Note: Descriptions are shown in the official language in which they were submitted.
W~ 93/1X797 PCT~/U~93/02772
Metho~ of intraoPerativelv detectina ~nd locatinp tumoural tissues.
S The invention relates to a method of intraoperatively detectin~ and locating tumoural
tissues in the body of a warm-blooded living being, tD a method of radioguid3d
sursery of said being, and to a radiopharmaceutical composition to be used for ths
latter rnethod. The invention ~urther relates to a labslled peptide compound to be
used in said composition and to a kit for preparing said composition.
The accurate staging of tumours, in particular malignant tumours, in general remains
-.; one of the most important clinical challenges. Often such tumours or their
me~astases are ex~rernely srnall ~<1 cm), and because o1 this small size they are not
readily det~ctable and distinguishable using conventional imaging techniqLIes. Even
the us~ of advanced imaging t~chniques, such as SPECT acquisition techniques, incornbination with tl~mour-selective imaging agents~ is frequentlv unable to show all
l~sions, b2cause of disturbing background activity that makes accuraee image
interpretation difficuît. Especially in the abdominal area it is often difficult ~o
distin~uish benign for mali9nant tissues usin~ conwntional irnagin~ methods. An
example of such a subgroup of tumours are gastro-enteropancreatic tumours that
produce hormones, whi~h result in sometimes life-threatening symptoms, e.g.
massive diarrhea. The obvious thetapy in these cases is to surgicalty remove these
tumours. However, their often small si~e makes imaging techniques no~ relia~le in
accurately locating the lesion, while the surgeon is simply not capable of findin~ the
tumours quickly and, mo!eover~ is in fact not sure that all lesions are operatively
removed.
A relatively new technigue provides surgical aid: a gamma detecting probe, e.g.
Neoprobe~, that can b~ used to detect sourc~s o~ 0amma radiation that are v0ry
small. After parenteral adrninistration of a radiolabelled substance, the surgeon can,
intraoperatively, USB this probe to find the lesions ih which uptake of ~his substanc~
has taken place. E.W. Martin and coworkers have investi~ated this new technique:e.g. Amer. J. Surgcty 156, 1988, 386-392; Antibody Immunocon. Radiopharm. 4,
1991, 339-358. These investigators have observed that antibodies or antibody
fr~gmentS, labelled with iodine-125, a low-energy gamm~ photon ernittor, are
promising substances to be used in th;s technique- They indicate that this technique
may successfullY target 80% of colorectal cancer and detect occult turnours in ~h~
abdomen in 20% of the surgical cases involving can~er of the colon. Although it is
generally reco~nized that this improvement in diagnosing enables the surgeon to
W~ 93/1~797 ~ ~ ~ PCr/US93/02772
.t 3
better resect tumour deposits, in particular those tumours and metastases which
cannot be seen or palpated, and so contributes to the chance of curing cancer
patients, the results of this technique are not yet satisfactory. The known
radioiabelled substances generally show an insufficiently selective tumour uptake
and, in particular, a not sufficiently fast blood clearance, so that the tumour to
background ratio is often inadequate for accurate detection.
It is the object of the invention to provide a method of inttaoperativsly detecting and
locating ~umoural tissues in the body of a warm-blooded liYing being by using a
radiolabelled substance showing an improved and more selective tumour uptake anda much faster blood clearance.
,~ ,
. This object can be achieved according to the present invention by a method as
mentioned ~bove, comprising (a) parenterally administering to said being a
- pharmaceutical composition comprising, in a quantity sufficient for detection by a
gamma detecting probe, a peptide compound labelled with a low-energy gamma
photon emit~ing radionuclide, said peptide compound being derived ~rom
a peptide selectsd from the following groups:
(i) peptidcs having ~ selective neurokinin 1 receptor affinity and having the
~eneral formula
R~~~A~)m~A2)n~A3)0-pro)p-A4~-As-phe-A6-A7-A8-NH-c~ H-F~2
O
~1)
wherein all of the symbols rn, n, o, p and q are 1,
or all but one of the symbols m, n, o, p and q are 1, and the remaining
symbol is 0;
R, is a hydrogen a~om or a C.,-(::4 alkyicarbonyl ~roup;
R2 is a carbamoyl group, a carboxy group, a C:~-C4 alkoxycarbonyl
~roup, a hydroxymethyl group or a C,-C~ alkoxymethyl group;
A, is Arg, Gly or 5-oxo-Pro (pGlu);
A2 is Pro or ~-AIa;
A3 is Lys or Asp;
A4 is Gln, Asn or 5-oxo-Pro;
A5 is Gln, Lys, Arg, N-acylated Arg or 5-oxo-Pro;
WO 93/18797 ~ PCI/US93/02772
or wherein As together with A3 forms a cystine moiety;
AQ jS Phe or Tyr;
A7 is Gly, Sar or Pro;
AB jS Leu or Pro; and
RB jS a straight or branched C2-C4 alkyl group, which group may be inter-
rupted by thio, suiphinyl or sulphonyl;
and their Tyr derivatives;
(ii) peptides having a selective somatostatin receptor affinity ~nd having the
general formula
, R1-B,-Cys-B2-~D)Trp-B3-B,,-Cys-NH-CH-R2 ~11)
R7
t5
:: wherein R, and. R2 have above meanings,
B, and B2 are e~ch independently Ph~, MePhe, EtPhe, Tyr, Trp and Nal,
E33 is L~s or MeLys,
B, is Thr or Val, and
R~ is a 1-hy~roxyethyl group or an indol-3-ylmethyl group;
and their Tyr detivatives:
and
iii) peptides selected from cytokines, growth factors and hormones, as well as their
derivatives and analogues;
~: ~
and then ~b~, after allowing the activ~ substance to be taken up in the ~umoutaltissues and a~ter blood clearanc~ of radioactivity, subjectin~ saicl being to a
radioimmunodetectlon techniqu~ in the relevant area of the body of said being, by
using a gamma detecting probe.
In ~he above ~escription of the invention the symbol Nal means a naphthylalanyl
:: ~ group, and Sar means a sarcosyl ~roup.
Sui~able examples of substituent R~ are (CH~)2S~0)5(:H3, wherein s is 0,1 or 2, and
CH2CH~CH3~
SL~itable examples of cytokines are tumour necrosis factor ~TNF~, in partic~llar TNF-
o~, interleukines (IL), in particular IL~ L-2, IL-4, IL-5 and IL-6, and interferons.
Wo 93/18797 PC~/US93fO?772
.. . . .
J 4
Suitable examples of growth factors are epiderrnal growth factor ~E5F), insulin-like
growth-factor (IGF~, in particular IGF-I ~somatedin C) and IGF-II, bombesin,
transformin~ growth factor (TGF), in particular TGF-~and TGF-J~, platelet-derived
growth factor, fibroblast grow~h factor and nerve growth factor.
Suitable examples of hormones are luteinizing hormone-releasing hormone (LttRH),gastrin, gasttin-releasitlg pep~ide, angiotensin, thyroid-stimulating hormone,
vasoactive intestinal polypeptide, prolactin, thyrotropin-releasing hormone, insulin,
adrenoeortico~ropic hormone ~ACTH), in particular ~-MSH ~melanocyte-stimulating
hormone) and J~-~methylsulfonyl)-L- o~-aminobutyryl-L- o~-9lutamyl-L-histidyl-L
phenylalanyl-D-lysyl-L-phenylalanine, cholecystokinin, corticotropin-releasing
hormona ~CRtl), ~rowth hormone-releasing hormone (GRH), arginine and Iysine
vasopressin, ox~ocin, glucagon, secretin, parathyroid hormone (PTH) and PTH
relat~d peptide.
By usin~ the above method of the invention, virtually all malignant turnours can be
detected and located and can be distinguished ~rom benign tiss~es, because ~hesetumours contain substantially large numbers of receptors for binding the above
peptide comp~und.
The above-defined peptides are composed of amino acids, of which at least one may
have the D-configuration. The peptidss may also comprise so-called pseudo peptide
bonds, viz. -CH2-NH bonds, in addition to the natural amide bonds, viz. -C0-NH-
bonds.
Labelled peptide compounds for external imaging aceording to conventional imaging
teehniques and consequently labelled with radioisotop~s suitable for ~his purpose,
such as Ga-6~, In-~ 11 and Tc-99m, are describ~d in literature. Peptide oompounds,
labelled in this manner and derived from peptides mentioned sub ~i) above, are the
subjeot of the non-prepublished European patent application no. ~1200955.2 in the
name of Applicants. Equaily labelled peptide compounds derived from peptides
mentioned sub (ii) and sub (iii) above are known ~rorn the published intsrnational
patent applicatiGns W0 90/06949 and W0 9t/01144, resp~c~ively.
3~ Suitable ~xamples of peptides sub (i) above, which can be used as indicated above,
are:
~1) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 lSubstanoe P),
W O 93/18797 ~ ~ P ~ IUS43/02772
(2) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met(02)-NH2,
(3~ H-13-Ala-Gln-Gln-Phe-Phe-Sar-Leu-Met(O~)-NH2,
(4) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2~
~51 H-Arg-Pro-Cys-Pro-Gln-Cys-Phe-Tyr-Pro-Leu-Met-NH2, and
Tyr~ deriva~ives thereof.
Suitable examples of peptides sub(ii) above, which can be used as indicated above,
are:
6) H-(D~Phe-C ys-Phe-(l))Trp-Lys-Thr-Cys-Thr-ol (Octreotide),
~7) H-(D)Phe-Cys-Thr~ )Trp-Lys~Val-Cys-Thr-NH~,
(8~ H-(D)Ph~-Cys-Tyr-(D)Trp-Lys-Val-Cys-Trp-NH2,
: ~9) H-(D)Trp-Cys-Phe ~D)Trp-Lys-Thr-Cys-Thr-NHa,
(10) H-(D)Phe-Cys-Phe-~D)Trp-Lys-Thr-Cys-Thr-NH;?,
(11) H-(D)Nal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-NH2,
'- i
(t2) H-(D)Nal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Nal-NH2,
i13) H-(D)Nal-~ys-Nal-~DjTtp-Lys-VaJ-Cys-Thr-NH2,
( 14~ H-(D3Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Nal-NH 2,
( 1 5~ H-~D~Phe-C ys-Tvr-~D)Trp-Lys-Thr-Cys-Thr-ol,
: 30 and Tyr derivatives thereof.
` Suitable examples of peptides sub (iii) above, which can be used as indicated
above, are: EGF, TGf-~, gastrin, bombesin and derivatives of these peptides.
Suitable low-energy gamm~ photon emitting radionuciides which can be used
as labels for the peptide compounds to be used in the method vf the present
invention should have a gannma energy of approx. 80 keV at most. Such
W0 ~3/18797 ~ , PC~r/US93J02772
radionuclides ar~ well tuned to the gamma detecting microprobe to be
manipulated by the surgeon and intended to register the emitted ~amma
radiation. Such a hand-held microprobe, e.g. Neoprobe 100d0, is at present
equipped with a rniniature cadmium telluride crystal detector. Such a detector
requires for optinnurn detection properties gamma energies in the range of
approx. 30-~0 KeV. Higher energies may cause excessive scattering so that
the accuracy of the detection is considerably decreased. After uptake of the
labelled peptide in ths tumoural tissues and after blood clearance of
radioactivity to avoid disturbing background activity, the surgeon can use his
~amma detecting probe during operation to be sure that he/she does not
overlook srnail-sized tumours. The weli-tuned label enables the surgeon to
accura~ely detec~ and locate such smalt tumours with the aid of ~he hand-held
microprobe in otder to guide the surgery treatment. Examptes of suitable
radionuclides fot labelling the above peptide compounds are 1-125, As-73, Sb-
119, Cs-131, Dy-159, W-181 and Hg-197.
The desir~d radioisotope should be firmly attached to the peptide molecule to
reducs l;he chance of detaching this label after adrninistratisn to ~he living
being. The peptide can be labelled with the desired ;sotope directly or
indirectly, i.e. via a so-called linker. Direct labelling 1113y be carried out, for
example, b~ introducing a halogen atom or radioactivs halogen atom, i.c.
iodine-125, in~o an activated aromatic group te.g. tyrosyl or irnidazolyl)
,present in the peptida, into the peptide molecule in a manner known per se,
if desired followed by exchange with 1-125. Tyrosine and histidine are suitable
amino aoids which, if present in ~h~ peptid~ molecule, allow an easy
substitution with ~radioactive) halogen. Often, however, the labelling procedureis performed via a suitable linker, bein~ capabie of reacting with an amino
group, pre~erably a terminal amino ~roup, of said peptide, and having a
~unctional group for binding said radioisotope. By using a suitable linker, the
desired isotope can ~eneratly better be introduced into the peptide rnolecule.
It is of advantage to attach the linker to ~ terminal amino group of the peptidemolecule, în order to affect the biological properties of this peptide as least as
possibl~.
Suitabla linkers ~Ot labelling the peptide with metal radionuclides are discussed
in detaii hereinafter.
WO g3/18797 ~ " PCr/US93/02772
A suitable linker for labelling the peptide with 1-125 is derived from tyrosine or
from Bolton-Hunter reagent, i.e. N-succinimidyl-3-(4-hydroxy-3-
halo ~ phenyl)propionate, wherein halo ~ means iodine- 1 25. Preferably,
however, the peptide is first reacted with tyrosine or with a "halo-deprived"
Bolton-Hunter reagent, viz. N-succinimidyl-3-~4-hydroxyphenyl)propionate,
after which the derivatised peptide, thus obtained, is substituted by the
desired haJogen radioisotope by an appropriate reaction. Both by using the
latter, more conveni2nt r~action route and by employing the former method,
the peptide can be labelled with the desired radioactive halogen isotope
without affecting its biolo~ical properties.
- The above radioiodinating reaction is preferably performed by reacting th2
peptide in question with a solution o~ an alkali metal radionuclide from 1-125
iodide, under the influence of a halide-oxidizing agent, such as chlotarnine T
or iodo~en. Alternatively, the above substi~ution reaction can be carried out
with a non-radioactive halogenide, after which halo-exchange with radioactive
halogen is performed, eØ as described in European patent 165630.
In geneta3i the tumour~ and metastases which can be detected and located by
using the method of the present invention, are small soft tissue tumours which
are firmly a~ached to the surrounding benign tissues. This makes the surgical
remova3 of such tumours after their detection often difficult. It is another
o~lect of the present invention to facilitate the therapeutic treatment of such
tumoural tissues and consequently to improve ~he radioguided surgery.
"~
1~ is a particular merit of the present invention to combine detection and
improved therapy. Consequently, the present invention also and in particular
relates to a method of radioguided surgery of a warrn-blooded living being,
which method, in addition to the method as discussed hereinbafore and
intended to detect and locate tumoural tissues, comprises (i~ parenterally
administering to said bein~ a pharmaceutical composition comprising, in a
quantity suff;cient ~or at least partial necrosis of turnoural tissues, a pep~ide
compound derived {rom a peptide as defined herein~efore and labelled with an
isotope with suffici~ntly high specific activity and ernitting Gorpuscular
radiation, preferably selected from the group consisting of Tadionuclides as
reviewed by Schubigsr et al. (in 6th Int. Symp. Rad;opharm. Chem., Boston
1986, paper no. t49) or by Volkert et al. (in ~). Nuc3. Med., 1991, Vol. 32 (1),
WO 93/187g7 PCI~ S93~02772
174-185), such as the radionuclides selected from the group consisting of P-
32, S-35, As-77, Y-90, Rb-105, Ag-111, Sn-121, Te-127, Re-186, Re-188,
Au-198, Au-199 and radionuclides of the lanthanide group emitting
corpuscular radiation; and then (ii), after allowing the active substance to be
taken up in the tumoural tissues and to cause an at least partial necrosis of
said tissues, subjecting said being to a surgical treatmen~.
Suitable examples of the last-mentioned lanthanide radionuclides are Pr-142,
Pr-143, Pm-î49, Pm-151, Srn-153, Gd-159, Tb-161, Dy-165, Ho-166, Er-
169, Tm-172, Yb-169, Yb-175 and Lu-177.
Because the above isotopes have sufficient corpuscular emissions to be useful
. for therapeutic purposes, an injected dose has a cell killing effect due to the
uptake in the tumoural tissues in question, leading to an at least partial
necrosis of the tumour cells. This enables the surgeon to more easily remove
: these tumours by excision during surgery. The overall result can be a
treatmsnt schedule, wherein an optimum use is made of this combination ~f
detection and therapy. After uptake of the "detecting" peptide compound i
the tumoural tissues, the gamma emissions permit the aecurate detection of
thess tissues with a microprobe. These tissues, however, have already been
nat~ackedn by the corpuscular radiation caused by uptake of the peptide
compound labelled with one of the above corpuscular radiation emitting
isotopes. The surgical treatment of these malignant tissues, i.~. the excision
of the already at least partially necrotic tissues, is therefore highly facilitated.
It is of advantage to use for both detection and ~or therapy th~ same
pharmaceuticai composition, comprising a peptide compound labelled with a
: low-ener~y gamma photon emitting radionuclide and a corpuscular radiation
emitting isotope as defined hereinbefore. In this manner, viz. by using exactly
the same peptide as a starting material, the surgeon can bs sure that the
specific affinity to the tumoural tissuss to be removed is exactly the same for
the diagnostic a~ent as for ~he therapeutic agent.
: : :
Surprisingly it has been found, that certa;n lanthanide radionuclides are very
suitabl~ to perform both functions at the same time, viz. have a suitable
gam~na energy f or detection and havo a corpuscular radiation emitting
effectiveness sufficient for tumour cell necrosis. By combining these both
WO 93/l B797 PCl /US93/02772
functions in one and the same radionuclids, it is guaranteed that the uptake in
ths tumour oells is optimal for both intended effects. In this connection
terbium-161 (Tb-161~ is pre-erninently suitable. This ianthanide radionuclide
combines an optimum gamma energy for microprobe detection, viz. in the
range of 25-74 keV, with a beta emission suitable for therapeutic treatment
of tumour cells, viz. in the range of 250-590 keV. In addition, the half-life ofterbium- 1 61 is extremely appropriate for the intended purpose, viz. 6.91 days.This means that sufficiently long after injection the blood clearance of
r2dioactivi~y is sufficiently complete to permit accurate detsction of the
tumoural tissu~s, while at the same time necrosis of the malignant tissue cells
has advanced sufficiently to allow easy excision of the detec~ed tumours.
- Finally, this preferred lanthanide radionuclide is readily accessible and can be
produced carrier-free by irradiation of highly enriched gadolinium Gd-160 in a
nuclear reactor.
Preferably the labeiled peptide cornpound to be used in the method of the
invention, for de~ecting purposes or both for detection and for therapy, i~
provided, directly or through a spacing group, with 3 chelatin~ gtoup. This
chelating group is attached by an amide bond to an amino group of said
p0ptide and is derived from ethylene diamine tetra-acetic acid ~FDTA), di-
ethylene triamine penta-acetic acid (DTPA~, ethyleneglycol-0,0'-bis(2-
aminoe~hyl)-N,N,N',N'-tetra-acetlc acid (EGTA), N,N-bislhydroxybenzyll-
sthyienediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic
acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra-ace~ic acid
~D~A), 1,4,8,11~tetra-azacyclotetradecane-N,N',N",N"'-tetra-acetic acid
~TETA), 1 ,2-di~minocyclohexane t~tra-acetic acid ~DCTA), substituted DTPA,
substituted EDTA, or ~rom a compound of the general formula
,_ ~R-- --
`~--S--Y ~
wherein R is a b~anched or non-branched, optionally substituted hydrocarbyl
radioal, which rnay be interrupted by one or more hetero-atoms
selected from N, 0 and S andlor by ons or more NH groups, and
WO 93/18797 P~lUS93/02772
1 0
Y is a group which is capable of reacting with an amino group of the
peptide and which ;s preferably seiected from the group consis~ing
of carbonyl, carbimidoyl, N-~C,-C~)alkylcarbimidoyl, N-hydroxycarbi-
n~idoyl and N-(Cl-C6)-alkoxycarbimidoyl;
and
wherein said optionally present spacing group has the general formula
~NH--R5 eo-- or ~H2-e3NH-X--
~lV) (V)
lQ
- wherein Rs is a C~ o alkylene group, a C,-C,0 alkylidene ~roup or a C2-C,O
, alkenylene group, and X is a thiocarbonyl group or a methylcarbonyigroup.
Examples of sui~able chela~ors of the general formula 111 are unsubstituted or
t S . subs~i~uted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular 2-
imino-4-rnerca ptomethylthiolane .
It has be~n observed, that the 2bove preferred peptide compound, provided
~; ~ with a chelating group, shows a very fast clearance. This is of great
; ~ ~ 20 advantage, because the use of this preferred peptide compound after labelling
allows surgery a very short time after administration, if desired ftom medical
considerations.
~: The invention further relates to a radiopharmaceutical composition to be used
~or the me~hod of radioguided sur~ery as defined abov~, which composition
comprises in addition to a pharmaceutically acceptable carrier and, if desired,
: at least one pharnnaceutically acceptable adjuvant, as îhe activ~ substance a
peptide compound labelled with a iow-energy gamma photon emitting
radionuclids and a corpuscuiar radiation emitting isotope as defined
hereinbe~ore. If desired, th0 composition can b~ brought into a form more
suitable for parenteral administra~ion, e.~. by adding a pharmaceutically
acceptable liquid catrier material. For parenteral administration the solution
should of co~rse be in a sterile condition.
The above oomposi~ion preferably comprises as the active substance a pep~ide
compound provided with a chelating ~roup as defined above, said chela~ing
group chelating a metal radionuclide. The preferred radionuciides of the
WO 93/18797 ,, . ! A. j ~; ) PCI'/US93/02772
lanthanide group have excellent characteristics for being chelated with the
above aminoacetic acid based chelating agents, thus assuring a good and
stable binding to the chelating group of the peptide compound. In this
chelating or complex-forming reaction, the radioisotope is presented to the
chelatin~ gro~.p comprising peptide compound in the form of a salt o~ a
suitable acid, e.g. a rnineral acid or acetic acid. The complex-formin~ reactioncan ~enerally be carried out in a simple manner and under conditions which are
not detrimental to the peptide.
The invention further relates to a labelled pep~ide compound to be used as an
active ingredient in the above composition, said peptide compound having a
-. selective affinity to endocrinically active tumours and being labelled with a
low-energy gamma photon emi~ting radionuclide and a corpuscular radiation
emitting iso~ope, as defined hereinbefore, preferably with a suitable lanthanideradionuclid~ having the above-defined radiation characteristics.
The invention finally relates to a so-called oold kit ~or preparing a
radiopharmaceutical composition, comprising ~i) a peptide provided with a
chelating group as defined hereinbefore, to which substance, if desired, an
inert pharrnaceutically acceptable carrier and/or formulating agentls) and/or
adjuvant(s) is/are added, (ii) a solution of a salt of a lanthanide radionuclidehaving ~he above-defined radiation characteristics, and ~iii) instructions for us~
with a prescription for reacting the in~redients present in the kit.
~uch a kit a5 described above can be delivered to the user. The user can easily
perform the labelling of the chelated peptide with the ianthanide radionuelide
by himself or herself in the clinical hospital or laboratory. The labelling
procedure is simple and does not require complicated manipulations, so that
the ussr is able to prepare the labelled composition ~rom the kit ingredients byusin~ the facilities that are at his or her disposal. The radionuclide is the only
ingredient in the kit having a restricted shelf life. Therefore, if desired, thelan~hanide radionuclide may be delivered separately and substituted af~er its
expiration date.
Ths invention will now be described in greater detail with reference to the
following specific Examples.
WO93/18797 PCT/US93/Q~772
t) I .~` 12
~X~P~ 1
.
A. Pre~aratlon of DTP~-Octreo~ida kit
The DTPA-Octreotide kit formulated on basis of sodium
acetate buffer with the final composition
3.89 mg sodium acetate
~- . . 0.029 mg acetic acid
. 10 ~g DTPA-Octreotide
per vial is prepared as follows:
~ 15: First the following solutions are made:
: - acetic acid solution 0.06M, by diluting 35.9 mg
glacial acetic acid to 100.0 ml with water;
2;0 ~ - sodium acetate solution 0.286M, by dissolving 3.B9 g
of sodium acetate 3H20 in 100 ml water.
To formulate the kit, 0.5 mg of DTPA-Octreotide is
:: ~
d:issolved in 4 ml of acetic acid solution, and 5 ml of
25~ sodium acetate solutîon are added.
:
To this mixture are added 16 ml water to make 25 ml of
~ final solution, which is subsequently filtered through a
:~ : 0.22~ bacterial filter. The filtrate is then dispensed in
0.5 ml portions per vial and ~ial5 are lyophiliæed. The
final freeze dried product is stored at 4C.
WO93/18797 ~ PC~/US93/0277
13
In a similar way, startin~ from 2.5 mg DTPA-Octreotide was
also prepared and a kit containing 50~g DTPA-Octreotide per
vlal .
S B. P~3L__n of Tb-161 solution
2 mg of enriched (98.1%) 160-Gd2O3 is irradiated for 48
hours in a nuclear reactor with thPrmal neutron flux 2 x
10l4 n/cm2 sec.
After ca 30 hours of cooling the sample is dissolved in 2
portions of 1 ml warm (70C) lON HCI directly in the
irradiation quartz ampo~le, the solutions are transferred
.to a 20 ml quartz beaker and combined with 3 portions of 1
ml washing water. The solution is 2 times evaporated with
lON ~Cl till dry and the dry rest is taken up in few ml of
O.O~N HCl and diluted ~o 10 ml with 0.02N HCl, Irradiation
: yield is ca 11.5 mCi Tb-161.
For the separation of Tb-1610 5 ml of the Gd/Tb s~ock
solu~ion in 0.02N HCl is evaporated to dry and taken up in
~, 200~1 of 0.02N HCl. Thi~ solution is loaded on a 0.8 x 12
; : : cm column of 5CX BioRad~ 50 W-X8, 200-40C mesh, in NH4t
fo~m. As an eluent is used a 0.2 M solution of a-hydroxy-
isobutyric acid, adjusted to pH 4.1 with ammonia.
Frac~ions ~f 1 ml of eluate are collected for radiodnuclide
identification. ~ombined fractions containing Tb-161 are
: made of 0.5N in HCl and run over a second small columQ of
BioRed~ 50W-X8 in H~ form. The loaded column is then
washed wi~h 0.5 and 1.5~ HCl, followed with water to re~ove
the exce~s of a-~ydroxy-isobutric acid. Tb 161 is finally
stripped from the column with 6N H~I. The strip solution
is again evaporated to dryness. The residue is taken up
into O.OOlN HCI (4 ml) and u~ed for analy~is and labelling
experiments.
:~ 35
W~3/t8797 - PCT/US93/02772
.~, ; 14
Analysis:
Radionuclide purity determined by ~ spectrometry ~ND 66 ~
spectrometer Ga/Li detector): substantiallyl 100%; no other
radionuclide detected. Radiochemical purity: Thin layer
chromatography -ITL SG ~Gelman) plates, ascending, solvent,
lM sodium acetate pH 5. Result: single peak on front,
98.9% Tb-161 activity.
C. Labellinq of_DTPA-Octreotide kit with Tb-161.
.
~; Several kiks of DTPA-Octreotide, prepared according to
Example 1 containing 10 or 50 ~g DTPA-Octreotide, are
labelled by addition of 0.5 ml of Tb-161 solution obtained
under B. The mixture is incuba~ed for 30 min. at room
temperature.
~nalysis:
ITLC as described above,
~ Tb-161-DTPA-Octreotide Rf ca 0.5-0.6
:: Free Tb-161 Rf ca 0.9-1.0
Hydrolysed Tb-161 Rf ca 0.0-0.1
~; 25 : HPLC: Column:: ~Bondapak~C 18 10~m, 3.9 x 300 m~
: Eluent: 0.07M acetate buffer pH 5.5 ~a),
~,,
~:: 100% MeOH ~b~, a and b mixed in ratio 6:4 vtv.
~:
Gradient: 40-80% b in 20 min.
Operation: Flow rate 1 ml/min., temperature 35C.
Detection: Dual, NaI crystal, W detec~or~at
280nm.
Results of labelling experiment: (at time interYal between
addition of Tb-161 activlty and analysis) LY = Labelling
yield.
WO93/1~797 ~ PCT/US93/02772
Time (h) LY 10 ~g LY 50
0.5 33% >92%
3 46% >92%
24 78.4% >93%
challenge exper}ment with serum (bovine), added at 24 h
.
4~ 76.4~ >95%
,, '
* Free Tb-161 was not detectable in any kit containing 50
~g DTPA-Octreotide.
Radiochemical purity - 50 ~g at 3 h - HPLC 96.2
'
~20 HPLC identification positivet because W spectrum and
~ : activity peaks of Tb-161 are found identical with those for
; In~ labelled DTPA-Oc~reotide used as control.
T~ obtain an injectab~e preparation with radiochemical
2:5 ~ puri~y > 98%,:the labelle~ soluti~n is purified over a Sep-PakRCl~ cartridge, which after loading is ~a~hed with water
~(5~ml) and eluted~with methanol (5 ml), the latter fraction
containing T~-161 Octreotide. Evaporation and dissolution
of the residue in physiologic saline solution give after
; :30 sterilization ~membrane filtration) the desired injectable
preparation.
:: :
~ ~XAMP~
. .
WO 93fl8797 PCT/US93/02772
1 6
Labellinq of DTPA-Qctreotide kit with Yb-175 and its use ir.
combination with detectinq a~ent DTPA-125-I-Tyr3-Octreotide
Labellinq of DTPA-Octreotide kit with Yb-175.
Ca 1 mg of enriched (97.8%) 174-Yb2O2 is irradiated for 48
hours in a nuclear reactor with thermal neutron flux 2 x
10l4 / m2 C
n c .~e .
After 30 hours cooling time the sample is dissol~ed
directly in the irradiation quartz ampoule in 2 X 1-ml
.- portions of warm (70C) concentrated HC1.
The obtained solution is withdrawn ~nd trasferred to a
small ~uartz beaker, the ampoule is washed with 3 x 1-ml
portions of water and the washings are combined with the
active solution. The solution of Yb-175 chlori~e is twice
evaporated to dryness with concentrated HCl and the res't is
taken up into 2 x 5-ml of 0.02N HCl, transferred to a 20 ml
volumetric fl sk and dilu~ed to the desired volume with
0.02N ~C'.
A 1.O ml aliquot of this solution is diluted t~ 50.0 ml
with 0.02N HCl to`obtain a stock solution of Yb-175
:~ 25 chloride, used for the labelling experiments. This
;~ solution (1 ml) has a specific activity of A00 ~Ci/~g Yb-
175, a radionuclide purity of > 99% and a radiochemical
purity of > 99.9%; both values are determined by the
- methods described in Example 1.
Several kits containing 10 ~g 9f DTPA-Octreotide prepared
a cording to Example 1 are labelled by addition of 1 ml of
the Yb-175 stock solution. The mixture is let to incubate
30 min. at room temperature. Samples for analysis are
taken at time intervals indicated by the results. Used
w~93/l~7s7 `~ PCT/VS93/02772
17
analytical methods are described in Example 1.
Results:
Radiochemical purity Yb-175:ITLC, lM Na-acetate Rf 0.9-1~0
99.5% :
Yb-17~ Octreotide: LY at 30 min. ITLC Rf 0.5-0.6 98.1%
SepPak~ 98.8%
: LY at 75 min. HPLC 90.7%
Id~ntity of Yb-175 labelled Octreotide is confirmed as
described abo~e for the Tb-161 labelled peptide compound.
Cha}lenge experiment with added (at 75 min.) serum
(bovine~: :
Yb-175 Octreotide: LY at 3 h. ITLC Rf 0.5-06 91.2%
::~ at 24 h. ITLC Rf 0.5-06 91.7
B. Preparation of DTPA-l-lZ5=~L~ eot~ide.
~:
~ DTPA-Tyr3-Octreotide of the fo~mula
:
DTPA- (D) Phe-~s-l~rr - (D) Trp-Lys-Thr-Cys-Throl
is prepared from Tyr3-Octreotide in a corresponding manner
as described in Int. Pat. Appln. WO 90/06949, Example 1,
and further iodinated with 125I sodium iodide, dissolved in
phosphate buffer in the presence of chloramine T. The
W093/~797 PCT/US93/0~772
~ i ,l 3 18
molar ratio of DTPA-Tyr3-Octreotide; chloramine T: 125-I is
1:4,6:0.6 The reaction is terminated with 10% BSA
solution. The labelled product of the abo~e formula
wherein Tyr = 125-I-Tyr, is p~rified by HPLC.
C. Combined use for detection and therapy.
To combine the therapeutical effect with the radioguided
surgery are used both preparations; Yb-175-Octreotide for
the desired therapeutic e~fect and DTPA-125-I-Tyr3-
Octreotide as the ~detecting~ agent.
Depending on the conditions, they can be used separately,
in this case by administering Yb-17S-Octreotide first to
cause partial or deep tumour necrosis, followed by
: administration of DTPA-125-I-Tyr3-Octreotide to guide the
tumours removal, or they can be administered simultaneously
as a mixture in an appropriate ratio. Such a mixture is
obtained by mixin~ both agents in the proper ratio. In
this case the difference in ~h of Yb-175 and I-125, viz.
: :~ 4.2 and 60.2 days respectiYely, gives sufficient time for
~: : therapeutic effect while at the moment of surgery the
: background radiation, originating from Yb-175 (having also
very low ~ abundance), is already sufficiently low as not
to diminish the sensitivity of the microprobe.
;
:
EXAMPLE III
Labellinq of DTPA-Octreotide kit with Ho-166 and its use in
ombination with Qctreotide labelled with Tb~161.
WO93/18797 ~ PCT/~S93/02772
19
A. Labellinq of DTPA-Octreotide kit with Ho 16~
Ca 1 mg of natural ~monoisotopic) 165-Ho2O3 is irradiated
for 48 hours in nuclear reactor with a thermal neutron flux
2 x 10l4 n/cm2.sec.
After 30 hours cooling time the sample is treated in
exactly the same w2y as described for 175-Yb in Example
II.A.
, 10
Ob~ained Ho-166 stock solution (1 ml) has a specific
activity 525 ~Ci/~ Ho-166, a radionuclide purity > 99.9~
and a radiochemica~ purity > 99.9%, both values determined
by the methods described in Example I.
Several kits, containing 10~g of DTPA-Octreotide prepared
according to Example I., are labelled by addition of 0.5 or
1 ml of Ho-166 stock solution. The mixture is let to
: incubate 30 min. at roo~ temperature. Samples for analysis
at time intervals indicated by the results. Used
analytical methods are described in Example I.
~:
.
Results for labelling with 0.5 ml Ho-166 stock solution:
Time (h) LY ~%) Free Ho-166 (%)
: 30
0.5 99.3% < 1
: 20 99.7 ~ 1
48 9~-100 ~ 1
72 98.9 ~.1
~093/1~7~7 PCT/US93/02772
~ 20
Radiochemical purity at 72 h - HPLC 99-100%i, identity
confirmed.
Results for labelling with 1.0 ml of Ho-166 stock solution:
Time (h) LY (~) Free Ho-166 ~%~
1 94.3 5.7
~ 92.6 7.
challenge tast with addition of serum lbovine) a~ 8 h.
,,
32 (S24) 89.0 11
56 (S48~ 88.7 11.3
Radiochemical purity at 56 h- HPLC:
: Labelled Ho-166-Oc~reotide 91.1%
2~0 Free Ho-166 B.9%
:
.
:: :
C. Combined use for_detection_and thera~
Similarly as described in Example II. is used a combination
o~ both preparations, Ho-166-Octreotide and Tb-161-
Oc~reo~ide, particularly when larger tumours are suspected.
Since the maximum range of beta particles of Ho~166 in
tissue is about 0.85 cm (vs. 0.15 cm for Yb-175) and the
dep~sited energy is for Ho-166 ~s.ràd/~lci.h - 1,42) five
3S times higher than or Yb-175 (~.rad~Ci.h - 0,27~, then the
WO93/18797 ,~ PCT/US93/02772
21
process of tumour necrotization proceeds more rapidly. At
the same time the favourable ratio of half-lives (Ho-166
26.9 h, Tb-~61 d) guarantees that after one T~ of Tb-161 no
more than 1.3~ of the originally bound Ho-166 activity
remains at the site of the tumour so that the sensitivity
of the microprobe cannot be influenced, particularly not
since the range of gamma rays emitted by Ho-166 (48-80 keV~
is comparable to that of Tb-161.
.
: :~ : :
.
:::
