 1.0
`1.9 ± 0.5
`1.9 ± 0.5
`116.0 ± 14
`116.0 ± 14
`
`1.0
`1.0
`0.63 ± O.lS
`0.63 _ 0.18
`0.50 ± 0.09
`0.50 ± 0.09
`
`1.0
`1.0
`0.74 ± 0.10
`0.74 ± 0.10
`0.51 ± O.OS
`0.51 _ 0.08
`
`1.0
`1.0
`13.7
`13.7
`0.031
`0.031
`
`1.0
`1.0
`6.40
`6.40
`0.07
`0.07
`
`aValues listed are mean ± standard error for n 2:: 3 or ± 1/2 range for n = 2 replicate experiments.
`"Values listed are mean _+ standard error for n --> 3 or + 1/2 range for n = 2 replicate experiments.
`bThe ratio of V max for a substrate to the V max of lometrexol with either mouse or hog liver FPGS.
`bThe ratio of Vm~ for a substrate to the Vm~ of lometrexol with either mouse or hog liver FPGS.
`cThe Vmax of a substrate relative to lometrexol divided by the Km of a substrate relative to lometrexol,
`CThe Vmax of a substrate relfitive to lometrexol divided by the Km of a substrate relative to lometrexol,
`the kinetics of a standard compound was measured in each experiment to allow accurate comparisons among
`the kinetics of a standard compound was measured in each experiment to allow accurate comparisons among
`substrate.
`substrate.
`
`versed the cytotoxicity of MT A, suggesting that at higher concentrations, inhibition of
`versed the cytotoxicity of MTA, suggesting that at higher concentrations, inhibition of
`DHFR and/or purine de novo biosynthetic enzymes were responsible for other secondary
`DHFR and/or purine de nov. biosynthetic enzymes were responsible for other secondary
`cytotoxic actions of the drug, a conclusion that is consistent with results from enzymatic
`cytotoxic actions of the drug, a conclusion that is consistent with results from enzymatic
`studies. Recent finding that H630-RlO cells (29) (resistant to 5FU with a 39-fold ampli(cid:173)
`studies. Recent finding that H630-R10 cells (29) (resistant to 5FU with a 39-fold ampli-
`fication ofTS protein) demonstrated a significantly reduced resistance to MTA (fivefold
`fication of TS protein) demonstrated a significantly reduced resistance to MTA (fivefold
`vs 6900-fold for raltitrexed) further support the conclusion that TS is not the sole molec(cid:173)
`vs 6900-fold for raltitrexed) further support the conclusion that TS isnot the sole molec-
`ular target for this novel agent.
`ular target for this novel agent.
`
`2.3. The Role of Polyglutamation and Folate Transport
`2.3. The Role of Polyglutamation and Folate Transport
`Polyglutamation plays an essential role in determining the overall biochemical and
`Polyglutamation plays an essential role in determining the overall biochemical and
`pharmacological properties of the classical antifolates. The formation of polyglutamates
`pharmacological properties of the classical antifolates. The formation of polyglutamates
`leads to the accumulation of polyglutamated metabolites to levels that are significantly
`leads to the accumulation of polyglutamated metabolites to levels that are significantly
`higher than could otherwise be achieved at steady state by the parent mon0glutamates,
`higher than could oilierwise be achieved at steady state by the parent monoglutamates,
`and thus serves as an important cellular retention mechanism for folates and antifolates.
`and thus serves as an important cellular retention mechanism for folates and antifolates.
`Studies have shown that MTA is an excellent substrate for mammalian FPGS. The sub- .
`Studies have shown that MTA is an excellent substrate for mammalian FPGS. The sub- .
`strate activity of MTA and several other antifolates for mouse and hog liver FPGS
`strate activity of MT A and several other antifolates for mouse and hog liver FPGS
`(15,17) are listed in Table 3. Both the substrate constants (Km) and toe relative first-order
`(15,17) are listed in Table 3. Both the substrate constants (Kin) and the relative first-0rder
`
`olygluta-
`olygluta-
`
`~d stud-
`~d stud-
`
`also in-
`also in(cid:173)
`mtaglu-
`:ntaglu(cid:173)
`:d rhTS
`rdrhTS
`~ogluta-
`togluta(cid:173)
`M)was
`M) was
`rs and
`FS and
`DHFR.
`DHFR.
`
`:ent an-
`:ent an(cid:173)
`ial pro-
`ial pro(cid:173)
`[e50 of
`1C5o of
`TS in-
`TS in(cid:173)
`in var-
`in var(cid:173)
`,’.cecal
`:ocecal
`ls from
`ls from
`.’reased
`Teased
`te (100
`Ie (100
`A (300
`A (300
`ius hy-
`us