S22i
`
`ITAL J GASTROENTEROL HEPATOL I 999;31 (SUPPL.2):S224-6
`
`New somatostatin analogues for radiotherapy of somatostatin
`receptor expressing tumours
`
`B. Stolz, P. Smith-Jones, R. Albert, G. Weckbecker, C. Bruns
`
`r;;nong various newly synthesized chelator-linked oc(cid:173)
`treotide analogues 90Y-[DOTA-DPhe 1, Thyr3 ]-octreotide
`(9°Y-SMT 487) was finally selected for clinical develop(cid:173)
`ment. In vitro, SMT 487 binds selectively with nanomolar
`qfjinity to the somatostatin receptor subtype 2 ( IC10=0.39
`
`nM±0.02). In vivo, 90Y-[DOTA-DPhe 1, Thyr3 J-octreotide
`shows a rapid blood clearance (T112 a<5 min) and high ac(cid:173)
`cumulation in somatostatin subtype 2 receptor expressing
`tumours. The in vivo administration of 90Y-[DOTA-DPhe 1
`,
`Thyr3 J-octreotide induces a rapid tumour shrinkage in
`three different somatostatin receptor positive tumour mod(cid:173)
`els: CA20948 rat pancreatic tumours grown in normal rats,
`AR42J rat pancreatic tumours and NCI-H69 human small
`cell lung cancer both grown in nude mice. The radiothera(cid:173)
`peutic efficacy of 90Y-SMT 487 was enhanced in combina(cid:173)
`tion with standard anticancer drugs, such as mitomycin C,
`which resulted in a tumour decrease of 70% of the initial
`volume. In the CA 20948 syngeneic rat tumour model, a
`single treatment with 10 ftCi/kg 90Y-SMT 487 resulted in the
`disappearance of 5 out of 7 twnours. Thus the new radio(cid:173)
`therapeutic agent showed its curative potential for the se(cid:173)
`lective treatment of SRIF receptor-expression tumours.
`Clinical Phase I studies with 90Y-SMT 487 were started in
`September 199.::J
`
`Ital J Gastroenterol Hepatol 1999;3 l (Suppl.2):S224-6
`
`Key words: endocrine tumours; somatostatin analogue; somato(cid:173)
`statin receptor.
`
`Introduction
`
`A wide variety of human tumours of different origin
`have a high incidence of somatostatin (SRIF) recep-
`
`Ftvm: ~artis Pharma AG, Basel, Switzerland.
`
`{
`
`Address for correspondence: Dr. B. Stolz, Project Head
`Oncology Research, Novartis Pharma AG, K 125 15 14 CH-
`4002 Basel, Switzerland. Fax: +41-61-3243576.
`E-nzail:~bara.stolz@plwrma.novartis.co~
`
`Acknowledgeme/lts: 17ie author is grateful to the following
`colleagues who contributed considerably with their excellent
`technical help to the above results: Ms Gabi Hofmann, Ms.
`Kerstin Pollehn, M,: Roland Haller, Mr. Helmut Knocht and Mr.
`Levente Ib/csvai.
`
`tors (e.g. pituitary tumours, endocrine pancreatic tu(cid:173)
`mours, carcinoids, small cell lung cancer, brain tu(cid:173)
`mours and human breast tumours 1 2). Those tumours
`most frequently express SRIF receptor subtype 2
`(ssh).
`The feasibility of targeting radionuclides to SRIF re-
`1111
`.
`ceptors has already been demonstrated by usmg
`n-
`[DTPA-DPhe1 ]-octreotide (111 In-SDZ 215-811, Oc(cid:173)
`treoscan® 111) for the imaging of ssh expressing tu(cid:173)
`mours 23.
`For selective radiotherapy of ssh expressing tu(cid:173)
`mours a !3-emitter labelled octreotide analogue may
`be targeted to ssh expressing tumours, where a cy(cid:173)
`totoxic radiation dose will be delivered to the tu(cid:173)
`mour cells. Receptor-mediated radiotherapy of SRIF
`receptor expressing tumours requires a peptide con(cid:173)
`jugate that consists of the peptide receptor ligand as
`the targeting moiety and a chelator that binds the ra(cid:173)
`dionuclide.
`In order to make such radiotherapeutic treatment of
`SRIF receptor-expressing tumours possible, we syn(cid:173)
`thesized a series of octreotide analogues that were able
`to tightly chelate beta emitting rare earth metals
`(e.g.90Y). Among those compounds were [DTPA-ben(cid:173)
`zyl-acetamide-DPhe1-Tyr3J-octreotide 4, [DTPA-ben(cid:173)
`zyl-acetamide-DPhe1]-octreotide, Tyr3 [DTPA-suc(cid:173)
`cinyl-acetamide-Ala-Pro-Phe-DPhe1-Tyr3]-octreotide 5,
`[DOTA-benzyl-acetamido-DPhe1]-octreotide, [DOTA(cid:173)
`benzyl-acetamide-DPhe1, Thyr3]-octreotide. Finally,
`[DOTA-DPhe1, Thyr3]-octreotide (SMT 487) 6 was se(cid:173)
`lected among various DOTA coupled octreotide ana(cid:173)
`logues because of its advantageous biodistribution
`properties and efficacy in tumour models.
`
`Somatostatin receptor binding and organ distribution
`
`In vitro, both octreotide and SMT 487 bound with
`subnanomolar affinity to the human SRIF receptor
`subtype 2 (hssh). Receptor binding experiments
`demonstrated that both octreotide (n-10) and SMT
`487 (n-3) inhibited the binding of [125I]Tyr3-oc(cid:173)
`treotide to CA 20948 rat pancreatic tumour mem(cid:173)
`branes in a monophasic manner, with ICso-values for
`ocfreotide of 0.22 nM±0.02 and for SMT 487 of 0.39
`nm±0.02.
`
`This material was copied
`at th,e N LM and may be
`Subjact US Co,pyright Laws
`
`NOVARTIS EXHIBIT 2087
`Par v. Novartis, IPR 2016-01479
`Page 1 of 3
`
`

`

`B. Stolz et al.
`
`S225
`
`3.0
`
`2.5
`
`0 µCl
`
`::::::::-
`CCI
`
`:p :s
`-0
`
`VJ
`
`~
`~ 1.5
`..J
`0 >
`0::
`=>
`0
`~ =>
`I-
`
`2.0
`
`1.0
`
`0.5
`
`0.0
`
`Pharmacokinetics and tissue distribution
`
`Ideally, a radiotherapeutic drug should be rapidly
`cleared from the blood with T 112 a<5 min. Long circu(cid:173)
`lation times would result in a high whole body irradia(cid:173)
`tion which may damage the bone marrow, the liver and
`other healthy, highly vascularized organs. A disadvan(cid:173)
`tage of labelled mAbs (MW in the 104-range) is their
`long circulation times of 48 to 72 h 7 • In contrast, pep(cid:173)
`tides (MW in the 1O3-range) are cleared much faster
`from the circulation, thus lowering the whole body ra(cid:173)
`diation exposure. The 90Y-SMT 487 is rapidly cleared
`from the blood with T39 alpha of 5 min and T41 beta of 76
`min. As a consequence, already two hours post injec(cid:173)
`tion of 90Y-SMT 487, the ratio of activity accumulation
`of
`tumour-to-normal
`tissue was
`high, with
`tumour/blood 42: I, tumour/muscle 117: I and tu(cid:173)
`mour/liver 20: 1 in AR42J tumour bearing mice.
`The radiotherapeutic dose was excreted mainly via the
`kidneys. A comparably high accumulation of radioac(cid:173)
`tivity in the kidneys was lowered by 60% of the initial
`value after co-administration of 2 g/kg of the cationic
`amino acid L-lysine 15 min prior to the radioligand.
`The kidney protective doses of L-lysine did not antag(cid:173)
`onize the anti-tumour effect of 90Y-SMT 487. On the
`basis of the pharmacokinetic and tissue distribution
`studies, dosimetric calculations were made and a ra(cid:173)
`diotherapeutically effective dose that yielded 0.4
`Gy/h/24 h to the tumour was calculated to be 500
`µCi/mouse.
`
`Therapeutic efficacy
`Proof-of-concept studies with the SRIF receptor ligand
`90Y-SMT 487 were carried out in three different tumour
`models: AR42J pancreatic tumour bearing nude mice,
`NCI-H69 human small cell lung cancer bearing nude
`mice and CA 20948 rat pancreatic tumour bearing nor(cid:173)
`mal rats (syngeneic model). The sstz shows high ho(cid:173)
`mology (>90%) in all three species (mouse, rat, man) 8•
`In AR42J tumour bearing nude mice, a single adminis(cid:173)
`tration of 500 µCi of 90Y-SMT 487 led to a tumour
`shrinkage by 60% of the initial volume within IO days.
`Similarly, 90Y-SMT 487 exhibited a potent inhibitory
`effect against the sstz receptor-expressing NCI-H69
`human small cell lung tumours grown in nude mice. A
`single dose of 600 µCi/mouse resulted in a tumour
`shrinkage by 50% of the initial volume within 10 days
`p.i. (Fig. I). In contrast, tumours in the control group
`showed rapid growth and the animals had to be sacri(cid:173)
`ficed after three weeks. The tumour shrinkage in mice
`treated with 600 µCi 90Y-SMT 487 resulted in a pro(cid:173)
`longed survival time of about three weeks when com(cid:173)
`pared to controls.
`The sstz receptor density and the high affinity status.of
`the receptors did not change even after multiple treat-
`
`600 µCl
`
`0
`
`10
`
`20
`
`30
`
`40
`
`TIME (days p.i.)
`
`Fig. 1. Radiotherapeutic effect of 90Y-SMT_487 i~ ~C_T-~69 human
`small cell lung cancer xenografts after a smgle 1v mJectlon of 600
`µCi 90Y-SMT 487. The initial tumour volume at day 0 was
`1203±102 mm3 (mean±SEM), n=5/group.
`
`ment cycles. Since no receptor downregulation oc(cid:173)
`curred during radiotherapy with 90Y-SMT 487, a _s~c(cid:173)
`ond dose of the radiotherapeutic agent was adm1ms-
`. 1 9oy
`tered. Both single and repeated treatments wit 1
`-
`SMT 487 resulted in a significant increase in the sur(cid:173)
`vival rate as a consequence of tumour shrinkage (per(cid:173)
`sonal unpublished observations).
`
`T
`!
`
`i
`:l:
`
`I
`]Jf ...
`'
`/Jl'
`
`I
`
`_.,,
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`T
`\
`
`-<>- 600 µCi 90Y-SMT 487
`-ooo µCi+ MMC
`-
`,.. -3 mg/kg Mitomycin C
`
`- - ,,. . -45 µg/Kg SMT 487
`
`0.0 +--1---+-+--t----t--;
`0
`10 20 30 40 50 60
`TIME (days p.i.)
`
`_j
`
`0 >
`0::
`:::>
`0
`~
`:::>
`I-
`
`Fig. 2. Radiotherapcutic effect of 600 µCi 90Y-SMT 487 combined
`with 3 mg/kg mitomycin in C in NCI-H69 human small cell lung
`cancer xenografts. 45 ~Lg/kg SMT 487 (control), 3 mg/kg mito(cid:173)
`mycin C and 600 µCi 90Y-SMT 487 were iv injected alone; in one
`group mitomycin C was injected together with 90Y-SMT 487. The
`initial tumour volume at day 0 was 1290±81 mmi (mean±SEM),
`n=6/group.
`
`This matarial was <<Ipeied
`at the NLM and may ti€
`5u bject US Copeyright Laws
`
`NOVARTIS EXHIBIT 2087
`Par v. Novartis, IPR 2016-01479
`Page 2 of 3
`
`

`

`S226
`
`SomaLostatin receptor expressing tumours: radiotherapy
`
`90Y-SMT 487 will most likely be used in combination
`wit? exis~i~~ anticancer drugs which preferably have
`rad1osens1tizmg properties (e.g. doxorubicin). There(cid:173)
`fore, the combination of 90Y-SMT 487 with doxoru(cid:173)
`bicin, 5-fluorouracil and mitomycin-C was tested in
`the AR42J and the NCI-H69 nude mouse tumour
`models. The single agent therapy with 90Y-SMT 487
`was superior to ~~nventional chemotherapeutic drugs
`such as doxorub1cm, 5-fluorouracil and mitomycin-C.
`~owever, the administration of 600 µCi 90Y-SMT 487
`rn combination with 3 mg/kg mitomycin-C enhanced
`the extent of tumour shrinkage and resulted in a de(cid:173)
`crease of 70% of the initial tumour volume in NCI(cid:173)
`H69 tumours (Fig. 2) (unpublished data).
`The maximum radiotherapeutic affect was achieved in
`the syngene~c <?A20948 rat tumour model 9• Complete
`tumour rem1ss10n was obtained in 5 out of 7 rats, 8
`:eeks after a single administration of 1 O µCi/kg of
`Y-SMT 487. No regrowth occurred during the sub(cid:173)
`seqt~ent observation period (8 months). Upon re-inoc(cid:173)
`ulation of tumour material into the cured rats, in 5 out
`of 7_ n_o_ tumour growth occurred, whereas in those rats
`exh1b1tmg tu_mour growth the tumour disappeared af(cid:173)
`ter three _to four weeks without any further treatment
`of t?e ammals. The latter result may indicate a possi(cid:173)
`ble 1m~t~ne m~diated affect of 90Y-SMT 487. This hy(cid:173)
`pothesis 1s subject to further research.
`
`Conclusions
`
`The preclinical rationale for the use of 90Y-octreotide
`analo~\1es f~r the _trea_tment of sst2 expressing tu(cid:173)
`~ours is ?ase~ _on m vitro and in vivo evidence of a
`d1rec~ antiprohferative effect resulting in tumour re(cid:173)
`gres~wn. The concept of using an 90Y labelled oc(cid:173)
`treotide for the treatment of malignant neoplasms was
`r .
`.
`'
`prove
`n prec_ m1cally m ssh expressing rodent tumour
`models .. This new radiotherapeutic drug combines
`several important prerequisites for an effective tu(cid:173)
`mour ~reatment with targeted radiolabelled peptides
`~os~ importantly, 90Y-SMT 487 has the potency of
`Cd~smg complete t~mour remission upon a single
`tredtment as shown, m a syngeneic rat tumour model.
`
`90
`Y-SMT 487 rep(cid:173)
`Receptor targeted radiotherapy with
`resents a new strategy for the treatment of ssh express(cid:173)
`ing tumours that have previously been diagnosed with
`OctreoScanl 11. Together the three compounds, oc(cid:173)
`treotide the diaanostic octreotide derivative Octreo(cid:173)
`Scanl Ii and the°radiotherapeutic octreotide analogue
`90Y-SMT 487, will be complementary in the diagnosis
`and treatment of ssh expressing tumours, especially
`those of neuroendocrine origin. Clinical Phase I stud(cid:173)
`ies with 90Y-SMT 487 are ongoing.
`
`References
`
`1 Reubi JC, Krenning E, Lamberts SWJ, Kvols L. In vitro detection
`of somatostatin receptors
`in human
`tumors. Metabolism
`1992;41: 104-10.
`2 Krenning EP, Kwekkeboom DJ, Bakk~r WH, Breei:iar~ WAP,
`Kooij PPM, Oei HY, et al. Somatostatm receptor _scmt1graphy
`with [111 In]-DTPA-DPhe 1 ]- and [123T-Thyr3]-octreotlde: the Rot(cid:173)
`terdam experience with more than 1,000 patients. Eur J Nucl Med
`! 993;20:283-92.
`3 Bruns C, Stolz B, Albert R, Marbach P, Pless J. OctreoScan 111
`for imaging of a SRIF receptor-positive islet _cell tumor i.n rat.. In:
`Steiffer EF, Reaven GM, editors. Somatostatm receptor-1mag111g.
`Harmon Metab Res Suppl I 993;27:5-11.
`4 Stolz B, Smith-Jones P, Albert R, Tolcsvai L, Briner U, Ruser G,
`et al. Somatostatin analogues for somatostatin-receptor mediated
`radiotherapy of cancer. Digestion I 996;57(Suppl. I): 17-21.
`5 Smith-Jones P, Stolz B, Albert R, Knecht H, Bruns C. Synthesis,
`biodistribution and renal handling of various chelate-somato(cid:173)
`statin conjugates with metabolizable linking groups. Nucl Med
`Biol 1997;24:761-9.
`6 Albert R, Pless J, Simeon C, Knecht H, Smith-Jones P. Direct
`synthesis of [DOTA-DPhe 1 ]-Octreotide and [DOTA-DPhe 1
`,
`Thyr3]-Octreotide (SMT487): Two chelators for systemic deliv(cid:173)
`ery of radiotherapeutical nuclides to somatostatin receptor posi(cid:173)
`tive tumors in man. Bioorg Med Chem Lett 1998;8:1207-10.
`7 De Nardo GL, Kroger LA, De Nardo SJ, Miers LA, Salako Q,
`Kukis DL, et al. Comparative toxicity studies ofYttrium-90 Mx(cid:173)
`DTPA and 2-IT-BAD conjugated monoclonal antibody (BrE-3).
`Cancer l 994;73(Suppl.): IO 12-22.
`8 Bruns C, Weckbocker C, Raulf F, Li.ibbcrt H, Hoyer D. Charac(cid:173)
`terization of somatostatin receptor subtypes. Ciba Found Symp
`1995; 190:89-101.
`9 Stolz B, Weckbecker G, Smith-Jones P, Albert R, Raulf F, Bruns
`C. The somatostatin receptor targeted radiotherapeutic [90Y(cid:173)
`DOTA DPhe 1, Thyr3]octreotide ([9°YJ SMT487) eradicates exper(cid:173)
`imental rat pancreatic CA 20948 tumours. Eur J Nucl Med
`I 998;25:668-74.
`
`This material wasuipied
`atthe N LM and may bi!
`~u bj,ect US Copyright Laws
`
`NOVARTIS EXHIBIT 2087
`Par v. Novartis, IPR 2016-01479
`Page 3 of 3
`
`