QJ NUCL MED 1999;43:356-66
`
`Therapy of neuroendocrine tumors
`with radiolabeled somatostatin-analogues
`
`M. DEJONG, W. A. P. BREEMAN, H. F. BE~ARD, P. P.M. KOOIJ, G. D. SLOOTER*, C. H. J. VAN EIJCK*
`D.J. KWEKKEBOOM, R. VALKEMA, H. R. MACKE**, E. P. KRENNING**
`
`Peptide receptor scintigraphy with the radioactive som(cid:173)
`atostatin-analogue [111Jn-DTPAO]octreotide (DTPA = dieth(cid:173)
`ylenetriaminepentaacetic acid) is a sensitive and spe(cid:173)
`cific technique to show in vivo the presence and abun(cid:173)
`dance of somatostatin receptors on various tumors.
`With this technique primary tumors and metastases of
`neuroendocrine cancers as well as of many other can(cid:173)
`cer types can be localised. A new application is the use
`of peptide receptor radionuclide therapy, administrat(cid:173)
`ing high doses of tllJn- or 90¥-labeled octreotide-ana(cid:173)
`logues.
`Preclinical. We investigated the radiotherapeutic effect
`of9oy. and 111Jn-labeled [DOTAO,Tyr3]octreotide (DOTA=
`tetraazacyclododecanetetraacetic acid) or [111In(cid:173)
`DTPA O]octreotide in lewis rats bearing the somatostatin
`receptor-positive rat pancreatic tumor CA20948 in A)
`the flank or B) in the liver.
`Patients. Thirty end-stage patients with mostly neuroen(cid:173)
`docrine progressing tumors were treated with [111In(cid:173)
`DTPA O]octreotide, up to a maximal cumulative patient
`dose of about 74 GBq, in a phase 1 trial.
`Preclinical results. A) Flank model: at least two 111MBq
`injections of(111In-DOTAO,Tyr3]octreotide were needed
`to reach tumor response, in 400/o of the animals complete
`tumor remission was found after a follow-up period of
`10 months. One or two injections of [90V-DOTAO,Tyr3]
`octreotide yielded transient stable disease.
`B) Liver model: we found that peptide receptor radionu(cid:173)
`clide therapy is only effective if somatostatin receptors
`are present on the tumors, and is therefore receptor(cid:173)
`mediated. High radioactive doses of 370 MBq (111In(cid:173)
`DTPAO]octreotide or 93 MBq [90V-DOTA0,Tyr3]octreotide
`can inhibit the growth of somatostatin receptor-posi(cid:173)
`tive metastases.
`
`Address reprint requests to: E. P. Krenning, Department of Nuclear
`Medicine, University Hospital Rotterdam, 3015 GD Rotterdam.
`E-mail: krenning@nuge.azr.nl
`
`·
`
`From the Department of Nuclear Medicine
`*Surgery, **Kantonspital Basel, Switzerland
`and ***Internal Medicine III
`University Hospital and Erasmus University
`Rotterdam, 7be Netherlands
`
`Clinical results. There w~ no major clinical side effects
`after up to 2 years treatment, except that a transient
`decline in platelet counts and lymphocyte subsets can
`occur. Promising beneficial effects on clinical symp(cid:173)
`toms, hormone production and tumor proliferation
`were found. Of the 21 patients with progressive disease
`at baseline and who received a cumulative dose of more
`than 20 GBq (111In-DTPAO]octreotide, 8 patients showed
`stabilisation of disease and 6 other patients a reduction
`in size of tumors. There is a tendency towards better
`results in patients whose tumors have a higher accu(cid:173)
`mulation of the radio ligand
`Conclusion. Radionuclide therapy with octreotide-deriv(cid:173)
`atives is feasible, both with 1111n and 9oy as rad.ionu(cid:173)
`clides.
`KEY woRDs: Neuroendocrine tumors - Somatostatin analogues
`and derivates- Radioisotopes, therapeutic use.
`
`T he inhibitory effect of somatostatin (SS) on ?<:>r(cid:173)
`
`mone secretion led to the concept of benef1oal
`effects of somatostatin in the treatment of diseases
`based on gland hyperfunction or overproduction of
`hormones by endocrine-active tumors. The tetradeca(cid:173)
`peptide ss14 itself is unsuitable for treatment, because
`of its very short half-life of minutes in man after
`intravenous injection and because of its diversity of
`action, such as lowering insulin levels. Successful
`efforts have been undertaken to synthesise more
`
`356
`
`THE QUARTERl-Y JOURNAL OF NUCLEAR MEDICINE
`
`December 1999
`
`Roxane Labs., Inc.
`Exhibit 1033
`Page 001
`
`

`
`THERAPY OF NEUROENDOCRINE TIJMORS WI1H RADIOLABELED SOMATOSTATIN-ANALOGUES
`
`DEJONG
`
`Octreotide
`
`D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr (ol)
`
`[Tyr3] octreotide
`
`D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr ( ol)
`
`DTPA
`
`DOTA
`
`HOOC-CH 2
`"'-
`
`/
`HOOC-CH 2
`
`CH 2-HOOC
`H 2C-HOOC
`I
`/
`N-(CH 2)z-N-(CH 2)z-N
`""'
`
`HOOC
`COOH
`\r-----1)
`
`[ ]
`
`(L___j'!
`HOOC
`COOH
`
`Fig. I.-Structures of octreotide, [Tyr3]octreotide, DlPA and DOTA.
`
`s.table somatostatin-analogues, resulting in octreotide
`(Fig. 1).
`Somatostatin receptors are integral membrane gly(cid:173)
`coproteins. Five different human somatostatin recep(cid:173)
`tor types have been cloned. All subtypes bind SS14
`with high affinity, while the affinity of numerous som(cid:173)
`atostatin-analogues differ considerably.t-3 Octreotide
`binds with high affinity to the somatostatin receptor
`subtype 2 (sst2), while this analogue has a lower affin(cid:173)
`ity for sst3 and sst5 and shows no binding to sst1 and
`sst4.t-4 Octreotide scintigraphy is, therefore, based on
`tpe visualisation of (an) octreotide-binding somatos(cid:173)
`tatin receptor(s), most probably the sst2•
`Peptide receptor scintigraphy with the radioactive
`somatostatin-analogue, (lttin-DTPAO]octreotide (Fig.
`1), is a sensitive and specific technique to show in vivo
`the presence and abundance of somatostatin receptors
`on various tumors. In general, mapping of the pres(cid:173)
`ence of various peptide receptors on the cell mem(cid:173)
`brane by peptide receptor scintigraphy may become
`an attractive, non-invasive, harmless, easy-to-perform
`tool for an individual therapeutic approach of the
`qtncer patient. 5-8 A new and fascinating application is
`the use of radiolabeled peptides for peptide receptor
`radionuclide-therapy, further referred to as radionu-
`
`elide-therapy. The success of the therapeutic strategy
`relies upon the amount of radioligand, which can be
`concentrated within tumor cells and the rates of inter(cid:173)
`nalisation, degradation and recycling of both ligand
`and receptor will among other things determine this.
`Binding of several peptide hormones to specific sur(cid:173)
`face receptors is generally followed by internalisa(cid:173)
`tion of the ligand receptor complex via invagination
`of the plasma membrane.9 10 The resulting intracellu(cid:173)
`lar vesicles, termed endosomes, rapidly acidify, which
`causes the ligand to dissociate from the receptor. The
`ligand may be delivered to the lysosome u and the
`receptor may recycle back to the plasma membrane.
`The whole process takes approximately 15-min,9 and
`a single receptor can deliver numerous ligand mole(cid:173)
`cules to the lysosomes. We have studied internalisa(cid:173)
`tion and degradation of radiolabeled [DTPAO]octreo(cid:173)
`tide and we detected internalisation of the radiophar(cid:173)
`maceutical in tumor cells, in accordance with the find(cid:173)
`ings of Andersson et at., tz this process was receptor(cid:173)
`specific and temperature dependent.t3 Receptor-medi(cid:173)
`ated internalisation of (Htln-DTPAO]octreotide will
`most probably result in degradation to lltln-DTPA-D(cid:173)
`Phe, this metabolite is not capable passing the lyso(cid:173)
`somal membrane .11
`Since 111In emits not only gamma-rays, which are
`visualised during scintigraphy, but also short-ranged
`Auger-electrons, an effect on tumor cell proliferation
`could be expected, as the radiotoxicity of Auger-elec(cid:173)
`trons is very high if the DNA of the cell is within the
`particle range.t4-16 tllln emits Auger- and conversion(cid:173)
`electrons having a tissue penetration of 0.02 to 10 Jlm
`and 200 to 500 J.Lm, respectively, and can therefore be
`used to investigate its antiproliferative effect in cancer.
`We reported a biological half-life for lllln of >700
`hours in tumor tissue.6 Therefore, Ill In-labeled [DTPAo]
`octreotide has an appropriate distribution profile in
`humans for scintigraphy and radionuclide therapy.t7
`ts Earlier we reported t9 that high radioactive doses
`with [lltln-DTPAO]octreotide for radionuclide-therapy
`could inhibit the growth of somatostatin receptor(cid:173)
`positive liver metastases in an animal model, and that
`radionuclide-therapy was only effective if somatos(cid:173)
`tatin receptors were present on the tumors. The effect
`of radionuclide-therapy with [lllln-DTPAO]octreotide
`could be reduced by pretreatment with excess unla(cid:173)
`beled octreotide, which indicates that receptor bind(cid:173)
`ing is essential for radionuclide-therapy.
`For radiotherapeutic applications, other radionu(cid:173)
`clides have also been proposed and investigated for
`
`Vol. 43- No.4
`
`THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE
`
`357
`
`_j
`
`Roxane Labs., Inc.
`Exhibit 1033
`Page 002
`
`

`
`DEJONG
`
`1HERAPY OF NEUROENDOCRINE TUMORS WITH RADIOLABELED SOMATOSTATIN-ANALOGUES
`
`coupling to octreotide-analogues. 9oy is a ~-particle
`emitter, the maximum energy of the electrons is 2.3
`MeV, their mean range is a few millimeters in tissue.
`90Y shows dissociation from DTPA-conjugated peptides
`in serum, resulting in hematopoietic toxicity in vivo,
`therefore, Tyr3-octreotide (Fig. 1), which has a higher
`binding affinity for sst2 than octreotide itself, has been
`derivatized with the chelator DOTA enabling stable
`radiolabeling with both 9oy and 111In. Preclinical and
`clinical studies with [DOTAO,Tyr3]octreotide showed
`favourable biodistribution and tumor uptake charac(cid:173)
`teristics. 2o 21
`In this paper preclinical and clinical studies on the
`radiotherapeutic effect of radiolabeled octreotide(cid:173)
`derivatives are described:
`Preclinical studies.-A) Flank model: experiments
`using 9oy_ and 111In-labeled [DOTAO,Tyr3)octreotide
`in Lewis rats bearing the somatostatin receptor-posi(cid:173)
`tive rat pancreatic tumor CA20948 in their flank.
`B) Liver model: timing and dosage characteristics of
`radionuclide therapy using [lllJn-DTPAO)octreotide
`and [90Y-DOTAO,Tyr3]octreotide in rats inoculated with
`CA20948 tumor cells in the portal vein of the liver.
`Clinical study.-The side-effects and antiprolifera(cid:173)
`tive effect of high, multiple radiotherapeutic doses of
`[lllJn-DTPAO] were investigated in a phase 1 study.
`We included end-stage patients with mainly a high
`tumor load of progressing somatostatin receptor-pos(cid:173)
`itive neuroendocrine tumors.
`
`Materials and methods
`
`Labeled peptides
`[DTPA o]octreotide (DTPA diethylenetriaminepenta(cid:173)
`acetic acid) and 111InC13 (DRN 4901, 370 MBq/ml in
`HCl, pH= 1.5- 1.9) were obtained from Mallinckrodt
`Medical BV (Petten, The Netherlands). [DTPAO]octreo(cid:173)
`tide was labeled with 1HinCl3 as has been described pre(cid:173)
`viously. 22 [DOTA o ,Tyr3]octreotide (DOTA = tetraazacyclo(cid:173)
`dodecanetetraacetic acid) was synthesised by Prof. H.
`Macke (Basel, Switzerland). 90Y- and tllin-labeling of
`[DOTA o ,Tyr3]octreotide was performed as described. 20
`
`Preclinical in vivo radionuclide therapy experiments
`using radiolabeled somatostatin-analogues
`A) Flank model.-The CA20948 pancreatic tumors
`were grown at the flank of Lewis rats. Male Lewis rat
`
`(Harlan, The Netherlands; 250-300 g) were injected
`subcutaneously into both flanks, each with 500 J..Ll of
`a cell suspension of the CA20948 tumor, prepared
`from 5 g crude tumor tissue in 100 ml saline. At the
`start of therapy, rats were anaesthetised, and the pep(cid:173)
`tides were injected into the dorsal vein of the penis.
`Five groups of 6 rats were formed. The four therapeu(cid:173)
`tic groups received either a single injection or two
`injections (one week apart) of either 111 MBq [111In(cid:173)
`DOTAO,Tyr3]octreotide or 111 MBq [90Y-DOTAO,Tyr3)
`octreotide. Specific activity of the peptides was 37
`MBq/1.2 J..Lg peptide. The control group received a
`concordant amount of unlabeled [DOTAO,Tyr3]octre(cid:173)
`otide (3.6 J..Lg).
`In an earlier study we investigated the effects of
`variation in injected peptide amount on tumor uptake
`in CA20948 pancreatic tumor-bearing rats for lllin(cid:173)
`labeled [DOTAO,Tyr3]octreotide, in order to find the
`peptide amount at which the highest uptake in the tar(cid:173)
`get was achieved. We found that uptake in the tumor
`as a function of injected amount peptide showed a bell
`shape with the highest uptake at 0.5 J..Lg peptide. At
`higher peptide amounts tumor uptake of radioactiv(cid:173)
`ity was decreased to about 50% of the maximum
`tumor uptake after injection of 4 J..Lg peptide [Eur J
`Nucl Med, July 1999:26(7):693-81 In the present study
`we had to inject 3.6 J..Lg peptide, this was adminis(cid:173)
`tered in 2 injections of 1.8 J..Lg each, 1 hour apart.
`The tumors were measured on the day of radiolig(cid:173)
`and administration and every 3-4 days thereafter.
`Tumor growth, animal condition and body weight
`were determined independently by two investigators.
`At progressed stage of the CA20948 tumor necrosis
`may occur. Besides loss of more than 10% of original
`body weight, tumor necrosis was an indication to sac(cid:173)
`rifice the rats.
`B) Liver model.-The tumor was transplanted and
`maintained in the liver after direct injection of tumor
`cells into the vena porta to produce artificial liver
`metastases. Therefore, tumors were excised from
`donor livers, cleaned from normal liver tissue and
`pressed through sieves with decreasing mess size.
`The suspension was washed twice in phosphate-buf(cid:173)
`fered saline. Viability was measured with trypan-blue
`exclusion. A suspension of 2.5x106 living cells/mL
`was used for injection into the vena porta. All rats
`were sacrificed 20-21 days after inoculation of tumor.
`Tumor growth was determined by two investigators
`
`358
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`THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE
`
`December 1999
`
`Roxane Labs., Inc.
`Exhibit 1033
`Page 003
`
`

`
`lHERAPY OF NEUROENDOCRINE TIJMORS WilH RADIO LABELED SOMATOSTATIN-ANAWGUES
`
`DEJONG
`
`counting and ranking the number of metastases (from
`0 to 5+ ), while blinded for treatment modality.
`Experiment 1 was designed to determine the minimal
`effective dosage of radionuclide-therapy: the rats
`were injected with 3.7, or 37 or 370 MBq (0.5 J..Lg)
`[lnin-DTPAO]octreotide on day 1. In experiment 2
`rats were treated with 370 MBq (0.5 J..Lg) [lllin(cid:173)
`DTPAO]octreotide on day 6 or 12. In experiment 3
`rats received radionuclide-therapy on day 7 or 14.
`In experiment 4 the rats were injected at day 1 with
`saline (A), or with vehicle (2 J..Lg [DOTAO,Tyr3]octreo(cid:173)
`tide) (B), or with 93 MBq (2 J.!g) [90Y-DOTAO,
`Tyr3]octreotide (C). Experi-ments 1-4 were performed
`twice. Rats were weighed 3 times/week, and their
`body weight was expressed as the percentage of
`their body weight at day 3.
`
`Statistical analysis
`Statistical analysis was performed using Mann(cid:173)
`Whitney "U" -test on categorised outcomes, Fisher's
`exact test on proportions and analysis of variance.
`Statistical significance was defined as p<0.05.
`
`Clinical in vivo radionuclide therapy experiments
`using radiolabeled somatostatin-analogues
`The typical dose per administration is 6000-7000
`MBq Illin incorporated in 40-50 J..Lg [DTPAO]octreotide.
`Doses were given with at least 2 week intervals
`between administrations and a total of 8 adminis(cid:173)
`trations is aimed at with extensions in a few patients
`to about 20 administrations. Radionuclide therapy
`with [lllJn-DTPAO]octreotide was applied after wit(cid:173)
`nessed informed consent by the patient and approv(cid:173)
`al by the medical ethics committee of our hospital.
`The following measurements, carried out prior to
`and between all administrations, served as parame(cid:173)
`ters of possible side-effects: the usual haematologi(cid:173)
`cal and chemical analyses of bone-marrow, liver,
`kidney and endocrine pancreatic (glucose or Hb A1c)
`function. Pituitary function (Free T4, post-menopau(cid:173)
`sal women: LH and FSH, men: testosterone) was
`assessed prior to and 4 weeks after the 4th and 8th
`administration of [tllin-DTPAO]octreotide; also pos(cid:173)
`sible effects on: 1) the endocrine activity of the
`tumors and/ or their production of specific serum
`markers, and 2) tumor-size ( CT or MRI) were inves(cid:173)
`tigated. Pituitary-adrenal-axis function testing (mety(cid:173)
`rapone test) prior to and after 8 administrations as
`
`well as long-term follow up with 3-4 month intervals
`was investigated if feasible.
`
`Dosimetry oj{111Jn-D1PAO}octreotide in humans
`Scoring of tumor radioactivity uptake in patients
`prior to the start of treatment with [l11In-DTPA0]octre(cid:173)
`otide was done visually by using scintigrams obtained
`24 hr after injection of a diagnostic dose (220 MBq)
`of [lllin-DTPAO]octreotide. The scoring grades used
`were: 4 =intense, 3 =clear (higher than liver uptake),
`2 = clear but faint (lower than or equal to liver
`uptake), 1 = equivocal and 0 = no accumulation.
`Patients were also scanned 3 and 7 days after each
`administration of the radiotherapeutic dose. Percen(cid:173)
`tage uptake of the administered dose in total body
`and in the most prominent tumor was calculated (data
`not shown). Uptakes decreased slowly or remained
`the same if the interval between the successive admin(cid:173)
`istrations was less than one month. In patients who
`had 6 or more administrations of 6000 to 7000 MBq
`of [111In-DTPAO]octreotide with intervals of maximal(cid:173)
`ly one-month between administrations, uptake in the
`tumor was still clearly visible after the last administra(cid:173)
`tion. Typical radiation doses to tissues with doses of
`6000-7000 MBq [111In-DTPAO]octreotide are: kidneys
`300-1400 (depending on the relative biological effec(cid:173)
`tiveness [RBE: 1- 20] for Auger electrons) cGy, spleen
`200 cGy, liver 50 cGy, bone marrow 13 cGy (target
`organ for gamma photons), thyroid gland 25 cGy
`and pituitary 70 cGy, the critical organs are kidneys
`and spleen. With these doses the estimated tumor
`radiation doses for a 10 g tumor (assumptions: 191>
`uptake; effective half-life is the physical half-life)
`1700 and 6700 cGy (RBE for Auger electrons 1 and 20,
`respectively) and for a 100 gram tumor (1% uptake)
`250 and 750 cGy, respectively.
`
`Results and discussion
`
`Preclinical in vivo radionuclide therapy experiments
`using radiolabeled somatostatin-analogues
`A) Flank model.-Figure 2 shows that the tumors of
`the rats in the control group grew excessively. Those
`rats had to be sacrificed on average 12 days post unla(cid:173)
`beled peptide injection. The treatment with a single
`dose of 111 MBq [n1In-DOTAO,Tyr3]octreotide did not
`induce tumor response, whereas two injections of
`
`Vol. 43 No.4
`
`THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE
`
`359
`
`Roxane Labs., Inc.
`Exhibit 1033
`Page 004
`
`

`
`DEJONG
`
`lHERAPY OF NEUROENDOCRINE TUMORS Willi RADIOLABELED SOMATOSTATIN-ANALOGUES
`
`Control
`
`30
`
`60
`
`90
`
`120
`
`0
`
`t
`
`111 In-, lllMBq
`
`1500 fJ
`
`1200
`900
`600
`300
`o~~~--~~----~--~~~----~---
`60
`120
`90
`30
`0
`
`t
`
`90Y-, 111MBq
`
`1500
`1200
`900
`600
`300
`Ol-~U.~~~~~----~~--~~---
`60
`120
`90
`30
`
`0 t
`
`111 In-, 2 x 111MBq
`
`1500
`1200
`900
`600
`300
`OL-~ . . ~~--~~--~--~--~~----
`120
`30
`90
`60
`0
`tt
`
`90Y-, 2 X 111MBq
`
`1500
`1200
`900
`600
`300
`0~~~----~~~~~~~~----
`120
`30
`90
`60
`tt
`
`0
`
`Days post therapy
`
`Fig. 2.--Effect of lllJn- or 90y-Iabeled [DOTA0,Tyr3]octreotide (one or
`two injections of 111 MBq/rat, indicated by the arrows at the horizon(cid:173)
`tal axis) on tumour growth in Lewis rats bearing CA20948 tumors in
`the flanks. Control animals received unlabeled peptide in the same
`amount as given in the radiolabeled administrations (3.6 !!g).
`
`TABLE I.-Effects of therapy with P11Jn-DTPAO]octreotideon count
`of tumor colonies in liver.
`
`Exp 1
`
`3.7 MBq
`37 MBq
`370 MBq
`Control
`
`Tumor colonies count
`
`0
`
`1+
`
`2+
`
`3+
`
`2
`
`4
`
`4
`
`4+
`
`5
`4
`
`5+
`
`1
`4
`
`4
`
`Rats (n)
`
`8
`8
`8*, **, §, 9f, 9f9T
`4
`
`*: p<O.OS versus Control, **: p<O.OS versus 37 MBq, §: p<O.OS versus 3.7
`MBq, <jf: p<O.OS versus 37 MBq, based on liver weight, <J<J: p<O.OS versus 3.7
`MBq, based on liver weight.
`
`111 MBq resulted in at least partial tumor remission.
`Of the animals in this group 40% reached complete
`tumor remission. Mean survival time in the latter group
`was 85 days post therapy (p<0.001 versus control),
`determined 140 days post therapy. After a single injec(cid:173)
`tion with [90Y-DOTAO,Tyr3]octreotide transient regres(cid:173)
`sion of the tumors was found and the rats lived 28 days
`post therapy (p<0.001 versus control). Two injections
`with 111 MBq [90Y-DOTAO,Tyr3]octreotide, one week
`apart, resulted in a mean survival of 65 days post
`therapy (p<0.001 versus control), no complete remis(cid:173)
`sion was reached.
`We and others have also studied several parameters
`like histology of various organs, blood cell count and
`endocrine parameters (manuscript in preparation),
`no obvious toxicology of the given radiotherapy has
`been found so far in rats.
`Liver model.-In experiment 1 we investigated
`radionuclide-therapy at day 1 after injection of 370, 37
`or 3.7 MBq [11 1In-D1PAO]octreotide (Table I). Figure 3
`shows examples of livers from control (left) and 37
`MBq-treated (right) rats. Twenty days after the direct
`injection of CA20948 tumor cells into the portal vein
`the rats were sacrificed. In all groups tumor colonies
`were counted. The 370 MBq dosage had significant(cid:173)
`ly more effect on tumor score and inhibited the
`increase of liver weight due to tumor growth more
`than the 37 or 3.7 MBq dose. Starting from day 14
`after therapy we also found a significant relation
`between the decrease of body weight (100% at day 3)
`and the liver weight and tumor score at sacrifice at day
`20 (data not shown). A significant predictive value
`for tumor growth was found at a loss of ;;:::9% of body
`weight (data not shown).
`Since a dose response was found, we continued our
`studies with 370 MBq for experiments 2 and 3. In
`
`360
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`lHE QUARTERLY JOURNAL OF NUCLEAR MEDICINE
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`December 1999
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`Page 005
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`THERAPY OF NEUROENDOCRINE TUMORS \X11TH RADIOLABELED SOMATOSTATIN-ANALOGUES
`
`DEJONG
`
`Fig. 3.-A) These photographs show examples of livers from the treated and untreated group. B) Right livers with no visual metastases after
`peptide receptor radionuclide therapy with 370 MBq (0.5 !lg) [11 1Jn-DTPAO]octreotide.
`
`experiment 2 radionuclide-therapy on day 6 or day 12
`induced a significant decrease in tmnor score versus the
`control group. The livers in the control group all had
`the highest tumor score (5+). There was significant less
`tumor growth and increase in liver weight in rats treat(cid:173)
`ed on day 6 or 12 versus controls (data not shown). In
`experiment 3 radionuclide-therapy on day 7 was also
`able to significantly reduce the tumor score and to
`reduce the liver weight versus control, this in contrast
`to radionuclide-therapy on day 14 (data not shown). In
`experiments 2 and 3 we thus confirmed the finding
`that tumor growth can be inhibited with radionuclide(cid:173)
`therapy,19 even 12 days after inoculation of the soma(cid:173)
`tostatin receptor-positive tumor in the portal vein. These
`findings suggest that even on established tumors reduc(cid:173)
`tion of tumor volume can be obtained and thereby
`support the results in case reports that also describe
`
`reduction of tumor volume.17 Liver weights of rats treat(cid:173)
`ed on day 12 were significantly lower compared to
`the liver weights of rats treated on day 6. One hypoth(cid:173)
`esis is that the tumor cell has a long lag-phase and
`starts proliferating fast after 1 week and thereby may
`
`TABLE H.-Effects of therapy with (90Y-DOTAo, Tyr3)octreotide on
`count of tumor colonies in liver.
`
`Exp 1
`
`A
`B
`c
`
`Tumor colonies count
`
`0
`
`1+
`
`2+
`
`3+
`
`2
`2
`3
`
`4+
`
`2
`
`5+
`
`4
`5
`
`Rats (n)
`
`8
`7
`7*
`
`A: control rats, B: rats received 2 pg [DOTAO, Tyr3]octreotide, and C: 93 MBq
`(2 pg) [90Y-DOTAo, Tyr3]octreotide, *: p <0.05 us A and B.
`
`Vol 43- No. 4
`
`THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE
`
`361
`
`Roxane Labs., Inc.
`Exhibit 1033
`Page 006
`
`

`
`DEJONG
`
`THERAPY OF NEUROENDOCRINE TUMORS WITH RADIOLABELED SOMATOSTATIN-ANALOGUES
`
`TABLE III.-Characterlstics of patients treated with [1 11 In-DTPA(cid:173)
`D-Phe'I }octreotide.
`
`Pathology
`
`Carcinoid
`NE Tumour
`Gastrinoma
`Vipoma
`Glucagonoma
`Med. Thyroid Cane.
`Pap. Thyroid Cane.
`Glomus tumour
`Pheochromocytoma
`Leiomyosarcoma
`
`N
`
`10
`7
`1
`1
`1
`4
`2
`1
`2
`
`Grade II
`
`Grade III
`
`Grade IV
`
`1(P)
`1(R)
`
`6(2*, P, 2S, R) 3(2•, S)
`3(2*, P)
`3(P, S, R)
`1(S)
`
`1(R)
`l(R)
`
`1(R)
`
`4(1•, 2P, S)
`2(1*, S)
`
`2(P, S)
`1(1*)
`
`Total
`
`21(+9*) 8(+3*)
`
`6(+4*)
`
`7(+2*)
`
`86%
`Pos Effect (S+R)
`67%
`50%
`67%
`P= progression; S= stable; R= reduction in tumoursize; •= << 20 GBq
`and/or no FU.
`
`become more radiosensitive. Another hypothesis is
`that the tumor treated on day 6 had more time to re(cid:173)
`establish and subsequently becomes heavier.
`Heterogeneity of receptor-expression on the tumor
`will cause incomplete responses during radionuclide(cid:173)
`therapy with lliin because of the short particle range of
`its Auger- and conversion-electrons. '"Therefore, we also
`investigated [90Y-DOTAO,Tyr3]octreotide in this model.
`In experiment 4 rats receiving 93 MBq (2 J..Lg) [9oy_
`DOTAO,Tyr3]octreotide (C) on day 1 had significant
`lower tumor score and lower liver weight due to less
`tumor growth than the control groups (Table II).
`Experiments 1-4 were repeated with concordant results.
`Our results show radiotherapeutic effects of both
`
`lliin- and 90Y-labeled somatostatin-analogues in two
`different rat tumor models. The flank model rats had
`small, but established tumors at the start of the ther(cid:173)
`apy (product of the largest tumor dimensions ~1 cm2),
`whereas in the liver tumor model the therapy was
`started just a few days post inoculation of the tumor
`cell suspension. Both Illin- and 90Y-labeled peptides
`had potential to induce tumor response in these mod(cid:173)
`els. In the liver model experiments all rats were sac(cid:173)
`rificed 20-21 days after inoculation of tumor, therefore
`no judgement with regard to cure could be done in
`this model.
`Anderson et al.23 found up to 1-2 week tumor
`growth delay in rats bearing the CA20948 tumor in the
`flank after radionuclide therapy with 1480-2220
`MBq/kg (64Cu-TETAO]octreotide. Stolz et al.24 obtained
`similar findings after treatment of CA20948 tumor(cid:173)
`bearing rats with [90Y-DTPAO-benzyl-acetamido,
`Tyr3]octreotide. Using 370 MBq/kg of [90Y-DOTAO,
`Tyr3]octreotide, which is comparable to our dose of
`111 MBq/rat using this same compound, the latter
`group observed complete tumor reduction in 5 out of
`7 rats, using the same model. 25 The discrepancy with
`our transient tumor response findings after 1 or 2
`injections of [90Y-DOTAO,Tyr3]octreotide might be
`explained by differences in tumor size and in radio(cid:173)
`sensitivity of the tumors maintained in both groups,
`although the same tumor model was used.
`
`Clinical in vivo radionuclide therapy experiments
`using radiolabeled somatostatin-analogues
`Thirty end-stage patients have been treated with
`(111In-DTPAO]octreotide (Table III). Twenty-one recei-
`
`10.0
`
`N=21
`
`:t---
`
`21
`
`!
`
`12
`
`!
`
`3
`
`-1
`
`375
`
`300
`
`225
`
`150
`
`75
`
`::g
`fa
`!!..
`a
`0
`~
`
`Pre
`
`M±SEM
`>70
`
`0
`
`;.;.
`~ 7.5
`"'
`"o
`u
`::I
`~ 5.0
`~
`..0
`::c:
`
`2.51
`0
`
`>50
`>20
`>20
`>50
`Cumulative dose (GBq) of
`Cumulative dose (GBq) of
`B
`A
`lll In-DTPA-octreotide
`lllin-DTPA-octreotide
`Fig. 4.-Course of haemoglobulin and blood cells during peptide receptor radionuclide treatment with the indicated cumulative d0ses of [111In-
`DTPAO]octreotide. N is number of patients (M±SEM).
`
`7.5
`
`N=21
`
`21
`
`12
`
`1500
`
`3
`
`<1.)-
`
`;.;.
`" '0 5.0
`~~
`g:.s
`u
`0...
`~ .§
`::1
`<1.) 2.5
`z
`
`....l
`
`0
`
`''~
`
`~~~~.~~I ___________ r __________ •
`
`1000
`
`500
`
`~
`.§
`:r
`0
`t"l
`'<
`0
`0
`.g
`
`('JO
`
`- Neutrophils
`-•- Leuco's
`-+- Lympho's
`
`Pre
`
`M±SEM
`>70
`
`0
`
`362
`
`THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE
`
`December 1999
`
`Roxane Labs., Inc.
`Exhibit 1033
`Page 007
`
`

`
`THERAPY OF NEUROENDOCRINE TUMORS WITH RADIOLABELED SOMATOSTATIN-ANALOGUES
`
`DEJONG
`
`200
`
`•
`
`150
`
`100
`
`50
`
`::E
`::1.
`'cd
`~
`u
`
`•
`•
`••
`•
`•• ••
`•• •
`••
`•••
`••••
`
`...
`...
`...
`......
`...
`......
`... ............
`.........
`·!··
`
`•
`•
`•
`
`...
`... , ... I
`... ; ...
`...
`
`c
`]
`s
`0 100
`'cd
`~
`u
`
`...
`...
`...
`...
`... ...
`... •!
`*
`... ...
`...
`...
`...
`... ...
`... ...
`
`... ......
`......
`
`...
`... ...
`... ......
`
`••
`•
`
`21
`
`12
`
`3
`
`•
`••••
`• ••
`• •
`•
`•••••
`•
`•
`•
`
`N=21
`Pre
`Pre
`>20
`>70
`>50
`>20
`>70
`>50
`A
`B
`Cumulative dose (GBq) of 111In-DTPA-octreotide
`Cumulative dose (GBq) of 111In-DTPA-octreotide
`Fig. 5.--Course of serum creatinine and creatinine clearance during peptide receptor radionuclide treatment with the indicated cumulative
`doses of [1 11In-DTPAO]octreotide. N is number of patients.
`
`N=21
`
`21
`
`12
`
`3
`
`0
`
`0
`
`Surgery Strepto- Strepto-
`doxo
`5FU
`
`800
`
`111In-pentetreotide
`
`1200
`
`++ + +
`
`>. 700 + + + + +
`
`~ 600
`"0
`I:;
`.:=9 500
`-~ 400
`~ 300
`(.) .a 200
`0
`100
`0
`
`0
`
`j fm am j j a s o n d j f m a m j j a s o n d j f
`I
`1994
`I
`1995
`I
`
`Fig. 6.-The endocrine effect of [111Jn-DTPAO]octreotide (or 111Jn(cid:173)
`pentetreotide) treatment (180 mCi or 6.7 GBq doses) in a patient
`with a very progressive, metastasised neuroendocrine tumor of the
`pancreas producing among other hormones insulin leading to pro(cid:173)
`found hypoglycemia, which forced permanent hospitalization for iv
`~lucose administration. Between the 4th and 5th administration of
`[ 11 In-DTPA O]octreotide the patient could be discharged for a short peri(cid:173)
`od in which no special precautions had to be taken to maintain an
`euglycemia. Some tumors showed regression and others an increase
`in size. The effect of radionuclide therapy in this patient has been
`scored in Table III as "progression". The patient died in February
`1996.
`
`ved a total cumulative dose of at least 20 G Bq (111 In(cid:173)
`DTPA0]octreotide. Of the nine patients treated with a
`total dose lower than 20 GBq, 7 had to stop prema(cid:173)
`turely because of progressive disease despite the treat(cid:173)
`ment with [111In-DTPAO]octreotide and 2 had not yet
`concluded the first course of four administrations.
`The tumor scores in the 7 patients with very progres(cid:173)
`sive disease were 2 (n = 3), 3 (n = 2) or 4 (n = 2). High,
`
`..... Volume
`...... Glucagon
`D 111 In-octreotide
`-+- GammaGT
`
`500
`
`400 ~
`
`~
`~
`300 ~
`~
`200 ~
`g
`:::1
`5
`100 b£
`
`OJ)
`
`E' 1000
`5
`t: 800
`0
`OJ)
`~
`(.)
`:::1
`
`On 600
`
`,-.,
`(.)
`~
`0 400
`>
`cs
`s
`~ 200
`
`0
`
`0
`
`6 8 1012 2 4 6 8 1012 2 4 6 8 9 11 1 3 5 7 9 12
`I 90
`I
`91
`I
`92
`I
`93
`94
`Month-year
`Fig. 7.-Effects of the first radionuclide therapy with [111 In-DTPA O]octre(cid:173)
`otide in a patient with metastasised glucagonoma. Other treatment
`modalities, e.g., surgery, high dose of octreotide without and with
`interferon-a were ineffective prior to the start of radionuclide thera(cid:173)
`PY in August 1992. Tumor volume is total abdominal tumor load
`based on measurements of all lesions shown on CT. Note that the
`decline in tumor size starts at a time point at which the total abdom(cid:173)
`inal tumor load is about one kilogram. The total reduction in tumor
`size was 35 o/o. The treatment protocol in this case is different from the
`present one described in section 3. The experience with the first two
`patients (second patient with glomus tumors) formed the basis of our
`present treatment protocol.
`
`multiple radiotherapeutic doses of (111Jn-DTPAO]octre(cid:173)
`otide were given to 21 patients up to total cumulative
`doses of 22 to 30 GBq and 12 patients 50 to 60 GBq
`per patient. Three patients received maximum doses
`of 75 GBq. No major side effects were noticed in the
`
`Vol. 43- No.4
`
`THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE
`
`363
`
`Roxane Labs., Inc.
`Exhibit 1033
`Page 008
`
`

`
`DEJONG
`
`THERAPY OF NEURO ENDOCRINE TUMORS WITH RADI OLABELED SOMATOSTATIN-ANALOGUES
`
`± 300cc
`
`NEUROENDOCRINE TUMOR
`70 %
`
`35 %
`
`1%
`
`Prior
`4/94
`
`Start
`10/95
`
`After 7 X PRRT
`4/96
`
`After 11 X PRRT
`8/96
`
`After hemihepa(cid:173)
`tectomy 11196
`
`Fig. 8.-Effects of radionuclide therapy with [11 1In-DTPAO]octreotide in a patient with a hepatic metastasis of a neuroendocrine tumor treat(cid:173)
`ed according to the described protocol. The upper panel shows transversal CT scans, except on 11/ 96 (MRI), and the lower panel illustrates
`transversal slices of lllJn-DTPAO]octreotide SPECT images. The d ecline in tumor size (300 cc) was after 7 treatment courses 30% and after 11
`courses