Title of Invention

TUMOR TARGETING AGENTS AND USES THEREOF.

Abstract This invention relates to novel tumor targeting motifs, units and agents, as well as tumor targeting peptides and analogues thereof. The targeting agents typically comprise at least one targeting motif, Aa-Bb-Cc, and at least one effector unic. The invention further relates to specific tumor targeting peptides, pharmaceutical and diagnostic composisitons comprising such peptides. Disclosed are also methods for diagnosing or treating cancer.
Full Text Tumor targeting agents and uses thereof
FIELD OF THE INVENTION
The present invention relates to tumor targeting agents comprising
at least one targeting unit and at least one effector unit, as well as to tumor
targeting units and motits. Further, the present invention concerns pharmaceutical
and diagnostic compositions comprising such targeting agents or targeting
units, and the use of such targeting agents and targeting units as Pharmaceuticals
or as diagnostic tools. The invention further relates to the use of such
targeting agents and targeting units for the preparation of pharmaceutical or
diagnostic compositions and for the preparation of reagents to be used in diagnosis
or research. Furthermore, the invention relates to kits for diagnosing or
treating cancer and metastases. Still further, the invention relates to methods
of removing, selecting, sorting and enriching cells, and to materials and kits for
use in such methods.
BACKGROUND OF THE INVENTION
Malignant tumors are one of the greatest health problems of man as
well as animals, being one of the most common causes of death, also among
young individuals. Available methods of treatment of cancer are quite limited,
in spite of intensive research efforts during several decades. Although curative
treatment (usually surgery in combination with chemothreapy and/or radiotherapy)
is sometimes possible, malignant tumors (cancer) still are one of the most
feared diseases of mankind, requiring a huge number of lives every year. In
fact, curative treatment is rarely accomplished if the disease is not diagnosed
early. In addition, certain tumor types can rarely, if ever, be treated curatively.
There are various reasons for this very undesirable situation but the
most important one is clearly the fact that nearly all (if not all) treatment sched-
ules (except surgery) lack sufficient selectivity. Chemotherapeutic agents
commonly used, such as alkylating agents, platinum compounds (e.g. cis-
platin), bleomycin-type agents, other alkaloids and other cytostatic agents in
general, do not act on the malignant cells of the tumors alone but are highly
toxic to other cells as well, being usually especially toxic to rapidly dividing cell
types, such as hematopoietic and epithelial cells. The same applies to radio
therapy.
In addition to the above mentioned complications, two further major
problems plague the non-surgical treatment of malignant solid tumors. First,
physiological barriers within tumors impede the delivery of therapeutics at effective
concentrations to all cancer cells. Second, acquired drug resistance resulting
from genetic and epigenetic mechanisms reduces the effectiveness of
available drugs.
The treatment of cancer patients with currently available, largely
non-selective, chemotherapeutic agents or radiotherapy results often also in
undesirable side effects. In order to improve the effect of chemotherapeutic
agents and to diminish the side effects it would be extremely important to identify
agents that are capable of targeting to specific organs or tissues or to tumor
tissues and to carry the desired cytotoxic or other drugs specifically to
these organs or tissues.
The same applies also to a specific field of cancer treatment,
namely neutron capture therapy, in which a non-radioactive nucleus (e.g. 10B,
157Gd or 6Li) is converted into a radioactive nucleus in vivo in the patient with
the aid of thermal (slow) neutrons from an external source. In this case, some
prior art agents are claimed to have some 2-3 fold selectivity for at least some
types of tumors, but the results obtained have been mainly disappointing and
negative. Specific targeting agents would offer remarkable advantages also in
this field.
Also in the diagnosis of cancer and of metastases, including the follow-up
of patients and the study of the effects of treatment on tumors and metastases,
more reliable, more sensitive and more selective methods and
agents would be a great advantage. This is true for all methods currently in
use, such as nuclear magnetic resonance imaging (NMR, MRI), X-ray meth-
ods, histological staining methods (for light microscopy and electron microscopy
and related methods, and in the future possibly also NMR, infrared, electron
spin resonance and related methods) and in general any imaging as well
as laboratory methods (histology, cytology, cell sorting, hematological studies,
FACS and so on) known by specialists in the field. Here, agents capable of
targeting an entity for detection (a spin label, a radioactive substance, a paramagnetic
contrast agent for NMR or a contrast agent for X-ray imaging or tomography,
a boron atom for neutron capture and so on) specifically or selectively
to tumor tissues, metastases or tumor cells and/or to tumor endothelium
would be a great advantage.
Solid tumor growth is angiogenesis-dependent, and a tumor must
continuously stimulate the growth of new microcapillaries for continued growth.

Tumor blood vessels are structurally and functionally different from their normal
resting counterparts. In particular, endothelial cells lining new blood vessels
are abnormal in shape, they grow on top of each other and project into the
lumen of the vessels. This neovascuiar heterogeneity depends on the tumor
type and on the host organ in which the tumor is growing. Therefore vascular
permeability and angiogenesisis are unique in every different organ and in tu-
mor tissue derived from the organ.
There are numerous publications disclosing peptides homing to dif-
ferent cell and tissue types. Some of these are claimed to be useful as cancer
targeting peptides. Among the earliest identified homing peptides described
are the integrin and NGR-receptor targeting peptides described by Ruoslahti et
al., in e.g., US Patent No 6,180,084. These peptides home to angiogenic vas-
culature and bind to the NGR-receptor.
When tumors switch to the angiogenic phenotype and recruit new
blood vessels, endothelial cells in these vessels express proteins on the lu-
minal surface that are not produced by normal quiescent vascular endothe-
lium. One such protein is avp3 integrin. US Patent publication, US 6,177,542,
discloses a peptide that can bind specifically to av|33 integrin. The tumor ves-
sel specific targets described are adhesion molecules that mediate binding of
endothelial cells to the vascular basement membrane. This peptide is a nine-
residue cyclic peptide containing an ArgGlyAsp (RGD) sequence. Pasqualini et
al., (1997) showed that when injected intravenously the peptide was able to
home to blood vessels of murine and human tumors in mice 40-80 fold more
efficiently than to those of control organs. It was suggested that RGD peptides
may be suitable tools in tumor targeting for diagnostic and therapeutic pur-
poses. However, integrin-binding peptides may interfere with cell attachment in
general, and are thus not suitable for clinical applications for selective tumor
targeting.
International Patent Publication WO 00/67771 provides endostatin
peptides comprising the amino acid sequence RLQD, RAD, DGK/R. Other ex-
amples of peptides that home to angiogenic vasculature are described in US
Patent Nos 5.817,750 and 5,955,572. These peptides recognize RGD.
US Patent 5.628.979 describes oligopeptides for in vivo tumor imag-
ing and therapy. The oligopeptides contain 4 to 50 amino acids, which contain
as a characteristic triplet the amino acid sequence Leu-Asp-Val (LDV). This
triplet is reported to provide the oligopeptide with in vivo binding affinity for
LDV binding sites on tumors and other tissues.
International Patent publication WO 99/47550 describes cyclic pep-
tides, containing an HWGF motif, that are specific inhibitors of MMP-2 and
MMP-9. They have also found that the cyclic decapeptide CTTHWGFTLC spe-
cifically inhibits the activities of these enzymes, suppresses migration of both
tumor cells and endothelial cells in vitro, homes to tumor vasculature in vivo,
and prevents the growth and invasion of tumors in mice. However, peptides
that act as inhibitors of MMPs show background binding to non-tumor tissues.
The fact that MMPs are expressed also in normal tissue throughout the body
also makes the administration of such peptides to humans or animals hazardous
and even fatal, since the activity of these enzymes is required for normal
tissue functions (Hidalgo and Eckhardt, 2001).
US Patent publication US 2002/0102265A1 describes a peptide,
TSPLNIHNGQKL, that targets squamous cell cancer cell lines, and becomes
internalized into cells in vitro. This peptide also targets experimental squamous
carcinomas in nude mice.
US Patent Nos. 5,622,699 and 6,068,829 disclose a family of peptides
comprising an SRL motif, which selectively home to brain.
International Patent publication WO 02/20769 discloses methods for
identifying tissue specific peptides by phage display and biopanning. Some of
the identified peptides are suggested to be tumor specific.
Although there are known homing peptides that bind to tumor vasculature,
there are still very scarce reports on targeting agents that actually
target tumor cells and tissues in vivo. Most of the previously described targeting
peptides are vasculature specific. Thus, there is still an established need
for new agents that target selectively to tumor tissue, tumor vasculature, or
both.
For therapeutic applications, targeting peptides have been conju-
gated to doxorubicin in an uncontrolled fashion, obviously resulting in mixtures
of products or at least in an undefined structure and possibly also resulting in
unefficient action and especially in difficulties in the identification, purification,
quality control and quantitative analysis of the agent, even the amount of
doxorubicin per peptide molecule remaining unknown (e.g. Arap et al., 1998).
The unspecific conjugation process might also impair the targeting functions of
the peptide.
Another very serious disadvantage of the prior art is that most of the
described targeting peptides appear to target to the tumor endothelium only
and not to the tumor mass itself. For example, the targeting peptide used by
Nicklin et al. (2000) directed adenovirus DNA transfection to resting endothelial
cells in vitro, under conditions that hardly could be applied in vivo.
The targeting units according to the present invention offer an advantage
over the prior art in that they seem to target to both the tumor endothelium
and the tumor cell mass. This fact provides the possibility to target and
destroy tumor endothellum suporting tumor growth as well as the tumor mass
itself. A major advantage of this approach comes from the fact that the endothelium
is a genetically stable tissue that will not acquire drug resistance but
will be irreversibly eliminated.
It is not known whether the prior art targeting peptides are universal
in the sense of being capable to target to any malignant tumor type. Thus, their
use as targeting therapeutic agents to a certain specified tumor may be com-
pletely useless, giving no therapeutic advantage or effect over the free thera-
peutic agent itself. An even more serious drawback is that the use of such tar-
geting agents in diagnostic procedures may not reveal all existing tumors and
the malignant process may remain unrecognized.
The present invention offers a significant improvement in view of the
prior art, since the targeting agents here described were found to target to all
of the various tumor types tested. Remarkably, they target, for example, sar-
comas, such as Kaposi"s sarcoma, ornithine decarboxylase (ODC) overex-
pressing, highly angiogenic tumors, carcinomas, and to human primary and
metastatic melanomas
Brief Description of the Invention
It is an object of the present invention to provide novel tumor and
angiogenic tissue targeting agents that comprise at least one targeting unit
and, optionally, at least one effector unit. In particular, the invention provides
targeting units comprising at least one motif that is capable of targeting both
tumor endothelium and tumor cell mass. Such targeting units, optionally cou-
pled to at least one effector unit, are therapeutically and diagnostically useful,
especially in the treatment and diagnosis of cancer, including metastases. Fur-
thermore the targeting agents according to the present invention are useful for
cell removal, selection, sorting and enrichment.
It is a second object of this invention to provide pharmaceutical and
diagnostic compositions comprising at least one targeting agent or at least one
targeting unit comprising at least one motif capable of specifically targeting tumors,
tumor cells and tumor endothelium.
Further, it is a third object of the invention to provide novel diagnostic and therapeutic
methods and kits for the treatment and/or diagnosis of cancer.
The present invention is based on the finding that a group of pep-
tides having specific amino acid sequences or motifs are capable of selectively
targeting tumors in vivo and tumor cells in vitro. Thus, the peptides of this in-
vention, when administered to a human or animal subject, are capable of se-
lectively binding to tumors but not to normal tissue in the body.
The present invention is also directed to the use of the targeting
agents and analogues thereof for the manufacture of a pharmaceutical or
diagnostic composition for treating or diagnosing cancer.
The targeting units of this invention may be used as such or coupled
to at least one effector unit. Such substances can destroy the tumors or hinder
their growth. The targeting units and targeting agents of this invention can tar-
get also metastases and therefore they may be used to destroy or hinder the
growth of metastases. As early diagnosis of metastases is very important for
successful treatment of cancer, an important use of the targeting units and tar-
geting agents of this invention is in early diagnosis of tumor metastases.
The present invention further encompasses salts, derivatives and
analogues of the targeting units and targeting agents, as described herein, as
well as uses thereof.
It is a further object of the present invention to provide diagnostic
and pharniaceutical composition-comprising targeting agents according to the
present invention, as well as therapeutic and diagnostic methods for the treatment
and diagnosis of cancer, utilizing targeting agents according to the present
invention. Also provided are kits for use in such methods or for research
purposes, as well as in cell sorting or removal.
Especially preferred embodiments of the present invention relate to
a group of small, cyclic tumor targeting peptides comprising a motif, LRS or
SRL, optionally coupled to an effector unit and other additional units, as de-
scribed in more detail herein.
ACCOMPANYING
BRIEF DESCRIPTION OF THE RAWINGS
Figure 1 is a graph showing the therapeutic effect of a targeting
agent comprising doxorubicin.
DETAILED DESCRIPTION OF THE INVENTION
For the purpose of this invention, the term "cancer" is used herein in
its broadest sense, and includes any disease or condition involving trans-
formed or malignant cells. In the art, cancers are classified into five major cate-
gories, according to their tissue origin (histological type): carcinomas, sarco-
mas, myleomas, and lymphomas, which are solid tumor type cancers, and leu-
kemias, which are "liquid cancers". The term cancer, as used in the present in-
vention, is intended to primarily include all types of diseases characterized by
solid tumors, including disease states where there is no detectable solid tumor
or where malignant or transformed cells, "cancer cells", appear as diffuse infil-
trates or sporadically among other cells in healthy tissue.
The terms "amino acitf"and "amino alcohol" are to be interpreted
herein to include also diamino, triamino, oligoamino and polyamino acids and
alcohols; dicarboxyl, tricarboxyl, oligocarboxyl and polycarboxyl amino acids;
dihydroxyl, trihydroxyl, oligohydroxyl and polyhydroxyl amino alcohols; and
analogous compounds comprising more than one carboxyl group or hydroxyl
group and one or more amino groups.
By the term "peptide" is meant, according to established terminol-
ogy, a chain of amino acids (peptide units) linked together by peptide bonds to
form an amino acid chain. Peptides may be cyclic as described below. For the
purposes of the present invention, also compounds comprising one or more D-
amino acids, p-amino acids and/or other unnatural amino acids (e.g. amino ac-
ids with unnatural side chains) are included in the term "peptide". For the pur-
poses of the present invention, the term "peptide" is intended to include pepti-
dyl analogues comprising modified amino acids. Such modifications may com-
prise the introduction or presence of a substituent in a ring or chain; the intro-
duction or presence of an "extra" functional group such as an amino, hy-
drazino, carboxyl, formyl (aldehyde) or keto group, or another moiety; and the
absence or removal of a functional group or other moiety. The term also in-
cludes analogues modified in the amino- and/or carboxy termini, such as pep-
tide amides and N-substituted amides, peptide hydrazides, /V-substituted hy-
drazides, peptide esters, and their like, and peptides that do not comprise the
amino-terminal -NH2 group or that comprise e.g. a modified amino-terminal
amino group or an imino or a hydrazino group instead of the amino-terminal
amino group, and peptides that do not comprise the carboxy-terminal carboxyl
group or comprise a modified group instead of it, and so on.
Some examples of possible reaction types that can be used to modify
peptides, forming "peptidy analogues", are e.g.,cycloaddition, condensation
and nucleophilic addition reactions as well as esterification, amide formation,
formation of substituted amides, N-alkylation, formation of hydrazides, salt
formation. Salt formation may be the formation of any type of salt, such as alkali
or other metal salt, ammonium salt, salts with organic bases, acid addition
salts etc. Peptidyl analogues may be synthesized either from the corresponding
peptides or directly (via other routes).
Compounds that are structural or functional analogues of the pep-
tides of the invention may be compounds that do not consist of amino acids or
not of amino acids alone, or some or all of whose building blocks are modified
amino acids. Different types of building blocks can be used for this purpose, as
is well appreciated by those skilled in the art. The function of these compounds
in biological systems is essentially similar to the function of the peptides. The
resemblance between these compounds and the original peptides is thus
based on structural and functional similarities. Such compounds are called
peptidomimetic analogues, as they mimic the function, conformation and/or
structure of the original peptides and, for the purposes of the present invention,
they are included in the term "peptide".
A functional analog of a peptide according to the present invention
; is characterized by a binding ability with respect to the binding to tumors, tumor
tissue, tumor cells or tumor endothelium which is essentially similar to that of
the peptides they resemble.
For example, compounds like benzolactam or piperazine containing
analogues based on the primary sequence of the original peptides can be
used (Adams et al., 1999; Nakanishi and Kahn, 1996a, 1996b; Houghten et al.,
1999; Nargund et al., 1998). A large variety of types of peptidomimetic sub-
stances have been reported in the scientific and patent literature and are well
known to those skilled in the art. Peptidomimetic substances (analogues) may
comprise for example one or more of the following structural components: re-
duced amides, hydroxyethylene and/or hydroxyethylamine isosteres, N-methyl
amino acids, urea derivatives, thiourea derivatives, cyclic urea and/or thiourea
derivatives, poly(ester imide)s, polyesters, esters, guanidine derivatives, cyclic
guanidines, imidazoyl compounds, imidazolinyl compounds, imidazolidinyl
compounds, lactams, lactones, aromatic rings, bicyclic systems, hydantoins
and/or thiohydantoins as well as various other structures. Many types of com-
pounds for the synthesis of peptidomimetic substances are available from a
number of commercial sources (e.g. Peptide and Peptidomimetic Synthesis,
Reagents for Drug Discovery, Fluka ChemieGmbH, Buchs, Switzerland, 2000
and Novabiochem 2000 Catalog, Calbiochem-Novabiochem AG, Laufelfingen,
Switzerland, 2000). The resemblance between the peptidomimetic compounds
and the original peptides is based on structural and/or functional similarities.
Thus, the peptidomimetic compounds mimic the properties of the original peptides
and, for the purpose of the present application, their binding ability is
similar to the peptides that they resemble. Peptidomimetic compounds can be
made up, for example, of unnatural amino acids (such as D-amino acids or
amino acids comprising unnatural side chains, or of b-amino acids etc.), which
do not appear in the original peptides, or they can be considered to consist of
or can be made from other compounds or structural units. Examples of synthetic
peptidomimetic compounds comprise N-alkylamino cyclic urea, thiourea,
polyesters, poly(ester imide)s, bicyclic guanidines, hydantoins, thiohydantoins,
and imidazoi-pyridino-inoles (Houghten et al. 1999 and Nargund et al., 1998).
Such peptidomimetic compounds can be characterized as being "structural or
functional analogues" of the peptides of this invention.
For the purpose of the present invention, the term "targeting unit"
stands for a compound, a peptide, capable of selectively targeting and selec-
tively binding to tumors, and, preferably, also to tumor stroma, tumor paren-
chyma and/or extracellular matrix of tumors. Another term used in the art for
this specific association is "homing". Tumor targeting means that the targeting
units specifically bind to tumors when administered to a human or animal body.
More specifically, the targeting units may bind to a cell surface, to a specific
molecule or structure on a cell surface or within the cells, or they may associ-
ate with the extracellular matrix present between the cells. The targeting units
may also bind to the endothelial cells or the extracellular matrix of tumor vas-
culature. The targeting units may bind also to the tumor mass, tumor cells and
extracellular matrix of metastases.
Generally, the terms "targeting" or "binding" stand for adhesion, at-
tachment, affinity or binding of the targeting units of this invention to tumors,
tumor cells and/or tumor tissue to the extent that the binding can be objectively
measured and determined e.g., by peptide competition experiments in vivo or
ex vivo, on tumor biopsies in vitro or by immunological stainings in situ, or by
other methods known by those skilled in the art. The exact mechanism of the
binding of targeting units according to the present invention is not known.
Tageting peptides according to the present invention are considered to be
"bound" to the tumor target in vitro, when the binding is strong enough to with-
stand normal sample treatment, such as washes and rinses with physiological
saline or other physiologically acceptable salt or buffer solutions at physiologi-
cal pH, or when bound to a tumor target in vivo long enough for the effector
unit to exhibit its function on the target.
The binding of the present targeting agents or targeting units to tu-
mors is "selective" meaning that they do not bind to normal cells and organs,
or bind to such to a significantly lower degree as compared to tumor cells and
organs.
Pharmaceutically and diagnostically acceptable salts of the target-
ing units and agents of the present invention include salts, esters, amities, hy-
drazides, N-substituted amides, N-substituted hydrazides, hydroxamic acid de-
rivatives, decarboxylated and N-substituted derivatives thereof. Suitable phar-
maceutically acceptable salts are readily acknowledged by those skilled in the
art.
TARGETING MOTIFS ACCORDING TO THE PRESENT INVENTION
It has now surprisingly been found that a three-amino-acid motif Dd-
Ee-Ff, wherein Dd-Ee-Ff is either Aa-Bb-Cc or Cc-Bb-Aa, and
Aa is isoleucine, leucine or tert-leucine, or a structural or functional analogue
thereof;
Bb is arginine, homoarginine or canavanine, or a structural or functional ana-
logue thereof; and
Cc is serine or homoserine, or a structural or functional analogue thereof,
targets and exhibits selective binding to tumors and cancers and tumor cells
and cancer cells.
Aa according to the present invention may comprise in its sidechain
a branched, non-branched or alicyclic structure with at least two siminal or different
atoms selected from the group consisting of carbon, silicon, halogen
bonded to carbon, ether-oxygens and thioether-sulphur. The analogue may be
selected from the group consisting of branched, non-branched or cyclic
non-aromatic, lipophilic and hydrophobic amino acids or amino acid analogues
or derivatives or structural and/or functional analogues thereof; amino acids or
carboxylic acids or amino acid analogues or derivatives or carboxylic acid analogues
or derivatives having one or more lipophilic carborane-type or other
lipophilic boron-containing side chains or other lipophilic cage-type structures.
Aa may be selected from the group consisting of:
1) a-amino acids whose side chain is one of the following:
- ethyl
- propyl
- 1 -methylpropyl (the side chain of isoleucine)
- 2-methylpropyl (the side chain of leucine)
- 2,2-dimethylpropyl
1 -ethylpropyl
tert-butyl
tert-pentyl
- 3-methylbutyl
- 2-methylbutyl
- methylbutyl
- ethylbutyl
- 2-ethylbutyl
cyclohexyl
- 2-methylcyclohexyl
cyclopentyl
- 2-methylcyclopentyl
- 3-methylcyclohexyl
- cyclobutyl
cyclopropyl
- 2-methylcyclopropyl
methoxyethyl
- methoxyethyl
methoxymethyl
- ethoxymethyl
- 2-ethoxyethyl
- 1-ethoxyethyl
- 2-methoxypropyl
- 2,2-dimethoxypropyl
- 1-methylpropyl
- 1-methylbutyl
- 1-methylpentyl
- 1,1-dimethylpropyl
- 1,1-dimethylbutyl
- 1,1-dimethylpentyl
- 1,2-dimethylpropyl
- 1-cyclopropylethyl
- 2-cyclopropylethyl
cyclopropylmethyl
- 1-cyclopropylethyl
- 1-cyclopropylpropyl
- 2-cyclopropylpropyl
3-cyclopropylpropyl
any cyclobutylalkyl
1 -ethylpropyl
1-methylethyl
- other mono-, di-, tri- or oligoalkyl-alkyl
other cyclic alkyl or substituted cyclic alkyl or alkyl that is substituted
with one or more substituted or unsubstituted cycloalkyl group(s)
and optionally one or more alkyl group(s)
- allyl
vinyl
1 -methylallyl
- 1-ethylallyl
1 -ethylvinyl
i-propenyl
1-methyl-1-propenyl
- methyl-1-propenyl
methyl-1-propenyl
1-ethyl-1-propenyl
- ethyl-1-propenyl
- ethyl-1-propenyl
- 1-methyl-1-butenyl
- methyl-1-butenyl
- methyl-1-butenyl
- 1 -ethyl-1 -butenyl
- 2-ethyl-1-butenyl
- ethyl-2-butenyl
- ethyl-2-butenyl
- ethyl-3-butenyl
- ethyl-3-butenyl
- ethyl-3-butenyl
2) any of the following carboxylic acids, including any optical isomers thereof :
4-methylpentanoic acid
- 3- methylpentanoic acid
4,4-dimethylpentanoic acid
- 3,4-dimethylpentanoic acid
- 3,3-dimethylpentanoic acid
3-methylhexanoic acid
- 4-methylhexanoic acid
5-methylhexanoic acid
- 2-ethylpentanoic acid
3-ethylpentanoic acid
4-ethylpentanoic acid
2-cyclopropylpentanoic acid
- 3-cyclopropylpentanoic acid
4-cyclopropylpentanoic acid
- 2-methylbutanoic acid
- 3-methylbutanoic acid
- 4-methylbutanoic acid
- 2-cyclopropylbutanoic acid
- 3-cyclopropylbutanoic acid
4-cyclopropylbutanoic acid
3) any optical and geometrical isomer of any of the following compounds:
- 2-amino-4-methyl-3-pentenoic acid
- 2-amino-4-methyl-4-pentenoic acid
- 2-amino-5-methyl-3-hexenoic acid
- 2-amino-5-methyl-4-hexenoic acid
- 2-amino-5-methyl-5-hexenoic acid
and
4) aminosubstituted (N-substituted) analogues of the amino-comprising compounds
of points 1 and 3 that bear at the amino group
- one methyl, ethyl, propyl, isopropyl or other alkyl group
- one cycloalkyl group
- one 9-fluorenylmethyloxycarbonyl (FMOC) group
- one benzyloxycarbonyl (Cbz) group
- one tert-butyloxycarbonyl (BOC) group
- two identical, similar and/or different groups selected from the ones men-
tioned above in this point (point 4).
Aa may also be an a-amino acid (either L- or D- amino acid) of the
formula R1 - CR2 (NH2) - COOH wherein the side chain R1 is selected from the
side chains listed above, and the side chains R2 is selected from the group
consisting of: hydrogen, methyl, ethyl, propyl.
Bb according to the present invention may be selected from the
group consisting of amino acids or structural or functional analogues thereof
containing one or more guanyl groups, aminodino groups or their analogues
and derivatives and structural or functional equivalents; one or more groups
containing at least two nitrogen atoms each and have or can gain a delocal-
ized positive charge.
Bb may be selected from the group of compounds of the following
formula:
wherein R1 - R5 is hydrogen or methyl, R2 and R3 may form -CH2-CH2- and
n is 1-6.
Preferably, Bb is the L- or D- form of
arginine,
homoarginine,
canavanine,
2-amino-8-guanidino-octanoic acid,
2-amino-7-guanidino-octanoic acid,
2-amino-6-guanidino-octanoic acid,
2-amino-5-guanidino-octanoic acid,
2-amino-7-guanidino-heptanoic acid,
2-amino-6-guanidino-heptanoic acid,
2-amino-5-guanidino-heptanoic acid,
2-amino-4-guanidino-heptanoic acid,
2-amino-5-guanidino-hexanoic acid,
2-amino-4-guanidino-hexanoic acid,
2-amino-3-guanidino-hexanoic acid,
2-amino-4-guanidino-pentanoic acid,
2-amino-3-guanidino-pentanoic acid.
Cc according to the present invention may be selected from the
group consisting of amino acids, amino alcohols, diamino alcohols, tri-, oligo-
and polyamino alcohols and amino acid analogues, derivatives and structural
or functional analogues thereof, comprising one or more hydroxyl group(s), es-
terified hydroxyl group(s), methoxyl group(s) and/or other etherified hydroxyl
(ether) groups.
Cc as defined above may be serine or homoserine or a structural or
functional analogue thereof, comprising at least one hydroxyl group; or may be
selected from the group consisting of:
any other monoaminocarboxylic acid comprising at least one alcoholic hydroxyl
group
any carboxylic acid comprising at least one alcoholic hydroxyl group
any other aminocarboxylic acid comprising an aliphatic or other side chain that
comprises one or more alcoholic hydroxyl (OH) function(s) and/or esterified
hydroxyl function(s).
Preferably, Cc is the L- or D- form of
serine,
homoserine,
2-amino-7-hydroxyheptanoic acid,
2-amino-5-hydroxypentanoic acid,
2-amino-6-hydroxyhexanoic acid,
2-amino-8-hydroxyoctanoic acid,
or any other hydroxy-2-aminocarboxylic acid.
Alternatively, the motif Aa-Bb-Cc, as a whole, according to the present
invention is a structural or functional analogue of a structure where Aa, Bb
and Cc are as defined above.
Preferred embodiments of the present invention include tumor targeting
motifs Aa-Bb-Cc selected from those given in Table 1 as well as structural
and functional analogues thereof.
Thus, typical and preferred characteristics of Aa include lipofilicity,
hydrophobicity and aliphatic character in at least one side chain, wheras Bb in-
cludes a delocalized positive charge and Cc has the ability of participating in
OH-binding.
Especially preferred motifs Dd-Ee-Ff according to the present inven-
tion are leucine-arginine-serine (LRS) and serine-arginine-leucine (SRL).
The motifs Dd-Ee-Ff according to the present invention may form
part of a larger structure, such as a peptide or some other structure. When the
compound or structure in question comprises more than one motif Dd-Ee-Ff,
the orientation and direction of the motifs may vary.
TARGETING UNITS ACCORDING TO THE PRESENT INVENTION
It has also been found that peptides and structural or functional ana-
logues thereof comprising a tumor targeting motif according to the present in-
vention target to and exhibit selective binding to tumor cells and tissues. Pep-
tides comprising a tumor targeting motif according to the present invention
and, optionally, up to four additional amino acid residues or analogues thereof,
likewise exhibit such targeting and selective binding and are especially pre-
ferred embodiments of the present invention.
Such peptides are highly advantageous for use as targeting units
according to the present invention, e.g., because of their small size and their
easier and more reliable and much cheaper synthesis, purification, analysis
and quality control.
Especially preferred targeting units according to the present invention
are peptides cyclized by an amide bond, such as a lactam bridge or by an
ester bond, such as a lacotone bridge.
Such cyclic targeting units have now been shown to selectively target
to tumors in vivo.
Preferred tumor targeting units according to the present invention
comprise a tumor targeting motif Dd-Ee-Ff as defined above, and additional
residues selected from the group consisting of:
natural amino acids;
unnatural amino acids;
amino acid analogues comprising maximally 30 non-hydrogen atoms and an
unlimited number of hydrogen atoms,; and
other structural units and residues whose molecular weight and/or formula
weight is maximally 270;
wherein
the number of said additional residues ranges from 0 to 4, preferably from 0 to
3, more preferably from 0 to 2, and most preferably is either 0 or 2.
Cyclic peptides are usually more stable in vivo and in many other
biological systems than are their non-cyclic counterparts, as is known in the
art. It has now, however, surprisingly been found that the targeting property of
the small peptides according to the present invention is more pronounced
when the targeting unit is cyclic or contained in a cyclic structure.
Preferred targeting units according to the present invention may
comprise a structure
wherein,
Dd-Ee-Ff is a tumor targeting motif according to the present inven-
tion, as described above,
Rr is any amino acid residue,
n and m are integers 0-4, preferably 0-3, more preferably 0-2,
whereby the sum of n and m does not exceed four, and,
Cy and Cyy are entities capable of forming a cyclic structure by
means of a lactam ring.
Lactams can be of several subtypes, such as "head to tail" (carboxy
terminus plus amino terminus), "head to side chain" and "side chain to head"
(carboxy or amino terminus plus one side chain amino or carboxyl group) and
"side chain to side chain" (amino groug of one side chain and carboxys group
of another side chaine).
Preferred targeting units are such, where Rr is any amino acid resi-
due, except histidine, lysine or tryptophane. Especially preferred are targeting
units wherein Rr is R or G.
Preferred structures are thus compounds of the general formula
as defined above, and wherein Cy and Cyy are residues capable of forming a
lactam bond, such as aspartic acid (D), glutamic acid (E), lysin (K), omithine
(0) or analogues thereof comprising no more than 12 carbon atoms.
Especially preferred targeting units according to the present inven-
tion having a cyclic structure by virtue of a lactam bridge, are :
DLRSK (SEQ ID NO: 1), DGRGLRSK (SEQ ID NO: 2), DRGLRSK (SEQ ID
NO: 3), DRYYNLRSK (SEQ ID NO: 4), DSRYNLRSK (SEQ ID NO: 5),
DLRSGRK (SEQ ID NO: 6), DLRSGRGK (SEQ ID NO: 7), OLRSE (SEQ ID
NO. 8), OLRSGRGE (SEQ ID NO. 9) and KLRSD (SEQ ID NO. 10),
as well as salts, esters, derivatives and analogues thereof, as defined above.
TARGETING AGENTS ACCORDING TO THE PRESENT INVENTION
It has also been found that targeting agents comprising at least one
tumor targeting unit according to the present invention, and at least one effec-
tor unit, target to and exhibit selective binding to cancer cells and tissues as
well as endothelial cells.
The tumor targeting agents according to the present invention may
optionally comprise unit(s) such as linkers, solubility modifiers, stabilizers,
charge modifiers, spacers, lysis or reaction or reactivity modifiers, internalizing
units or intemalization enhancers or membrane interaction units or other local
route, attachment, binding and distribution affecting units. Such additional units
of the tumor targeting agents according to the present invention may be cou-
pled to each other by any means suitable for that purpose.
Many possibilities are known to those skilled in the art for linking
structures, molecules, groups etc. of the types in question or of related types,
to each other. The various units may be linked either directly or with the aid of
one or more identical, similar and/or different linker units. The tumor targeting
agents of the invention may have different structures such as any of the non-
limiting types schematically shown below:
where EU indicates "effector unit" and TU indicates "targeting unit" and n, m
and k are independently any integers except 0.
In the targeting agents according to the present invention, as in
many other medicinal and other substances, it may be wise to include spacers
or linkers, such as amino acids and their analogues, such as long-chain
omega-amino acids, to prevent the targeting units from being "disturbed", steri-
cally, electronically or otherwise hindered or "hidden" by effector units or other
unit of the targeting agent.
In targeting agents according to the present invention it may be useful
for increased activity to use dendrimeric or cyclic structures to provide a
possibility to incorporate multiple effector units or additional units per targeting
unit.
Preferred targeting agents according to the present invention comprise
a structure
TU is a targeting unit according to the present invention as defined abover;
and
Ef and Eff are selected from the group consisting of:
effector units, linker units, solubility modifier units, stabilizer units, charge modifier
units, spacer units, lysis and/or reaction and/or reactivity modifier units, internalizing
and/or internalization enhancer and/or membrane interaction units
and/or other local route and/or local attachment/local binding and/or distribution
affecting units, adsorption enhancer units, and other related units; and
peptide sequences and other structures comprising at least one such unit; and
peptide sequences comprising no more than 20, preferably no more than 12,
more preferably no more than 6, natural and/or unnatural amino acids; and
natural and unnatural amino adds comprising no more than 25 non-hydrogen
atoms and an unlimited number of hydrogen atoms;
as well as salts, esters, derivatives and analogues thereof.
EFFECTOR UNITS
For the purposes of this invention, the term "effector unit" means a
molecule or radical or other chemical entity as well as large particles such as
colloidal particles and their like; liposomes or microgranules. Suitable effector
units may also consitute nanodevices or nanochips or their like; or a combina-
tion of any of these, and optionally chemical structures for the attachment of
the constituents of the effector unit to each or to parts of the targeting agents.
Effector units may also contain moieties that effect stabilization or solubility
enhancement of the effector unit.
Preferred effects provided by the effector units according to the pre-
sent invention are therapeutical (biological, chemical or physical) effects on the
targeted tumor; properties that enable the detection or imaging of tumors or
tumor cells for diagnostic purposes; or binding abilities that relate to the use of
the targeting agents in different applications.
A preferred (biological) activity of the effector units according to the
present invention is a therapeutic effect. Examples of such therapeutic activities
are for example, cytotoxicity, cytostatic effect, ability to cause differentiation
of cells or to increase their degree of differentiation or to cause phenotypic
changes or metabolic changes, chemotactic activities, immunomodulating activities,
pain relieving activities, radioactivity, ability to affect the cell cycle, abil-
ity to cause apoptosis, hormonal activities, enzymatic activities, ability to trans-
fect cells, gene transferring activities, ability to mediate "knock-out" of one or
more genes, ability to cause gene replacements or "knock-in", antiangiogenic
activities, ability to collect heat or other energy from external radiation or electric
or magnetic fields, ability to affect transcription, translation or replication of
the cell"s genetic information or external related information; and to affect
post-transcriptional and/or post-translational events.
Other preferred therapeutic approaches enabled by the effector
units according to the present invention may be based on the use of thermal
(slow) neutrons (to make suitable nuclei radioactive by neutron capture), or the
administration of an enzyme capable of hydrolyzing for example an ester bond
or other bonds or the administration of a targeted enzyme according to the
present invention.
Examples of preferred functions of the effector units according to
the present invention suitable for detection are radioactivity, paramagnetism,
ferromagnetism, ferrimagnetism, or any type of magnetism, or ability to be detected
by NMR spectroscopy, or ability to be detected by EPR (ESR) spectros-
copy, or suitability for PET and/or SPECT imaging, or the presence of an im-
munogenic structure, or the presence of an antibody or antibody fragment or
antibody-type structure, or the presence of a gold particle, or the presence of
biotin or avidin or other protein, and/or luminescent and/or fluorescent and/or
phosphorescent activity or the ability to enhance detection of tumors, tumor
cells, endothelial cells and metastases in electron microscopy, light microscopy
(UV and/or visible light), infrared microscopy, atomic force microscopy or tun-
neling microscopy, and so on.
Preferred binding abilities of an effector unit according to the present
invention include, for example:
a) ability to bind to a substance or structure such as a histidine or other tag,
b) ability to bind to biotin or analogues thereof,
c) ability to bind to avidin or analogues thereof,
d) ability to bind to an enzyme or a modified enzyme,
e) ability to bind metal ion(s) e.g. by chelation,
f) ability to bind a cytotoxic, apoptotic or metabolism affectin substance or a
substance capable of being converted in situ into such a substance,
g) ability to bind to integrins and other substances involved in cell adhesion,
migration, or intracellular signaling,
h) ability to bind to phages,
i) ability to bind to lymphocytes or other blood cells,
j) ability to bind to any preselected material by virtue of the presence of
antibodies or structures selected by biopanning,
k) ability to bind to material used for signal production or amplification,
I) ability to bind to therapeutic substance.
Such binding may be the result of e.g. chelation, formation of cova-
lent bonds, antibody-antigen-type affinity, ion pair or ion associate formation,
specific interactions of the avidin-biotin-type, or the result of any type or mode
of binding or affinity.
One or more of the effector units or parts of them may also be a part
of the targeting units themselves. Thus, the effector unit may for example be
one or more atoms or nuclei of the targeting unit, such as radioactive atoms or
atoms that can be made radioactive, or paramagnetic atoms or atoms that are
easily detected by MRI or NMR spectroscopy (such as carbon-13). Further ex-
amples are, for example, boron-comprising structures such as carborane-type
lipophilic side chains.
The effector units may be linked to the targeting units by any type of
bond or structure or any combinations of them that are strong enough so that
most, or preferably all or essentially all of the effector units of the targeting
agents remain linked to the targeting units during the essential (necessary)
targeting process, e.g. in a human or animal subject or in a biological sample
under study or treatment.
The effector units or parts of them may remain linked to the target-
ing units, or they may be partly or completely hydrolyzed or otherwise disinte-
grated from the latter, either by a spontaneous chemical reaction or equilibrium
or by a spontaneous enzymatic process or other biological process, or as a re-
sult of an intentional operation or procedure such as the administration of hy-
drolytic enzymes or other chemical substances. It is also possible that the en-
zymatic process or other reaction is caused or enhanced by the administration
of a targeted substance such as an enzyme in accordance with the present in-
vention.
One possibility is that the effector units or parts thereof are hydro-
lyzed from the targeting agent and/or hydrolyzed into smaller units by the ef-
feet of one or more of the various hydrolytic enzymes present in tumors (e.g.,
intracellularly, in the cell membrane or in the extracellular matrix) or in their
near vicinity.
Taking into account that the targeting according to the present in-
vention may be very rapid, even non-specific hydrolysis that occurs every-
where in the body may be acceptable and usable for hydrolysing one or more
effector unit(s) intentionally, since such hydrolysis may in suitable cases (e.g.,
steric hindrance, or even without any such hindering effects) be so slow that
the targeting agents are safely targeted in spite of the presence of hydrolytic
enzymes of the body, as those skilled in the art very well understand. The for-
mation of insoluble products and/or products rapidly absorbed into cells and/or
bound to their surfaces after hydrolysis may also be beneficial for the targeted
effector units and/or their fragments etc. to remain in the tumors or their closest
vicinity.
In one preferred embodiment of the invention, the effector units may
comprise structures, features, fragments, molecules or the like that make pos-
sible, cause directly or indirectly, an "amplification" of the therapeutic or other
effect, of signal detection, of the binding of preselected substances, including
biological material, molecules, ions, microbes or cells.
Such "amplification" may, for example, be based on one or more of
the following non-limiting types:
- the binding, by the effector units, of other materials that can further bind
other substances (for example, antibodies, fluorescent antibodies, other "la-
belled" substances, substances such as avidin, preferably so that several
molecules or "units" of the further materials can be bound per each effector
unit;
- the effector units comprise more than one entity capable of binding e.g. a
protein, thus making direct amplification possible;
- amplification in more than one steps.
Preferred effector units according to the present invention may be
selected from the follwing group:
_ cytostatic or cytotoxic agents
_ apoptosis causing or enhancing agents
_ enzymes or enzyme inhibitors
_ antimetabolites
_ agents capable of disturbing membrane functions
_ radioactive or paramagnetic substances
._ substances comprising one or more metal ions
- substances comprising boron, gadolinium, litium
- substances suitable for neutron capture therapy
- labelled substances
- intercalators and substances comprising them
- oxidants or reducing agents
- nucleotides and their analogues
- metal chelates or chelating agents.
In a highly preferred embodiment of the invention, the effector unit
comprises alpha emittors.
In further preferred embodiments of the invention, the effector units
may comprise copper chelates such as frans-bis(salicylaldoximaro) copper(ll)
and its analogues, or platinum compounds such cisplatin, carboplatin.
Different types of structures, substances and groups ar known that
can be used to cause or enhance e.g., intemalization into cells, including for
example RQIKIWFQNRRMKWKK; Penetratin (Prochiantz, 1996), as well as
stearyl derivatives (Promega Notes Magazine, 2000).
As an apoptosis-inducing structure, for example, the peptide se-
quence KLAKLAK that interacts with mitochondria! membranes inside cells,
can be included Ellerby et al. (1999).
For use in embodiments of the present invention that include cell
sorting and any related applications, the targeting units and agents of the in-
vention can, for example, be used
a) coupled or connected to magnetic particles,
b) adsorbed, coupled, linked or connected to plastic, glass or other solid, po-
rous, fibrous material-type or other surface(s) and the like,
c) adsorbed, covalently bonded or otherwise linked, coupled or connected into
or onto one or more substance(s) or material(s) that can be used in columns
and related systems
d) adsorbed, covalently bonded or otherwise linked, coupled or connected into
or onto one or more substance(s) or material(s) that can be precipitated, centri-
fuged or otherwise separated or removed.
OPTIONAL UNITS OF THE TARGETING AGENTS ACCORDING TO THE
PRESENT INVENTION
The targeting agents and targeting units of the present invention
may optionally comprise further units, such as:
linker units coupling targeting units, effector units or other optional units of the
present invention to each other;
solubility modifying units for modifying the solubility of the targeting agents or
their hydrolysis product;
stabilizer units stabilizing the structure of the targeting units or agents during
synthesis, modification, processing, storage or use in vivo or in vitro;
charge modifying units modifying the electrical charges of the targeting units or
agents or their starting materials;
spacer units for increasing the distance between specific units of the targeting
agents or their starting materials, to release or decrease steric hindrance or
structural strain of the products;
reactivity modifyer units;
internalizing units or enhancer units for enhancing targeting and uptage of the
targeting agents;
adsorption enhancer units, such as fat or water soluble structures enhancing
absorption of the targeting agents in vivo; or
other related units.
A large number of suitable linker units are known in the art. Exam-
ples of suitable linkers are:
1. for linking units comprising amino groups: cyclic anhydrides, dicarboxylic or
multivalent, optinally activated or derivatized, carboxylic acids, compounds
with two or more reactive halogens or compounds with at least one reactive
halogen atom and at least one carboxyl group;
2. for linking units comprising carboxyl groups or derivatives thereof: com-
pounds with at least two similar or different groups such as amino, substi-
tuted amino, hydroxyl, -NHNH2 or substituted forms thereof, other known
groups for the purpose (activators may be used);
3. for linking an amino group and a carboxyl group: for example amino acids
and their activated or protected forms or derivatives;
4. for linking a formyl group or a keto group to another group are: a compound
comprising e.g. at least one -N-NH2 or-0-NH2 or =N-NH2 or their like;
5. for linking several amino-comprising units: polycarboxylic substances such
as EDTA, DTPA and polycarboxylic acids, anhydrides, esters and acyl
halides;
6. for linking a substance comprising an amino group to a substance
comprising either a formyl group or a carboxyl group: hydrazinocarboxylic
acids or their like, preferably so that the hydrazino moiety or the carboxyl
group is protected or activated, such as 4-(FMOC-hydrazino)benzoic acid;
7, for linking an organic structure to a metal ion: substances that can be
coupled to the organic structure (e.g. by virtue of their COOH groups or
their NH2 groups) or that are integral parts of it, and that in addition
comprise a polycarboxylic part for example an EDTA- or DTPA-like
structure, peptides comprising several histidines or their like, peptides
comprising several cysteines or other moieties comprising an -SH group
each, and other chelating agents that comprise functional groups that can
be used to link them to the organic structure.
A large variety of the above substances and other types of suitable
linking agents are known in the art.
A large number of suitable solubility modifier units are known in the
art. Suitable solubility modifier units comprise, for example:
- for increasing aqeous solubility: molecules comprising SO3, O-SO3 COOH,
COO", NH2, NH3+, OH groups, guanidino or amidino groups or other ionic and
ionizable groups and sugar-type structures;
- for increasing fat solubility or solubility in organic solvents: units comprising
(long) aliphatic branched or non-branched alkyl and alkenyl groups, cyclic non-
aromatic groups such as the cyclohexyl group, aromatic rings and steroidal
structures.
A large number of units known in the art can be used as stabilizer
units, e.g. bulky structures (such as tert-butyl groups, naphthyl and adamantyl
and related radicals etc.) for increasing steric hindrance, and D-amino acids
and other unnatural amino acids (including b-amino acids, w-amino acids,
amino acids with very large side chains etc.) for preventing or hindering enzymatic
hydrolysis.
Units comprising positive, negative or both types of charges can be
used as charge modifier units, as can also structures that are converted or can
be converted into units with positive, negative or both types of charges.
Spacer units may be very important, and the need to use such units
depends on the other components of the structure (e.g. the type of biologically
active agents used, and their mechanisms of action) and the synthetic procedures
used
Suitable spacer units may include for example long aliphatic chains
or sugar-type structures (to avoid too high lipophilicity), or large rings. Suitable
compounds are available in the art. One preferred group of spacer units are co-
amino acids with long chains. Such compounds can also be used (simultaneously)
as linker units between an amino-comprising unit and a carboxyl-
comprising unit. Many such compounds are commercially available, both as
such and in the forms of various protected derivatives.
Units that are susceptible to hydrolysis (either spontaneous chemical
hydrolysis or enzymatic hydrolysis by the body"s own enzymes or enzymes
administered to the patient) may be very advantageous in cases where
it is desired that the effector units are liberated from the targeting agents e.g.
for internalization, intra- or extracellularl DNA or receptor binding. Suitable
units for this purpose include, for example, structures comprising one or more
ester or acetal functionality, Various proteases may be used for the purposes
mentioned. Many groups used for making pro-drugs may be suitable for the
purpose of increasing or causing hydrolysis, lytic reactions or other decomposition
processes.
The effector units, the targeting units and the optional units according
to the present invention may simultaneously serve more than one function.
Thus, for example, a targeting unit may simultaneously be an effector unit or
comprise several effector units; a spacer unit may simultaneously be a linker
unit or a charge modifier unit or both; a stabilizer unit may be an effector unit
with properties different from those of another effector unit, and so on.An effector
unit may, for example, have several similar or even completely different
functions.
In one preferred embodiment of the invention, the tumor targeting
agents comprise more than one different effector units. In that case, the effector
units may be, for example, diagnostic and therapeutic units. Thus, for example,
it is preferred to use, for boron neutron capture therapy, such agents
whose effector units, in addition to comprising boron atoms, also can be detected
or quantified in the patient in vivo after administration of the agent, in
order to be able to ascertain that the agent has accumulated adequately in the
tumor to be treated, or to optimize the timing of the neutron treatment, and so
on. This goal may be achieved e.g. by using such a targeting agents according
to the invention that comprise an effector unit comprising boron atoms (preferably
isotope-enriched boron) and groups detectable e.g. by NMRI. Likewise,
the presence of more than one type of therapeutically useful effector units may
also be preferred. In addition, the targeting units and targeting agents may, if
desired, be used in combination with one or more "classical" or other tumor
therapeutic modalities such as surgery, chemotherapy, other targeting modalities,
radiotherapy, immunotherapy etc.
PREPARATION OF TARGETING UNITS AND AGENTS ACCORDING TO
THE PRESENT INVENTION
The targeting units according to the present invention are preferably
synthetic peptides. Peptides can be synthesized by a large variety of well-
known techniques, such as solid-phase methods (FMOC-, BOC-, and other
protection schemes, various resin types), solution methods (FMOC, BOC and
other variants) and combinations of these. Even automated apparatuses/devices
for the purpose are available commercially, as are also routine
synthesis and purification services. All of these approaches are very well
known to those skilled in the art. Some methods and materials are described,
for example, in the following references:
Bachem AG, SASRIN™ (1999), The BACHEM Practise of SPPS
(2000), Bachem 2001 catalogue (2001), Novabiochem 2000 Catalog (2000),
Peptide and Peptidomimetic Synthesis (2000) and The Combinatorial Chemistry
Catalog & Solid Phase Organic Chemistry (SPOC ) Handbook 98/99. Peptide
synthesis is exemplified also in the Examples.
As known in the art, it is often advisable, important and/or
necessary to use one or more protecting groups, a large variety of which are
known in the art, such as FMOC, BOC, and trityl groups and other protecting
groups mentioned in the Examples. Protecting groups are often used for
protecting amino, carboxyl, hydroxyl, guanyl and -SH groups, and for any
reactive groups/functions.
As those skilled in the art well know, activation often involves
carboxyl function activation and/or activation of amino groups.
Protection may also be orthogonal and/or semi/quasi/pseudo-
orthogonal. Protecting and activating groups, substances and their uses are
exemplified in the Examples and are described in the references cited herein,
and are also described in a large number of books and other sources of
information commonly known in the art (e.g. Protective Groups in Organic
Synthesis, 1999).
Resins for solid-phase synthesis are also well known in the art, and
are described in the Examples and in the above-cited references.
Cyclic structures according to the present invention may be synthesized,
for example, by methods based on the use of orthogonally protected
amino acids. Thus, for example, one amino acid containing an orthogonally
protected "extra" COOH function (e.g the (-allyl ester of
N-(-FMOC-L-glutamic-acid, i.e., "FMOC-Glu-Oall"), or the (-tert-butyl ester of
N-(-FMOC-L-glutamic acid (TMOC-Glu-OtBu), or the
(-4{N-t1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzyl
ester of N-(-FMOC-L-glutamic acid ("FMOC-Glu-Odmab") or the
(-2-phenylisopropyl ester of N~(-FMOC-L-glutamic acid
("FMOC-Glu(O-2-PhiPr)-OH"), or related derivatives of other dicarboxylic
amino acids, such as aspartic acid; or resin-bound forms of any of the afore-
mentioned), and one amino acid with an orthogonally protected "extra" amino
group (e.g N-(-FMOC-N-(-4-methyltrityl-L-lysine ("FMOC-Lys(Mtt)-OH") or the
corresponding derivative of ornithine or some other diaminocarboxylic acid or a
resin-bound form of one of these; resin-bound forms, however, not simultane-
ously with resin-bound forms of the orthogonally protected amino acids with
"extra" COOH), may be incorporated in the structure and, after deprotection,
the carboxyl and amino groups may be reacted, usually using activator(s). This
type of methodology is well known and is described, for example, in the following
references Novabiochem Catalog (2000), pp. 19-21 and 33 and specifically
B9-B15, and in the references therein, Bachem 2001 catalogue (2001), pp.
31-32, Chan et ai. (1995), Yue et al. (1993) and Hirschmann et al. (1998).
Suitable starting materials are available commercially, and further
ones can be made by methods known in the art. D-amino acid derivatives can
also be used in this methodology. Instead of "truly" orthogonal protective
groups, also quasiorthogonal/semi-orthogonal/pseudoorthogonal protecting
groups can be employed, as those skilled in the art understand.
Cyclic products made according to the above described methods
are usually especially stable in biological milieu, and are thus preferred. This
type of structures may be produced by any of the methods for the production
of such structures (chemical, enzymatic or biological). Many such methods are
well known for those skilled in the art. Cyclic structures of this type can be
syntesized chemically with the aid of solid-phase synthesis but they can likewise
be synthesized using solution methods or a combination of both, as those
skilled in the art well know. Amino acids with an "extra" carboxyl or amino func-
tion suitable for cyclization purposes (when adequately protected) include (as
non-limiting possibilities), for example, those with the structures shown below:
In solution cyclizations of any type, dilute solutions are normally advantageous,
as is well known by those skilled in the art.
The targeting units and agents according to the present invention
may also be prepared as fusion proteins or by other suitable recombinant DNA
methods known in the art. Such an approach for preparing the peptides according
to the present invention is preferred especially when the effector units
and/or other optional units are peptides or proteins. One example of a useful
protein effector unit is glutathion-S-transferase (GTS).
ADVANTAGES OF THE TARGETING UNITS AND TARGETING AGENTS OF
THIS INVENTION
There are acknowledged problems related to peptides intended for
diagnostic or therapeutic use. One of these problems stem from the length of
the sequence; the longer it grows, the more difficult or even impossible the
synthesis of the desired product becomes, especially if there are other
synthetic problems such as the presence of difficult residues that require
protection-deprotection and/or cause side reactions etc. The tendency to side-
rections, and possible synthesis termination (that not only decreases the yield
of the desired product if this is formed at all, but also gives rise to products with
a wrong length of the peptide chain) and formation of serious amounts of
harmful by-products is drastically increased by the presence in the desired
sequence of any amino acid(s) that require(s) side-chain protection (e.g., basic
side-chains such as those of lysine, histidine and tryptophan) and (of course)
also deprotection. All of these problems also make, as those skilled in the art
very well know, the purification of the desired peptides very much more difficult
and may make production of adequately purified material impossible.
As compared to known products that contain long and difficult-to-
make sequences with problematic amino acid residues, the peptides of the
present invention are clearly superior, as described in more detail below.
Thus, the products and methods of the present invention and their
use offer highly significant and very important advantages over the prior art.
The targeting units of this invention can be synthesized easily and
reliably. An advantage as compared to many prior art peptides is that the
targeting units and motifs of this invention do not need to comprise the
problematic basic amino acids lysine and histidine, nor tryptophan, all of which
may cause serious side-reactions in peptide synthesis, and, due to which the
yield of the desired product might be lowered radically or even be impossible
to obtain in adequate amounts or with adequate quality.
When present, histidine, lysine and tryptophan must be adequately
protected using suitable protecting groups that remain intact during the
synthesis prodecures. This may be very difficult and at least increases the
costs and technical problems. Also costs are remarkably increased by the
reagents and work-load and other costs of the deprotection steps and the
costs per unit of desired product may be increased.
Because of their smaller size and thus drastically less steps in the
\ synthesis, the peptides of the present invention are much easier and cheaper
to produce than targeting peptides of the prior art.
As histidine is not needed in the products of the present invention,
the risk of racemization of it is of no concern.
It is a great advantage not only for the economic synthesis of the
products of the present invention but also for the purification and analysis and
quality control that any racemization of histidine is outside consideration. It
also makes any administration to humans and animals safer and more
straightforward.
Because of their smaller size, the peptides of the present invention
can also be purified much more reliably and easily and with much less labor
and apparatus-time, and thus with clearly lower costs. Overall costs are thus
drastically reduced and better products can be obtained and in greater
amounts. Furthermore, the reliability of the purification is much better, giving
less concern of toxic remainders and of fatal or otherwise serious side-effects
in therapeutic and diagnostic applications.
Shorter synthesis protocols with relatively few steps produce less
impurities, making the peptides of the present invention highly advantageous.
The risks of toxic and even fatal impurities, allergens etc. are dramatically
lowered and, in addition, purification is easier.
The analysis and thus the quality control of the products of the
present invention is easier and less costly, than that of the longer and more
"difficult" peptide sequences. This increases the reliability of the analyses and
of quality control.
As residues such as lysine are not present in the targeting unit,
there is no the risk of the effector units being inadequately connected to such
residues. This is a remarkable advantage.
The effector unit can easily be linked to the peptides and peptidyl
analogues and peptidomimetic substances of the present invention using
(outside the targeting motif) for example protected lysine or ornithine as there
is no risk of simultaneous reaction of any lysine residue in the targeting motif.
For cyclization of the peptides of the present invention, protected
lysine or ornithine can be used, as the targeting units do not contain such
amino acids. This is an enormous advantage
The lactam bridge is clearly superior to the commonly used
disulphide bridge. This is due to a number of reasons, such as:
1. Lactams are highly stable against reducing agents such as thiols
that easily destroy disulphide bridges and can cause various undesired
reactions, for example dimerization, polymerization and formation of disulphide
bridges to other thiols;
2. Lactams can be synthesized in a highly controlled fashion for
example by using orthogonal protection;
3. Lactams are more stable than disulphides in biological milieu.
4. The purification of lactams is easier than with disulphides.
5. Because of lack of reaction with macromolecules having SH-
groups lactones probable are less prone to cause allergic reactions.
In solid synthesis of targeting agents according to the present
invention, the effector units and optional additional units may be linked to the
targeting peptide when still connected to the resin without the risk that the
removal of the protecting groups will cause destruction of additional unit.
Similar advantage applies to solution syntheses.
Another important advantage of the present invention and its products,
methods and uses according to it is the highly selective and potent targeting
of the products.
As compared to targeted therapy using antibodies or antibody fragments,
the products and methods of in the present invention are highly advantageous
because of several reasons. Potential immunological and related risks
are also obvious in the case of large biomolecules. Allergic reactions are of
great concern with such products, in contrats to small synthetic molecules such
as the targeting agents, units and motifs of the present invention.
As compared to targeting antibodies or antibody fragments, the
products and methods described in the present invention are highly advantageous
because their structure can be easily modified if needed or desired.
Specific amino acids such as histidine, tryptophan, tyrosine, threonine can be
omitted if dersired, and very few functional groups are necessary. On the other
hand, it is possible, without disturbing the targeting effect, to include various
different structural units, to specific desired properties that are of special value
in specific applications.
USE OF TARGETING AGENTS ACCORDING TO THE PRESENT INVENTION
The targeting units and targeting agents according to the present
invention are useful in cancer diagnostics and therapy, as they selectively target
to tumors in vivo, as shown in the examples. The effector unit may be chosen
according to the desired effect, detection or therapy. The desired effect
may also be achieved by including the effector in the targeting unit as such.
For use in radiotherapy the targeting unit itself may be e.g., radioactively labelled.
The present invention also relates to diagnostic compositions comprising
an effective amount of at least one targeting agent according to the
present invention. In addition to the targeting agent, a diagnostic composition
according to the present invention may, optionally, comprise carriers, solvents,
vehicles, suspending agents, labelling agents and other additives commonly
used in diagnostic compositions. Such diagnostic compositions are useful in
diagnosing tumors, tumor cells and metastasis.
A diagnostic composition according to the present invention may be
formulated as a liquid, gel or solid formulation, preferably as an aqueous liquid,
containing a targeting agent according to the present invention in a concentra-
tion ranging from about 0.00001 mg/l to 25 x 107 mg/l. The compositions may
further comprise stabilizing agents, detergents, such as polysorbates and
Tween, as well as other additives. The concentrations of these components
may vary significantly depending on the formulation used. The diagnostic
compositions may be used in vivo or in vitro.
The present invention also includes the use of the targeting agents
and targeting units for the manufacture of pharmaceutical compositions for the
treatment of cancer.
The present invention also relates to pharmaceutical compositions
comprising a therapeutically effective amount of at least one targeting agent
according to the present invention. The pharmaceutical compositions may be
used to treat, prevent or ameliorate cancerdiseases, by administering an
therapeutically effective dose of the pharmaceutical composisiton comprising
targeting agents or targeting units according to the present invention or therapeutically
acceptable salts, esters or other derivatives thereof. The compositions
may also include different combinations of targeting agents and targeting
units together with labelling agents, imaging agents, drugs and other additives.
A therapeutically effective amount of a targeting agent according to
the present invention may vary depending on the formulation of the pharma-
ceuticakl composition. Preferably, a composition according to the present invention
may comprise a targeting agent in a concentration varying from about
0.00001 mg/1 to 250 g/l, more preferably about 0,001 mg/\ to 50 g/l, most preferably
0,01 mg/l to 20 g/l.
A pharmaceutical composition according to the present invention is
useful for administration of a targeting agent according to the present invention.
Pharmaceutical compositions suitable for peroral use, for intravenous or
local injection, or infusion are particularly preferred. The pharmaceutical compositions
may be used in vivo or ex vivo.
The preparations may be lyophilized and reconstituted before ad-
ministration or may be stored for example as a solution, solutions, suspen-
sions, suspension-solutions etc. ready for administration or in any form or
shape in general, including powders, concentrates, frozen liquids, and any
other types. They may also consist of separate entities to be mixed and, pos-
sibly, otherwise handled and/or treated etc. before use. Liquid formulations
provide the advantage that they can be administered without reconstitution.
The pH of the solution product is in the range of about 1 to about 12, prefera-
bly close to physiological pH. The osmolality of the solution can be adjusted to
a preferred value using for example sodium chloride and/or sugars, polyols
and/or amino acids and/or similar components. The compositions may further
comprise pharmaceutically acceptable excipients and/or stabilizers, such as
albumin, sugars and various polyols, as well as any acceptable additives, or
other active ingredients such as chemotherapeutic agents.
The present invention also relates to methods for treating cancer,
especially solid tumors by administering to a patient in need of such treatment
a therapeutically efficient amount of a pharmaceutical composition according
to the present invention.
Therapeutic doses may be determined empirically by testing the
targeting agents and targeting units in available in vitro or in vivo test systems.
Examples of such tests are given in the examples. Suitable therpeutically ef-
fective dosage may then be estimated from these experiments.
For oral administration it is important that the targeting units and
targeting agent are stable and adequately absorbed from the intestinal tract.
The pharmaceutical compositions according to the present invention
may be administered systemically, non-systemically, locally or topically, par-
enterally as well as non-parenterally, e.g. subcutaneously, intravenously, in-
tramuscularly, perorally, intranasally, by pulmonary aerosol, by injection or in-
fusion into a specific organ or region, buccaily, intracranically or intraperito-
neally.
Amounts and regimens for the administration of the tumor targeting
agents according to the present invention can be determined readily by those
with ordinary skill in the clinical art of treating cancer. Generally, the dosage
will vary depending upon considerations such as: type of targeting agent em-
ployed; age; health; medical conditions being treated; kind of concurrent
treatment, if any, frequency of treatment and the nature of the effect desired;
gender; duration of the symptoms; and, counterindications, if any, and other
variables to be adjusted by the individual physician. Preferred doses for ad-
ministration to human patients targeting targeting units or agents according to
the present invention may vary from about 0.000001 ng to about 40 mg per kg
of body weight as a bolus or repeatedly, e.g., as daily doses.
The targeting units and targeting agents and pharmaceutical com-
positions of the present invention may also be used as targeting devices for
delivery of DNA or RNA or structural and functional analogues thereof, such as
phosphorothioates, or peptide nucleic acids (PNA) into tumors and their metas-
tases or to isolated cells and organs in vitro; i.e. as tools for gene therapy both
in vivo and in vitro. In such cases the targeting agents or targeting units may
be parts of viral capsids or envelopes, of liposomes or other "containers" of
DNA/RNA or related substances, or may be directly coupled to the DNA/RNA
or other molecules mentioned above.
The present invention also includes kits and components for kits for
diagnosing, detecting or analysing cancer or cancer cells in vivo and in vitro.
Such kits comprise at least a targeting agent or targeting unit of this invention
together with diagnostic entities enabling detection. The kit may comprise for
example a targeting agent and/or a targeting unit coupled to a unit for detection
by e.g. immunological methods, radiation or enzymatic methods or other
methods known in the art.
Further, the targeting units and agents of this invention as well as
the targeting motifs and sequences can be used as lead compounds to design
peptidomimetics for any of the purposes described above.
Yet further, the targeting units and agents as well as the targeting
motifs and sequences of the present invention, as such and/or as coupled to
other materials, can be used for the isolation, purification and identification of
the cells, molecules and related biological targets.
The following non-limiting examples illustrate the invention further.
EXAMPLES
A list of reagents used in the examples below and reagent suppliers
is included after the last numbered example.
EXAMPLE 1
SYNTHESIS OF TARGETING UNIT (PEPTIDE) LRS
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS (leucyl arginyl serine), was synthesized
by means of manual synthesis as described in Example 2 below,
The following reagents were employed as starting materials (in this
order):
Fmoc-Ser(tBu) Resin
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
After the last cycle of the coupling process, a small sample of the
resin (comprising the still fully protected peptide) was subjected to the treatment
described in Example 2 for Fmoc removal (steps 1-10 in that Example),
after which the sample of peptide was cleaved from the resin by three hours"
treatment with the cleavage mixture described in Example 2, and isolated as
described in the same Example.
Then, the product (LRS) was identified with the aid of its positive
mode MALDI-TOF mass spectrum, in which the M+1 ion of LRS was clearly
predominant.
MALDI-TOF data (LRS):
calculated molecular mass = 374.44
observed signals:
375.30 M+H
397.22 M+Na
EXAMPLE 2
GENERAL PROCEDURES FOR PEPTIDE SYNTHESIS: MANUAL SOLID
PHASE SYNTHESES. MASS SPECTRAL MEASUREMENTS.
All synthetic procedures were carried out in a sealable glass funnel
equipped with a sintered glass filter disc of porosity grade between 2 and 4, a
polypropene or phenolic plastic screw cap on top (for sealing), and two PTFE
key stopcocks: one beneath the filter disc (for draining) and one at sloping an-
gle on the shoulder of the screw-capped neck (for argon gas inlet).
The funnel was loaded with the appropriate solid phase synthesis
resin and solutions for each treatment, shaken powerfully with the aid of a
"wrist movement" bottle shaker (Gallenkamp) for an appropriate period of time,
followed by filtration effected with a moderate argon gas pressure.
The general procedure of one cycle of synthesis (= the addition of
one amino acid unit) was as follows:
The appropriate Wang resin (Applied Biosystems), loaded with ap-
proximately 1 mmol of Fmoc-peptide (= peptide whose amino-terminal amino
group was protected with the 9-f!uorenylmethyloxycarbonyl group) consisting
of two or more amino acid units, or with approximately 1 mmol of the appropri-
ate Fmoc-amino acid (i.e., amino acid carrying the aforementioned protecting
group; approximately 2g of resin, 0.5 mmol/g) was treated in the way described
below, each treatment step comprising shaking for 2.5 minutes with 30 ml of
the solution or solvent indicated and filtration if not mentioned otherwise.
"DCM" means shaking with dichloromethane, and "DMF means
shaking with A/,N-dimethylformamide (DMF may be replaced by NMP, i.e. N-
methylpyrrolidinone).
The steps of the treatment were:
1. DCM, shaking for 10-20 min
2. DMF
3. 20% (by volume) piperidine in DMF for 5 min
4. 20% (by volume) piperidine in DMF for 10 min
5. to 7. DMF
8. to 10. DCM
11. DMF
12. DMF solution of 3 mmol of activated amino acid (preparation de-
scribed below), shaking for 2 hours
13. to 15. DMF
16. to 18. DCM
After the last treatment (18) argon gas was led through the resin for
approximately 15 min and the resin was stored under argon (in the sealed re-
action funnel if the synthesis was to continue with further units).
Activation of the 9-fluorenylmethyloxycarbonyl-N-protected amino
acid (Fmoc-amino acid) to be added to the amino acid or peptide chain on the
resin was carried out, using the reagents listed below, in a separate vessel
prior to treatment step no. 12. Thus, the Fmoc-amino acid (3 mmol) was dis-
solved in approximately 10 ml of DMF, treated for 1 min with a solution of 3
mmol of HBTU dissolved in 6 ml of a 0.5 M solution of HOBt in DMF, and then
immediately treated with 3 ml of a 2.0 M DIPEA solution for 5 min.
The activation reagents used for activation of the Fmoc-amino acid
were as follows:
HBTU= 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate, CAS No. [94790-37-1], Applied Biosystems Cat. No. 401091, molecular
weight: 379.3 g/mol
HOBt = 1-Hydroxybenzotriazole, 0.5 M solution in DMF, Applied Biosystems
Cat. No. 400934
DIPEA = N.N-Diisopropylethylamine, 2.0 M solution in N-methylpyrrolidone,
Applied Biosystems Cat. No. 401517
The procedure described above was repeated in several cycles using
the appropriate different Fmoc-amino acids, carrying suitable protecting
group(s), to produce a resin-bound source of the appropriate peptide (i.e., a
"resin-bound" peptide). The procedure provides also a practical way of connecting
certain effector and/or spacer and/or linker units and so on, for instance biotin
or the Fmoc-Ahx (= 6-(Fmoc-amino)-hexanoyl) moiety, to the resin-bound
peptide.
Cleavage from the resin was carried out using the following reagent
mixture:
trifluoroacetic acid (TFA) 92.5 vol-%
water 5.0 vol-%
ethanedithiol 2.5 vol-%.
After the removal of the protecting Fmoc group via steps 1. to 10.
(as described in the general procedure above), the resin was treated with three
portions of the above reagent mixture (each about 15 ml for 1 g of the resin),
each for one hour. The treatments were carried out under argon atmosphere in
the way described above. The TFA solutions obtained by filtration were then
concentrated under reduced pressure using a rotary evaporator and were recharged
with argon. Some diethyl ether was added and the concentration repeated.
The concentrated residue was allowed to precipitate overnight under
argon in dietyl ether in a refrigerator. The supernatant ether was removed and
the precipitate rinsed with diethyl ether. For mass spectrum (MALDI-TOF+) determination,
a sample of the precipitate was dissolved in solvents adequate for
the spectral method, followed by filtration and, as needeed, dilution of the filtered
solution. Further purification was done using reversed phase high-
performance liquid chromatographic (HPLC) methods by means of a "Waters
600" pump apparatus using a C-18 type column of particle size 10 micrometers
and a linear eluent gradient whose composition was changed during 30
minutes from 99.9% water/0.1% TFA to 99.9%,acetonitrile/0.1% TFA. The dimensions
of the HPLC columns were 25 cm x 21.2 mm (Supelco cat. no.
567212-U) and 15 cm x 10 mm (Supelco cat. no. 567208-U). Detection was
based on absorbance at 218 nm and was carried out using a "Waters 2487"
instrument.
The cleavage mixture described above also simultaneously re-
moved the following protecting groups: trityl (Trt) as used for cysteine -SH protection;
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) as used for
protection of side chain of arginine; the tert-butyl group (as an ester group on
the carboxyl function; OtBu) as used for protection of the side-chain carboxyi
group of glutamic acid and/or aspartic acid, and can normally be used also for
removal of these protecting groups on analogous structures (thiol, guanyl, car-
boxyl). It did not cause Fmoc removal.
The cleavage procedure described above can be carried out also
without the removal of the Fmoc group, to produce the amino terminal N-
Fmoc-derivative of the peptide, or for a peptide linked to an effector unit (com-
prising no Fmoc).
MASS SPECTRAL METHOD EMPLOYED
Matrix Assisted Laser Desorption lonization - Time of Flight (MALDI -TOF)
Type of the intrument:
Bruker Biflex MALDI TOF mass spectrometer
Supplier of the instrument:
, Bruker Daltonik GmbH
Fahrenheitstrasse 4
D-28359 Bremen
Germany
MALDI-TOF POSITIVE ION REFLECTOR MODE
External standards:
Angiotensin II and ACTH(18-39)
Matrix:
alpha-cyano-4-hydroxycinnamic acid (saturated solution in aqueous 50% ace-
tonitrile containing 0.1% of trifluoroacetic acid).
The sample, together with the matrix, was dried onto the target plate
under a gentle stream of warm air.
MALDI-TOF NEGATIVE ION REFLECTOR MODE:
External standards:
cholecystokinin and glucagon
Matrix:
2,4,6-trihydroxyacetophenone (3 mg/ml in 10 mM ammonium citrate in 50%
acetonitrile).
The sample, mixed with the matrix, was immediately dried onto the
target plate under vacuum.
SAMPLE PREPARATION
The specimen was mixed at a 10-100 picomol/microliter concentration
with the matrix solution as described.
"Shooting" by nitrogen laser at wawelength 337 nm. The voltage of
the probe plate was 19 kV in the positive ion reflector mode and -19 kV in the
negative ion reflector mode.
GENERAL REMARKS ABOUT THE SPECTRA (CONCERNING POSITIVE
ION MODE ONLY)
In all cases the M+1 (i.e. the one proton adduct M+H+) signal with
its typical fine structure based on isotope satellites was clearly predominant. In
almost all cases, the M+1 signal pattern was accompanied by a similar but
markedly weaker band of peaks at M+23 (Na+ adduct). In addition to the
bands at M+1 and M+23, also bands at M+39 (K+ adduct) or M+56 (Fe+ adduct)
could be observed in many cases.
In case of substances with a low molecular mass, the "matrix signals"
(signals due to the constituents of the matrix/ "the ionization environment")
have been omitted (i.e., signals at 294 and 380 Da have been omitted).
The calculated molecular mass values reported within synthesis examples
correspond to the most abundant isotopes of each element, i.e., the
"exact masses". The interpretations given for signals are only tentative.
EXAMPLE 3
GENERAL PROCEDURES FOR I2-PROMOTED CYCLIZATION OF PEPTI-
DE/TARGETING UNIT OR TARGETING AGENT ON RESIN (FOR PEPTIDES
AND TARGETING UNITS AND TARGETING AGENTS COMPRISING CYS-
TEINES)
The resin (1 g) was swelled on CH2C12 (15 ml) and stirred for 20
minutes. The solvent was removed by filtration and the resin was treated once
with DMF (15 ml) for three minutes. After filtration, the resin-bound peptide (or
targeting agent) was treated with iodine (5 molar equivalents) in DMF (10 ml)
for 1 hour.
The DMF-iodine solution was removed by filtration and the residue
was washed three times with DMF (15 ml) and three times with CH2CI2 (15ml)
for 3 minutes each time.
In case that a "plain" peptide (without the Fmoc group) was to be
prepared, the Fmoc group was removed and the peptide was released from
the resin according to the general procedure described in Example 2 and purified
by reversed phase HPLC. In the case of targeting agents comprising no
Fmoc group, the product was released from the resin and purified analogously.
Material used:
Iodine
CAS No.7553-56-2
molecular weight: 253.81
Merck Art. No. 4760
EXAMPLE 4
SYNTHESIS OF TARGETING UNIT (PEPTIDE) DLRSK
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of manual
synthesis as described in Example 2 above.
The following reagents were employed as starting materials (in this
order):
Fmoc-Lys(Mtt)-resin
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-Asp(2-phenylisopropylester)-OH
After the last cycle of the coupling process.the still resin-bound targeting
unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 17. After the cyclization process
(macrolactam formation) a small sample of the resin (containing the still fully
protected cyclized peptide) was subjected to the treatment described in Example
2 for Fmoc removal (steps 1-10 in that Example), after which the sample of
peptide was cleaved from the resin by three hours" treatment with the cleavage
mixture described in Example 2, and isolated as described in the same Example.
Then, the product (DLRSK macrolactam) was identified with the aid
of its positive mode MALDI-TOF mass spectrum, in which the M+1 ion of cyclic
DLRSK was clearly predominant.
MALDI-TOF data (cyclic DLRSK):
calculated molecular mass = 599.34
observed signals:
600.42 M+H
622.40 M+Na
638.29 M+K
Fmoc-DLRSK macrolactam
Cyclic Fmoc-DLRSK was prepared and identified in analogous
manner to cyclic DLRSK with the exeption of the final Fmoc removal that was
omitted in this case.
MALDI-TOF data (cyclic Fmoc-DLRSK):
calculated molecular mass = 821.41
observed signals:
822.60 M+H
844.62 M+Na
EXAMPLE 5
SYNTHESIS OF TARGETING UNIT (PEPTIDE) DGRGLRSK (CYCLIC BY
VIRTUE OF LACTAM BRIDGE)
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of man-
ual synthesis as described in Example 2 above.
The following reagents were employed as starting materials (in this
order):
Fmoc-Lys(Mtt) Resin
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-Gly-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-Gly-OH
Fmoc-Asp(2-phenylisopropyl ester)-OH
After the last cycle of the coupling process, the still resin-bound tareting
unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 17. After the cyclization process
(macrolactam formation) a small sample of the resin (containing the still fully
protected cyclized peptide) was subjected to the treatment described in Example
2 for Fmoc removal (steps 1-10 in that Example), after which the sample of
peptide was cleaved from the resin by three hours" treatment with the cleavage
mixture described in Example 2, and isolated as described in the same Example.
Then, the product (DGRGLRSK macrolactam) was identified with
the aid of its positive mode MALDI-TOF mass spectrum, in which the M+1 ion
of cyclic DGRGLRSK was clearly predominant.
MALDI-TOF data (cyclic DGRGLRSK):
calculated molecular mass = 869.48
observed signal:
870.48 M+H
EXAMPLE 6
SYNTHESIS OF TARGETING UNIT (PEPTIDE) DRGLRSK (CYCLIC BY VIRTUE OF LACTAM BRIDGE)
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of manual
synthesis as described in Example 2 above.
The following reagents were employed as starting materials (in this
order):
Fmoc-Lys(Mtt) Resin
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-Gly-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-Asp(2-phenylisopropyl ester)-OH
After the last cycle of the coupling process, the still resin-bound tar-
geting unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 17. After the cyclization process
(macrolactam formation) a small sample of the resin (containing the still fully
protected cyclized peptide) was subjected to the treatment described in Exam-
ple 2 for Fmoc removal (steps 1-10 in that Example), after which the sample of
peptide was cleaved from the resin by three hours" treatment with the cleavage
mixture described in Example 2, and isolated as described in the same Example.
Then, the product (DRGLRSK macrolactam) was identified with the
aid of its positive mode MALDI-TOF mass spectrum, in which the M+1 ion of
cyclic DRGLRSK was clearly predominant.
MALDI-TOF data (cyclic DRGLRSK):
calculated molecular mass = 812.46
observed signal:
813.34 M+H
EXAMPLE 7
SYNTHESIS OF TARGETING UNIT (PEPTIDE) DRYYNLRSK (CYCLIC BY
VIRTUE OF LACTAM BRIDGE)
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of manual
synthesis as described in Example 2 above. The following reagents were
employed as starting materials (in this order):
Fmoc-Lys(Mtt) Resin
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-L-Asn-OH
Fmoc-L~Tyr(tBu)-OH in two subsequent cycles
Fmoc-L-Arg(Pbf)-OH again
Fmoc-Asp(2-phenylisopropylester)-OH
After the last cycle of the coupling process, the still resin-bound tar-
geting unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 17. After the cyclization process
(macrolactam formation) a small sample of the resin (containing the still fully
protected cyclized peptide) was subjected to the treatment described in Exam-
pie 2 for Fmoc removal (steps 1 -10 in that Example), after which the sample of
peptide was cleaved from the resin by three hours" treatment with the cleavage
mixture described in Example 2, and isolated as described in the same Exam-
ple.
Then, the product (DRYYNLRSK macrolactam) was identified with
the aid of its positive mode MALDI-TOF mass spectrum, in which the M+1 ion
of cyclic DRYYNLRSK was clearly predominant.
Mass spectral data (cyclic DRYYNLRSK):
calculated molecular mass = 1195.61
observed signal:
1096.28 M+H
EXAMPLE 8
SYNTHESIS OF TARGETING UNIT (PEPTIDE) DSRYNLRSK (CYCLIC BY
VIRTUE OF LACTAM BRIDGE)
The functionally protected, resin bound. targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of man-
ual synthesis as described in Example 2 above. The following reagents were
employed as starting materials (in this order):
Fmoc-Lys(Mtt) Resin
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-L-Asn-OH
Fmoc-L-Tyr(tBu)-OH
Fmoc-L-Arg(Pbf)-OH again
Fmoc-L-Ser(tBu)-OH again
Fmoc-Asp(2-phenylisopropylester)-OH
After the last cycle of the coupling process, the still resin-bound tar-
geting unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 17. After the cyclization process
(macrolactam formation) a small sample of the resin (containing the still fully
protected cyclized peptide) was subjected to the treatment described in Exam-
ple 2 for Fmoc removal (steps 1-10 in that Example), after which the sample of
peptide was cleaved from the resin by three hours" treatment with the cleavage
mixture described in Example 2, and isolated as described in the same Exam-
ple.
Then, the product (DSRYNLRSK macroiactam) was identified with
the aid of its positive mode MALDI-TOF mass spectrum, in which the M+1 ion
of cyclic DSRYNLRSK was clearly predominant.
Mass spectal data (cyclic DSRYNLRSK):
calculated molecular mass = 1119.58
observed signal:
1120.24 M+H
EXAMPLE 9
SYNTHESIS OF TARGETING UNIT (PEPTIDE) AHXDLRSK
The targeting unit has the structure:
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of man-
ual synthesis as described in Example 2 above.
The following reagents were employed as starting materials (in this
order):
Fmoc-Lys(Mtt)-resin
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-Asp(2-pheny!isopropyiester)-OH
Fmoc-6-aminohexanoic acid
After the last cycle of the coupling process, the still resin-bound tar-
geting unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 17. After the cyclization process
(macrolactam formation) a small sample of the resin (containing the still fully
protected cyclized peptide) was subjected to three hours" treatment with the
cleavage mixture described in Example 2. By that way a sample of peptide
was cleaved from the resin and the protecting groups of side chains of that
sample were removed with the exeption of the final Fmoc removal that was
omitted in this case. The sample was isolated as described in the same Example.
Then, the product (DLRSK macrolactam) was identified with the aid
of its positive mode MALDI-TOF mass spectrum, in which the M+1 ion of cyclic
DLRSK was clearly predominant.
MALDI-TOF data (cyclic Fmoc-AhxDLRSK):
calculated molecular mass = 938.50
observed signal:
939.50 M+1
EXAMPLE 10
SYNTHESIS OF TARGETING AGENT FMOC2DAP-DLRSK (DAP= DIA-
MINOPROPIONYL), COMPRISING THE EFFECTOR UNIT DIAMINOPROPI-
ONIC ACID COUPLED (LINKED DIRECTLY, WITHOUT SPECIFIC LINKER
UNITS) VIA ITS CARBOXYL GROUP TO THE N-TERMINAL AMINO GROUP
OF THE PEPTIDE DLRSK BY VIRTUE OF AN AMIDE BOND, AND ALSO
COMPRISING THE TARGETING UNIT DLRSK, THAT IS CYCLIC BY VIRTUE
LACTAM BRIDGE
The targeting agent was synthesized using manual synthesis as de-
scribed in Example 2 above (analogously to the synthesis in Example Example
4 above, including cyclization). Next, the sequence DLRSK was continued with
DL-2,3-Bis(Fmoc-amino)propionic acid by means of the general coupling met-
hods described in Example 2.
The preparation of DL-2,3-Bis(Fmoc-amino)propionic acid:
DL-2,3-diaminopropionic acid monohydrochloride was dissolved in
15 mL of aqueous 10% Na2CO3 solution. Then 7 mL of dioxane was added
and the reaction mixture cooled to +4°C. Fmoc-chloride in 20 mL of dioxane
was added and the reaction mixture stirred for one hour at +4°C. After contin-
ued stirring at room temperature overnight the reaction mixture was extracted
with ethyl acetate that was then evaporated. The residue was triturated with n-
hexane and washed with small amount of hot ethyl acetate to afford white solid
that was dried in vacuo overnight.
Reagents used:
DL-2,3-diaminopropionic acid monohydrochloride
Fmoc-chloride; 9-fluorenylmethyl chloroformate 98%; C.A.S. no: 28920-43-6
Acros, cat no.: 170940250
MALDI-TOF data (Fmoc2Dap-DLRSK, cyclic):
calculated molecular mass = 1129.53
observed signal:
1130.32 M+H
EXAMPLE 11
GENERAL PROCEDURE EMPLOYED IN THE SYNTHESES OF BIOTI-
NYLATED COMPOUNDS [TARGETING AGENTS COMPRISING ONE D-
BIOTIN (VITAMIN H) AS AN EFFECTOR UNIT]
The appropriate protected peptide was synthesized on using solid-
phase synthesis according to the general procedure described in Example 2.
The peptide was not deprotected and also not removed from the resin. The re-
sin-bound peptide was added to the reaction flask. The resin was swelled us-
ing CH2CI2 (15 ml) and stirred for 20 minutes. The solvent was removed by fil-
tration and the resin was treated once with DMF for three minutes. The peptide
was deprotected using 20% piperidine solution in DMF (20ml) and shaking
therewith for 5, and the process was repeated using (now shaking for 10 min-
utes). The resin was washed three times with DMF (15 ml) and three times
with CH2CI2 (15ml) and once with DMF (15 ml) for three minutes each time.
D-biotin (3 molar equivalents) in DMF (10 ml) (heterogenous sus-
pension) was treated in a separate vessel with a 0.5 M solution of
HBTU/HOBT in DMF (3 molar eq.) for one minute. Into the vessel was added a
2 M solution of di-isopropylethylamine in NMP (6 molar eq.). After the addition,
the reaction mixture became homogenous. The mixture was added to the re-
action apparatus and the apparatus was shaken for 2 hours.
The reaction mixture was then filtered and the residue was washed
three times with DMF (15 ml) and three times with CH2CI2 (15ml) for 3 min-
utes each time.
In case that the peptide was to be both biotinylated as described
herein and cyclized by an iodine treatment as described in Example 3, the cy-
clization was performed after the biotinylation procedure.
Material used:
D-Biotin (Vitamin H)
CAS No. 58-85-5
molecular weight: 244.3
Sigma B-4501
99%
EXAMPLE 12
SYNTHESIS OF TARGETING AGENT BIO-LRS (BIO = D-BIOTIN = VITAMIN
H), COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED (LINKED DI-
RECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CARBOXYL GROUP
TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE LRS BY VIRTUE
OF AN AMIDE BOND, AND ALSO COMPRISING THE TARGETING UNIT
LRS
The targeting agent was synthesized using manual synthesis as de-
scribed in Example 2 above (analogously to the synthesis in Example Example
1 above) and using the biotinylation procedure described in Example 11 above
as the final coupling step. In this final coupling process, D-biotin was employed
instead of a protected amino acid. D-biotin was not protected but was em-
ployed as such. The product was isolated and purified in the manner indicated
in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1
ion clearly predominant).
MALDI-TOF data (Bio-LRS):
calculated molecular mass = 600.31
observed signals:
601.34 M+H
623.23 M+Na
639.25 M+K
EXAMPLE 13
SYNTHESIS OF TARGETING AGENT BIO-DLRSK (BIO = D-BIOTIN = VITA-
MIN H), COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED (LINKED
DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CARBOXYL
GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE DLRSK
BY VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING THE TARGET-
ING UNIT DLRSK, THAT IS CYCLIC BY VIRTUE AN AMIDE BOND BE-
TWEEN THE SIDE CHAINS OF D AND K
The targeting agent was synthesized using manual synthesis as de-
scribed in Example 2 above (analogously to the synthesis in Example Example
4 above, including cyclization) and using the biotinylation procedure described
in Example 11 above as the final coupling step. In this final coupling process,
D-biotin was employed instead of a protected amino acid. D-biotin was not pro-
tected but was employed as such. The product was isolated and purified in the
manner indicated in Example 2 and identified by positive-mode MALDI-TOF
spectroscopy (M+1 ion clearly predominant).
MALDI-TOF data (Bio-DLRSK cyclic):
calculated molecular mass = 825.42
observed signals:
826.49 M+H
848.35 M+Na
EXAMPLE 14
SYNTHESIS OF TARGETING AGENT BIO-DGRGLRSK (BIO = D-BIOTIN =
VITAMIN H), COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED
(LINKED DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CAR-
BOXYL GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE
DGRGLRSK BY VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING
THE TARGETING UNIT DGRGLRSK, THAT IS CYCLIC BY VIRTUE AN AM-
IDE BOND BETWEEN THE SIDE CHAINS OF ASPARTIC ACID AND LYSINE
The targeting agent was synthesized using manual synthesis as described
in Example 2 above (analogously to the synthesis in Example 5 above,
including cyclization) and using the biotinylation procedure described in Example
11 above as the final coupling step. In this final coupling process, D-biotin
was employed instead of a protected amino acid. D-biotin was not protected
but was employed as such. The product was isolated and purified in the man-
ner indicated in Example 2 and identified by positive-mode MALDI-TOF spec-
troscopy (M+1 ion clearly predominant).
MALDI-TOF data (Bio-DGRGLRSK cyclic):
calculated molecular mass = 1095.56
observed signal:
1096.51 M+H
EXAMPLE 15
SYNTHESIS OF TARGETING AGENT BIO-DRGLRSK (BIO = D-BIOTIN = VI-
TAMIN H), COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED
(LINKED DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CAR-
BOXYL GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE
DRGLRSK BY VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING THE
TARGETING UNIT DRGLRSK, THAT IS CYCLIC BY VIRTUE AN AMIDE
BOND BETWEEN THE SIDE CHAINS OF ASPARTIC ACID AND LYSINE
The targeting agent was synthesized using manual synthesis as de-
scribed in Example 2 above (analogously to the synthesis in Example 6 above,
including cyclization) and using the biotinylation procedure described in Exam-
ple 11 above as the final coupling step. In this final coupling process, D-biotin
was employed instead of a protected amino acid. D-biotin was not protected
but was employed as such. The product was isolated and purified in the man-
ner indicated in Example 2 and identified by positive-mode MALDI-TOF spec-
troscopy (M+1 ion clearly predominant).
MALDI-TOF data (Bio-DRGLRSK, cyclic):
calculated molecular mass = 1038.56
observed signal:
1039.59 M+H
EXAMPLE 16
SYNTHESIS OF TARGETING AGENT BIO-DRYYNLRSK (BIO = D-BIOTIN =
VITAMIN H), COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED
(LINKED DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CAR-
BOXYL GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE
DLRSK BY VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING THE
TARGETING UNIT DRYYNLRSK, THAT IS CYCLIC BY VIRTUE OF AN AM-
IDE BOND BETWEEN THE SIDE CHAINS OF ASPARTIC ACID AND LYSINE
The targeting agent was synthesized using manual synthesis as described
in Example 2 above (analogously to the synthesis in Example 7 above,
including cyclization) and using the biotinylation procedure described in Example
11 above as the final coupling step. In this final coupling process, D-biotin
was employed instead of a protected amino acid. D-biotin was not protected
but was employed as such. The product was isolated and purified in the man-
ner indicated in Example 2 and identified by positive-mode MALDI-TOF spec-
troscopy (M+1 ion clearly predominant).
Mass spectral data (Bio-DRYYNLRSK cyclic):
calculated molecular mass = 1421.69
observed signals:
1422.34 M+H
EXAMPLE 17
GENERAL METHOD FOR THE CYCLIZATION OF A PEPTIDE AND/OR
TARGETING UNIT AND/OR TARGETING AGENT AND/OR TARGETING
MOTIF AND/OR TARGETING MOTIF, AND/OR PART THEREOF, IN THE
FORM OF A LACTAM (AS MACROLACTAM; BY VIRTUE OF A PEPTIDE
BOND BETWEEN LYSINE AND ASPARTIC ACID THOSE WERE INCLUDED
IN THE SEQUENCE AT THE ENDS OF AN "INTERMEDIARY" SEQUENCE)
The uncyclized, fully protected, resin-bound peptides were prepared
manually by means of the general method described in Example 2 above.
Prior to the cyclization, a selective, one-process, dismantling of the
side-chain protecting groups of lysine and aspartic acid [the said groups were:
4-methyltrityl on the iysine unit and 2-phenylisopropyl (ester) on the aspartic
acid unit] was carried out with diluted TFA (4 % in dichloromethane). The cycli-
zation involved a condensation between the side-chain carboxyl group of the
aspartic acid unit and the 6-amino group (side-chain amino group) of the lysine
unit. Activation was by a PyAOP/HOAt/DIPEA reagent mixture (for details and
abbreviation explanation, see below) or, alternatively, by the
HBTU/HOBt/DIPEA mixture described in Example 2. The equipment, common
solvents, and practical techniques were similar to those described in Example
2.
The initially fully protected resin-bound peptide (0.3 mmol) was
shaken under argon atmosphere at room temperature with different solutions
(about 10 mL) for the periods of time indicated below, followed by filtration:
1. dichloromethane, for 20 min.
2. 4 % (by volume) trifuoroacetic acid in dichloromethane, for 15 min.
3.0.2 M DIPEA in 1:10 mixture of NMP and dichloromethane, for 3 min.
4. dichloromethane, for 3 min.
5. dichloromethane, for 3 min.
6. dichloromethane, for 3 min.
7. DMF,for 3 min.
8. activation, for 4 hours, according to the description below:
A mixture of PyAOP and HOAt, or alternatively a mixture of HBTU
and HOBt, 3 molecufar equivalents of both components with respect to the
resin-bound peptide (thus, 0.9 mmol both) in DMF (7 mL), was shaken with the
resin for 1 min without filtration, followed by addition of 6 molecular equivalents
of 2 M DIPEA in NMP.
After step 8 above, the procedures continued as described in Ex-
ample 2, starting from step 13.
The reagents for activation in this type of cyclization were:
PyAOP = 7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluoro-
phosphate
CAS No. 156311-83-0
PE Biosystems Cat. No. GEN076531
Molecular Weight: 521.4 g/mol
HOAt - 1-Hydroxy-7-azabenzotriazole
0.5 M solution in DMF
Applied Biosystems Cat. No. 4330631
DIPEA = N,N-Diisopropylethylamine
2.0 M solution in N-methyIpyrrolidinone
Applied Biosystems Cat. No. 401517
For materials in the "HBTU and HOBt1 alternative, see the materials
indicated in Example 2.
Starting materials for the "special" amino acid units (aspartic acid
and lysine), between which the "extra" peptide bond was formed:
Fmoc-Lys(Mtt) Resin
0.68 mmol/g
Bachem Cat. No. D-2565.0005
Fmoc-Asp(2-phenylisopropylester)-OH
Molecular weight: 473.53 g/mol
Bachem Cat. No. B-2475.0005
EXAMPLE 18
SYNTHESIS OF TARGETING AGENT BIO-DSRYNLRSK (BIO - D-BIOTIN =
VITAMIN H), COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED
(LINKED DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CAR-
BOXYL GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE
DLRSK BY VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING THE
TARGETING UNIT DSRYNLRSK, THAT IS CYCLIC BY VIRTUE OF AN AM-
IDE BOND BETWEEN THE SIDE CHAINS OF ASPARAGINE AND LYSINE
The targeting agent was synthesized using manual synthesis as de-
scribed in Example 2 above (analogously to the synthesis in Example 8 above,
including cyclization) and using the biotinylation procedure described in Exam-
ple 11 above as the final coupling step. In this final coupling process, D-biotin
was employed instead of a protected amino acid. D-biotin was not protected
but was employed as such. The product was isolated and purified in the man-
ner indicated in Example 2 and identified by positive-mode MALDI-TOF spec-
troscopy (M+1 ion clearly predominant).
Mass spectral data (Bio-DSRYNLRSK cyclic):
calculated molecular mass = 1345.66
observed signals:
1346.32 M+H
EXAMPLE 19
SYNTHESIS OF TARGETING AGENT BIO-AHXDLRSK (BIO = D-BIOTIN =
VITAMIN H), COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED
(LINKED DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CAR-
BOXYL GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE
AHXDLRSK BY VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING
THE TARGETING UNIT AHXDLRSK, THAT IS CYCLIC BY VIRTUE AN AMIDE
BOND BETWEEN THE SIDE CHAINS OF D AND K
The targeting agent was synthesized using manual synthesis as described
in Example 2 above (analogously to the synthesis in Example 9 above,
including cyclization) and using the biotinylation procedure described in Exam-
ple 11 above as the final coupling step. In this final coupling process, D-biotin
was employed instead of a protected amino acid. D-biotin was not protected
but was employed as such. The product was isolated and purified in the man-
ner indicated in Example 2 and identified by positive-mode MALDI-TOF spec-
troscopy (M+1 ion clearly predominant).
MALDI-TOF data (Bio-AhxDLRSK cyclic):
calculated molecular mass = 938.50
observed signal:
939.50 M+H
EXAMPLE 20
SYNTHESIS OF TARGETING AGENT BIO-K-AHXDLRSK (CYCLIC BY VIR-
TUE OF AN AMIDE BOND BETWEEN ASPARTIC ACID AND TERMINAL
LYSINE; BIO = D-BIOTIN = VITAMIN H), COMPRISING ONE EFFECTOR
UNIT D-BIOTIN COUPLED (LINKED VIA ONE PLUS ONE LINKER UNITS
AND/OR SPACER UNITS AND/OR AS ONE LARGER SPACER AND/OR
LINKER UNIT) VIA ITS CARBOXYL GROUP TO THE N-TERMINAL AMINO
GROUP OF THE LYSINE RESIDUE (UNIT) AND THIS IN TURN BY VIRTUE
OF AN AMIDE BOND TO THE AMINO GROUP OF AHX (6-
AMINOHEXANOIC ACID) AND THIS BY VIRTUE OF AN AMIDE BOND TO
THE AMINO TERMINUS OF THE PEPTIDE DLRSK, AND ALSO COMPRIS-
ING THE TARGETING UNIT DLRSK
The synthesis was carried out as follows: The fully protected resin-
bound cyclized targeting unit (peptide with spacer/linker unit) AhxDLRSK was
prepared as described in Example 9 above. Next, the sequence AhxDLRSK
was continued with one lysine unit (protected with Fmoc-group on N-terminal
amino group and with Boc-group on side branch amino group) by means of the
general coupling methods described in Example 2. The reagent used as start-
ing material:
Fmoc-L-Lys(tBoc)-OH
Finally the still resin-bound and fully protected K-AhxDLRSK was
biotinylated according to the general method described in Example 11 . Purifi-
cation by HPLC gave 30% of the theoretical as overall yield. Identification of
the product:
positive mode MALDI-TOF mass spectrum: M+1 ion clearly predominant.
MALDI-TOF data (Blo-K-AhxDLRSK, cyclic):
calculated molecular mass = 1066.60
observed signal:
1067.5 M+H
EXAMPLE 21
SYNTHESIS OF TARGETING AGENT BIO4-K3-K-AHXDLRSK (CYCLIC BY
VIRTUE OF AN AMIDE BOND BETWEEN ASPARTIC ACID AND TERMINAL
LYSINE; BIO = D-BIOTIN = VITAMIN H), COMPRISING FOUR IDENTICAL
EFFECTOR UNITS D-BIOTIN COUPLED (LINKED VIA A DENDRIMERIC
STRUCTURE THAT CAN BE CONSIDERED AS A COMBINATION OF
LINKER UNITS AND/OR SPACER UNITS AND/OR AS ONE LARGER
SPACER AND/OR LINKER UNIT) EACH VIA ITS CARBOXYL GROUP TO
ONE AMINO GROUP OF A LYSINE RESIDUE (UNIT), EITHER THE N-
TERMINAL AMINO GROUP OR THE SIDE-CHAIN AMINO GROUP, AND
THE DENDRIMERIC STRUCTURE (TWO LYSINES EACH CARRYING TWO
EFFECTOR BIOTIN UNITS, THESE LYSINES BEING COUPLED VIA THE
CARBOXYL FUNCTIONS TO ONE FURTHER LYSINE AND THIS IN TURN
BY VIRTUE OF AN AMIDE BOND TO THE N-TERMINAL AMINO GROUP OF
ONE LYSINE (HAVING THE SIDE CHAIN UNCOUPLED) THAT IS SIMI-
LARLY LINKED TO AHX (6-AMINOHEXANOIC ACID) AND THIS BY VIRTUE
OF AN AMIDE BOND TO THE AMINO TERMINUS OF THE PEPTIDE
DLRSK, AND ALSO COMPRISING THE TARGETING UNIT DLRSK
The product has the formula shown below:
and can be stated to comprise a four-fold biotinylated four-branch linker/spacer
unit on the amino terminus of K-AhxDLRSK.
The synthesis was carried out as follows: The fully protected resin-
bound, "on resin" cyclized targeting unit (peptide with two spacer/linker units) K-
AhxDLRSK was prepared as described in Example 20 above. The branched
structure comprising the four biotins and the three lysines was conctructed by
means of the general coupling methods described in Example 2 so that the
sequence K-AhxDLRSK was continued first with one lysine unit (protected with
one Fmoc-group on each of its two amino groups). Then, the procedure (lysine
addition) was repeated using doubled amounts of coupling reagents and the
doubly protected (Fmoc groups) lysine, in order to couple two more lysine
units, one of them on the side-chain amino and one on the amino-terminal
amino group of the first-coupled lysine unit. Reagent used (in addition to the
materials described in the referred Examples):
Fmoc-L-Lys(Fmoc)-OH
Biotinylation was done according to the general method described
in Example 11 using 12 molecular equivalents of coupling reagents and biotin,
employing the resin-bound branched peptide, to afford a stucture comprising
four biotin units. Purification by HPLC gave 44% of the theoretical as overall
yield.
Identification of the product:
positive mode MALDI-TOF mass spectrum: M+1 ion clearly predominant.
MALDI-TOF data (Bio4-K3-K-AhxDLRSK, cyclic):
calculated molecular mass = 2129.12
observed signal:
2129.89 M+H (the strongest isotopomer is 2130.9)
EXAMPLE 22
SYNTHESIS OF TARGETING AGENT B104-K3-K[DTPA]-AHXDLRSK (CY-
CLIC BY VIRTUE OF AN AMIDE BOND BETWEEN ASPARTIC ACID AND
TERMINAL LYSINE; BIO = D-BIOTIN ¦ VITAMIN H; DTPA = DIETHYL-
ENETRIAMINEPENTAACETIC ACID MINUS ONE OH), COMPRISING TWO
TYPES OF EFFECTOR UNITS: FOUR IDENTICAL EFFECTOR UNITS D-
BIOTIN COUPLED (LINKED VIA A DENDRIMERIC STRUCTURE THAT CAN
BE CONSIDERED AS A COMBINATION OF LINKER UNITS AND/OR
SPACER UNITS AND/OR AS ONE LARGER SPACER AND/OR LINKER
UNIT) EACH VIA ITS CARBOXYL GROUP TO ONE AMINO GROUP OF A
LYSINE RESIDUE (UNIT), EITHER THE N-TERMINAL AMINO GROUP OR
THE SIDE-CHAIN AMINO GROUP, AND THE DENDRIMERIC STRUCTURE
(TWO LYSINES EACH CARRYING TWO EFFECTOR BIOTIN UNITS, THESE
LYSINES BEING COUPLED VIA THE CARBOXYL FUNCTIONS TO ONE
FURTHER LYSINE AND THIS IN TURN BY VIRTUE OF AN AMIDE BOND
TO THE N-TERMINAL AMINO GROUP OF ONE LYSINE (HAVING THE SIDE
CHAIN COUPLED VIA AMIDE BOND TO DTPA) THAT IS SIMILARLY
LINKED TO AHX (6-AMINOHEXANOIC ACID) AND THIS BY VIRTUE OF AN
AMIDE BOND TO THE AMINO TERMINUS OF THE PEPTIDE DLRSK, AND
ALSO COMPRISING THE TARGETING UNIT DLRSK
The product has the formula shown below:
and can be stated to comprise a four-fold biotinylated five-branch linker/spacer
unit, carrying Dtpa-moiety on one branch, on the N-terminus of peptide
AhxDLRSK.
The synthesis was carried out as follows: The isolated and purified
targeting agent Bio4K3-K-AhxDLRSK was prepared as described in Example
21 above. The product thus obtained was then treated with 10 molecular
equivalents of diethylenetriaminepentaacetic dianhydride in DMF solution (0.01
M solution as calculated on the basis of the biotinylated peptide) for 18 hours.
After this treatment, the volume was doubled by addition of water to the DMF
solution, and the solution was put aside and allowed to stay still for 4 hours.
Finally, the solvents were evaporated in vacuo and the residue was mixed in
water containing 0.1% trifluoroacetic acid and was filtered and the filtrate was
purified by reversed-phase HPLC. The product was identified by its M+1 peak
in the MALDI-TOF mass spectrum.
Identification of the product:
positive mode MALDI-TOF mass spectrum: M+1 ion clearly predominant.
MALDI-TOF data (Bio4-K3-K[Dtpa]-AhxDLRSK, cyclic):
calculated molecular mass = 2504.24
observed signal:
2505.29 M+H
EXAMPLE 23
SYNTHESIS OF TARGETING AGENT AOA-DLRSK (AOA = AMINO-
OXYACETYL = NH2OCH2CO), COMPRISING THE EFFECTOR UNIT
AMINO-OXYACETIC ACID COUPLED (LINKED DIRECTLY, WITHOUT SPE-
CIFIC LINKER UNITS) VIA ITS CARBOXYL GROUP TO THE N-TERMINAL
AMINO GROUP OF THE PEPTIDE DLRSK BY VIRTUE OF AN AMIDE
BOND, AND ALSO COMPRISING THE TARGETING UNIT DLRSK, THAT IS
CYCLIC BY VIRTUE LACTAM BRIDGE
The targeting agent was synthesized using manual synthesis as de-
scribed in Example 2 above (analogously to the synthesis in Example Example
4 above, including cyclization). Next, the sequence DLRSK was continued with
amino-oxyacetic acid by means of the general coupling methods described in
Example 2.
Reagent used:
Boc-amino-oxyacetic acid; Boc-NH-OCH2-COOH
MALDI-TOF data (Aoa-DLRSK, cyclic):
calculated molecular mass = 674.37
observed signals:
673.54 M+H
EXAMPLE 24
SYNTHESIS OF TARGETING AGENT CBP-DLRSK [CBP= 5-(1-O-
CARBORANYU-PENTANOYL], COMPRISING THE EFFECTOR UNIT 5-(1-O-
CARBORANYL)-PENTANOIC ACID COUPLED (LINKED DIRECTLY, WITH-
OUT SPECIFIC LINKER UNITS) VIA ITS CARBOXYL GROUP TO THE N-
TERMINAL AMINO GROUP OF THE PEPTIDE DLRSK BY VIRTUE OF AN
AMIDE BOND, AND ALSO COMPRISING THE TARGETING UNIT DLRSK,
THAT IS CYCLIC BY VIRTUE LACTAM BRIDGE
The targeting agent was synthesized using manual synthesis as de-
scribed in Example 2 above (analogously to the synthesis in Example Example
4 above, including cyclization). Next, the sequence DLRSK was continued with
5-(1-o-carboranyl)-pentanoic acid by means of the general coupling technique
described in Example 2 with the exeption of PyAoP (instead of HBTU) and
HOAT (instead of HOBt) and reaction time 4 hours in the treatment step 12 of
Example 2.
Reagent used:
5-(1-o-carboranyl)-pentanoic acid, Katchem, Prague, Czech Republic,
F.W.244.34 g/mol
MALDI-TOF data (Cbp-DLRSK, cyclic):
calculated molecular mass = 817.60 (basis B10, abund. 20%), 827.57 (basis
B11 abund 80%)
average molecular weight = 826.01 g/mol
observed signals:
Multiplet with highest peaks at 826.55 and 827.55 : M+H
Multiplet with highest peaks at 848.45 and 849.50 : M+Na
EXAMPLE 25
SYNTHESIS OF TARGETING AGENT AMF-DLRSK [AMF= 4-AMINO-10-
METHYLFOLIC ACYL], COMPRISING THE EFFECTOR UNIT 4-AM1NO-10-
METHYLFOLIC ACID COUPLED (LINKED DIRECTLY, WITHOUT SPECIFIC
LINKER UNITS) VIA ITS CARBOXYL GROUP TO THE N-TERMINAL AMINO
GROUP OF THE PEPT1DE DLRSK BY VIRTUE OF AN AMIDE BOND, AND
ALSO COMPRISING THE TARGETING UNIT DLRSK, THAT IS CYCLIC BY
VIRTUE OF LACTAM BRIDGE
The targeting agent was synthesized using manual synthesis as described
in Example 2 above (analogously to the synthesis in Example 4 above,
including cyclization). Next, the sequence DLRSK was continued with 4-amino-
10-methylfolic acid by means of the general coupling technique described in
Example 2 with the exceptions of PyAOP (instead of HBTU) and HOAT (instead
of HOBt) and reaction time 5 hours and equimolar ratio of reagents to
resin-bound peptide (peptide/PyAOP/HOAT/DIPEA = 1:1:1:2) in the treatment
step 12 of Example 2.
Reagent used:
4-amino-10-methylfolic acid hydrate; (+)amethopterin; methotrexate
CAS No. 59-05-2
Formula weight: 454.4 g/mol
Sigma Cat. No. A-6770
MALD1-TOF data (Amf-DLRSK, cyclic):
calculated molecular mass = 1035.50
observed signals:
1036.35 M+H
EXAMPLE 26
SYNTHESIS OF TARGETING AGENT DNM-AOA-DLRSK (DNM= DAUNO-
MYCINE, AOA = AMINO-OXYACETYL = NH2OCH2CO), COMPRISING THE
EFFECTOR UNIT DAUNOMYCINE COUPLED VIA ITS CARBONYL GROUP
BY OXIME LIGATION TO THE AMINO-OXY GROUP OF AOA-DLRSK [A
TARGETING AGENT (DERIVATIVE OF PEPTIDE) COMPRISING THE
LINKER (LIGATION) UNIT AMINO-OXYACETIC ACID COUPLED (LINKED
DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CARBOXYL
GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE DLRSK
BY VIRTUE OF AN AMIDE BOND], AND ALSO COMPRISING THE TARGET-
ING UNIT DLRSK, THAT IS CYCLIC BY VIRTUE LACTAM BRIDGE
The targeting agent was synthesized by stirring daunomycine with
equimolar amount of Aoa-DLRSK described above in Example 23 in methanol
solution (concentration 0.0025M) at room temperature in dark for three days.
The product was isolated by evaporation of solvents and purified by HPLC.
Reagent used:
Daunomycin hydrochloride
CAS No. 20830-81-3
Molecular weight: 564.0 g/mol
ICN Biomedicals, Aurora, Ohio, USA
Cat. No. 44583
MALDI-TOF data (Dnm-Aoa-DLRSK, cyclic):
calculated molecular mass = 1181.52
observed signal:
1182.41 M+H
EXAMPLE 27
SYNTHESIS OF TARGETING AGENT DXRB-AOA-DLRSK (DXRB=
DOXORUBICINE, AOA = AMINO-OXYACETYL = NH2OCH2CO), COMPRISING
THE EFFECTOR UNIT DOXORUBICINE COUPLED VIA ITS CARBONYL
GROUP BY OXIME LIGATION TO THE AMINO-OXY GROUP OF AOA-
DLRSK [A TARGETING AGENT (DERIVATIVE OF PEPTIDE) COMPRISING
THE LINKER (LIGATION) UNIT AMINO-OXYACETIC ACID COUPLED
(LINKED DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CAR-
BOXYL GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE
DLRSK BY VIRTUE OF AN AMIDE BOND], AND ALSO COMPRISING THE
TARGETING UNIT DLRSK, THAT IS CYCLIC BY VIRTUE LACTAM BRIDGE
The targeting agent was synthesized by stirring doxorubicine with
equimolar amount of Aoa-DLRSK described above in Example 23 in methanol
solution (concentration 0.0025M) at room temperature in dark for three days.
The product was isolated by evaporation of solvents and purified by HPLC.
Reagent used:
Doxorubicin hydrochloride
CAS No. 25316-40-9
Molecular weight: 580.0 g/mol
Fluka Cat. No. 44583
MALDI-TOF data (Dxrb-Aoa-DLRSK, cyclic):
calculated molecular mass = 1197.52
observed signals:
1198.17 M+H
1198.17 M+H
EXAMPLE 28
SYNTHESIS OF TARGETING UNIT (PEPTIDE) DLRSGRK THAT IS CYCLIC
BY VIRTUE OF LACTAM BRIDGE
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of man-
ual synthesis as described in Example 2 above.
The following reagents were employed as starting materials (in this
order):
Fmoc-Lys(Mtt)-resin
Fmoc-L-Arg(Pbf)-OH
Fmoc-Gly-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-Asp(2-phenylisopropylester)-OH
After the last cycle of the coupling process.the still resin-bound targeting
unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 17. After the cyclization process
(macrolactam formation) a small sample of the resin (containing the still fully
protected cyclized peptide and being suitable starting material for further synthesis
e.g. biotinylation) was subjected to the treatment described in Example
2 for Fmoc removal (steps 1-10 in that Example), after which the sample of
peptide was cleaved from the resin by three hours" treatment with the cleavage
mixture described in Example 2, and isolated as described in the same Example.
Then, the product (cyclic DLRSGRK) was identified with the aid of
its positive mode MALDI-TOF mass spectrum, in which the M+1 ion of cyclic
DLRSK was clearly predominant.
MALDI-TOF data (cyclic DLRSGRK):
calculated molecular mass = 812.46
observed signal:
813.69 M+H
EXAMPLE 29
SYNTHESIS OF TARGETING AGENT BIO-DLRSGRK (BIO = D-BIOTIN = VITAMIN H),
COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED
(LINKED DIRECTLY, WITHOUT SPECIFIC LINKER UNITS) VIA ITS CAR-
BOXYL GROUP TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE
DLRSK BY VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING THE
TARGETING UNIT DLRSGRK, THAT IS CYCLIC BY VIRTUE OF AN AMIDE
BOND BETWEEN THE SIDE CHAINS OF ASPARAGINE AND LYSINE
The targeting agent was synthesized using manual synthesis as described
in Example 2 above (analogously to the synthesis in Example 28
above, including cyclization) and using the biotinylation procedure described in
Example 11 above as the final coupling step. In this final coupling process, D-
biotin was employed instead of a protected amino acid. D-biotin was not protected
but was employed as such. The product was isolated and purified in the
manner indicated in Example 2 and identified by positive-mode MALDI-TOF
spectroscopy (M+1 ion clearly predominant).
MALDI-TOF data (Bio-DLRSGRK cyclic):
calculated molecular mass = 1038.54
observed signals:
1039.74 M+H
1061.76 M+Na
1077.60 M+K
EXAMPLE 30
SYNTHESIS OF TARGETING UNIT (PEPTIDE) DLRSGRGK. SYNTHESIS
OF TARGETING AGENT BIO-DLRSGRGK (BIO = D-BIOTIN = VITAMIN H),0
COMPRISING THE EFFECTOR UNIT D-BIOTIN COUPLED (LINKED DIRECTLY,
WITHOUT SPECIFIC LINKER UNITS) VIA ITS CARBOXYL GROUP
TO THE N-TERMINAL AMINO GROUP OF THE PEPTIDE DLRSGRGK BY
VIRTUE OF AN AMIDE BOND, AND ALSO COMPRISING THE TARGETING
UNIT DLRSGRGK. CYCLIZATION OF THE TARGETING AGENT BIO-
DLRSGRGK IN THE FORM OF A LACTAM (AS MACROLACTAM)
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRSGRG, was synthesized by means of
manual synthesis as described in Example 2 above.
The following reagents were employed as starting materials (in this
order):
Fmoc-Lys(Mtt) Resin
Fmoc-Gly-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-Gly-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-Asp(2-phenylisopropylester)-OH
The targeting agent was synthesized using the biotinylation proce-
dure described in Example 11 above as the final coupling step. In this final
coupling process, D-biotin was employed instead of a protected amino acid. D-
biotin was not protected but was employed as such. The biotinylated, still resin-bound
targeting agent was subjected to the cyclization process in which an
extra amide bond is formed as described in Example 17. The product was isolated
and purified in the manner indicated in Example 2 and identified by positive-mode
MALDI-TOF spectroscopy (M+1 ion clearly predominant).
MALDI-TOF data (Bio-DLRSGRGK cyclic):
calculated molecular mass = 1065.56
observed signal:
1096.55 M+H
EXAMPLE 31
IN VIVO TARGETING OF TUMORS IN MICE
In this example in vivo targeting of the targeting units prepared in
the previous examples is shown for three different types of primary tumors, fi-
brosarcoma, melanoma and for melanoma metastases in lung. It is shown that
the tested targeting units according to the present invention selectively target
to primary tumors and to metastases in vivo but not to normal tissues or organs.
CELL LINES AND TUMOR-BEARING MICE
The following tumor cell lines were used to produce experimental
tumors in mice:
"ODC sarcoma cells", (OS), originally derived from tumors that were
formed in nude mice to which had been administered NIH3T3 mouse fibro-
blasts transformed by virtue of ornithine decarboxylase (ODC) overexpression
and have been described earlier (Auvinen et al., 1997);
A human melanoma cell line C8161 (M) described by Welch et al.
(1991).
The cell lines were cultured in Dulbecco"s Modified Eagle"s Medium
(DMEM; Bio-Whittaker) supplemented with 5-10% fetal calf serum (FCS; Bio-
Whittaker), 1% L-glutamine (Bio-Whittaker) and 1% penicillin/streptomycin
(Bio-Whittaker).
EXPERIMENTAL TUMOR PRODUCTION
For production of experimental tumors, the cells listed above (OS,
KS and melanoma: 0.5 x 106 cells were injected subcutaneously into both
flanks of nude mice of the strains Balb/c Ola Hsd-nude, NMRI/nu/nu or
Athymic-nu (all mice of both strains were from Harlan Laboratories). Tumors
were harvested when they had reached a weight of about 0.4 g.
Metastases (mostly formed in the lungs) were produced by injection
of melanoma cells i.v. into Balb/c Ola Hsd-nude mice. The mice were kept 4-6
weeks, and then targeting experiments were performed.
Tumor-bearing or metastase-bearing mice were anesthesized by
administering 0.02 ml/g body weight Avertin [10 g 2,2,2-tribromoethanol
(Fluka) in 10 ml 2-methyl-2-butanol (Sigma Aldrich)] intraperitoneally (i.p.).
IN VIVO TARGETING AND DETECTION OF TARGETING
For localization of the targeting peptides OS and melanoma tumor-
bearing NMRI nude mice were anesthesized and 1 or 2 mg of biotinylated syn-
thetic targeting peptide (prepared in Example 12) was injected i.v. 5-10 min af-
ter the i.v. injections, the mice were perfused via the heart using a winged infu-
sion 25G needle set (Terumo) with 50 ml DMEM. Then, their organs were dis-
sected and frozen in liquid nitrogen. Tumors and control organs (liver, kidney,
spleen, heart, brain) were dissected and frozen in liquid nitrogen.
Biotinylated peptides (targeting agents) were detected on 10 mi-
crometer cryosections using AB (avidin-biotin) -complex containing avidin, and
biotinylated HRP (Vectastain ABC-kit, cat no. PK6100; Vector Laboratories)
with diaminobenzidine (DAB substrate kit, cat no. 4100, Vector Laboratories).
The results of the experiments are shown in Table 2
EXAMPLE 32
IN VIVO EFFECT OF TARGETING AGENT COMPRISING CYTOTOXIC EFFECTOR
UNIT
In this experiment the targeting agent, Dxrb-Aoa-DLRSK prepared
in Example 24, comprising a cytotoxic effector unit, doxorubicin, linked by
oxime ligation to the cyclic targeting unit DLRSK comprising the targeting motif
LRS was used to demonstrate in vivo targeting and therapeutic effect on
melanoma tumors.
1 million C8161M/T1 melanoma cells were injected subcutaneously
into flank of eight Athymic-nu mice and tumours were allowed to grow for one
week. The mice were then divided into three groups those received the follow-
ing treatments:
- Group DMEM: two mice, DMEM only
- Group Dox: two mice, 1,43 mg/kg doxorubicin dissolved in DMEM
- Group pept + dox: four mice, 1,43 mg/kg Dxrb-Aoa-DLRSK (doxorubicin
linked to targeting mofif LRS) dissolved in DMEM (dose equimolar to Group
Dox)
Treatments were administered i.v. twice a week (Tuesdays and Fridays),
total of five doses were injected. Tumours were measured with a calliper
in two perpendicular directions on each injection day and on the day the
animals were sacrificed. Tumour volume was calculated by the formula for ellipsoid:
Volume=(length x width2) x 0.5
The result of the experiment is shown in Figure 1 and confirms that
a targeting agent according to the present invention selectively targets to
melanoma tumor in vivo and significantly increases the therapeutic effect of
doxorubicin.
Reagent used:
Doxorubicin hydrochloride, CAS No. 25316-40-9, Molecular weight: 580.0
g/mol, Fluka Cat. No. 44583
EXAMPLE 33
GENERAL METHOD FOR THE CYCLIZATION OF A PEPTIDE OR RELATED
SUBSTANCE BY VIRTUE OF AN AMIDE BOND BETWEEN D-ORNITHINE
AND GLUTAMIC ACID THOSE ARE INCLUDED IN THE SEQUENCE AT THE
ENDS OF AN "INTERMEDIARY" SEQUENCE: FORMATION OF "HEAD-TO-
SIDE-CHAIN MACROLACTAM", /E,"GLU(D-ORN)-RING"
The uncyclized, fully protected, resin-bound peptides are prepared
manually by means of the general method described above.
Prior to the cyclization, a selective, one-process, dismantling of particular
protecting groups of ornithine and gutamic acid [the said groups are: 2-
N-Fmoc on the ornithine unit and 5-(2-trimethylsilylethyl ester) on the glutamic
acid unit] is carried out with tetrabutylammonium fluoride solution in DMF. The
cyclization involves a condensation between the side-chain carboxyl group of
the glutamic acid unit and the 2-amino group (N-terminal amino group) of the
ornithine unit. Activation is by a PyAOP/DIPEA reagent mixture (for details and
abbreviation explanation, see below) instead of the HBTU/HOBt/DIPEA mix-
ture described in Example 2. The equipment, common solvents, and practical
techniques are similar to those described in Example 2.
This method can be modified for lysine (instead of ornithine) and
aspartic (instead of glutamic) acid unitst by empoying respective derivatives of
those amino acids.
The initially fully protected resin-bound peptide (0.3 mmol) is shaken
under argon atmosphere at room temperature with different solutions (about
10 mL) for the periods of time indicated below, followed by filtration:
1. Dichloromethane, for 20 min.
2. 1 M tetrabutylammonium fluoride in DMF, for 20 min.
3.-5. DMF, for 1 min (three treatments).
6.-8. DCM, for 1 min (three treatments).
9. DMF, for 1 min.
10. 0.9 mmol of PyAOP (3 molecular equivalents with respect to the
resin-bound peptide) in DMF (7 mL), is shaken with the resin for 1
min without filtration.
11. Addition of 6 molecular equivalents of 2 M DIPEA (thus, 1.8 mmol)
in NMP, followed by shaking for 4 hours.
After the steps above , the resin is washed etc. as described in the
general procedure for (manual) peptide synthesis (the steps after addition of
activated amino acid).
The reagent for deprotection prior to cyclization is:
Tetrabutylammonium fluoride trihydrate, CAS No. 87749-50-6, molecular
weight: 315.51 g/mol, Acros Organics Cat. No. 221080500.
The reagents for activation in this type of cyclization are:
PyAOP = 7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluoro-
phosphate, CAS No. 156311-83-0, PE Biosystems Cat. No. GEN076531, Molecular
Weight: 521.4 g/mol
DIPEA = N,N-Diisopropylethylamine, 2.0 M solution in N-methylpyrrolidinone,
Applied Biosystems Cat. No. 401517
Starting materials for the "special" amino acid units (glutamic acid
and ornithine), between which the "extra1 amide bond is formed:
Fmoc-D-Om(Mtt)-OH; 2-A/-Fmoc-5-N-(4-methyltrityl)-D-ornithine, molecular
weight: 610.8 g/mol, Novabiochem Cat. No. 04-13-1012.
Fmoc-L-Glu(OTMSEt)-ONa; N-2-Fmoc-glutamic acid 5-(2-trimethylsilylethyl)
ester sodium salt, molecular weight: 468.60 g/mol, Novabiochem Cat. No. 04-
12-1231.
EXAMPLE 34
SYNTHESIS OF TARGETING UNIT (PEPTIDE) D-ORNLRSE-AMIDE, CYCLIC
BY VIRTUE OF AN AMIDE BOND BETWEEN THE SIDE CHAIN OF
GLUTAMIC ACID UNIT AND THE a-AMINO GROUP OF D-ORNITHINE
The functionally protected, resin bound targeting unit (protected
peptide), comprising targeting motif LRS, was synthesized by means of man-
ual synthesis as described in Example 2 above, in which the the "empty" resin
was deprotected prior to the first coupling in the same manner as described for
the the pre-loaded resins (steps 1-11 in Example 2).
The following reagents were employed as starting materials (in this
order):
Rink amide MBHA Resin
Fmoc-L-Glu(OTMSEt)-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-D-Orn(Mtt)-OH
After the last cycle of the coupling process, the still resin-bound targeting
unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 33. Next, a sample of peptide was
cleaved from the resin by three hours" treatment with the cleavage mixture described
in Example 2, and isolated as described in the same example.
Then, the product was identified with the aid of its positive mode
MALD1-TOF mass spectrum, in which the M+1 ion of cyclic D-OrnLRSE-amide
was clearly predominant.
MALDI-TOF data (cyclic D~OrnLRSE-/VH2 ):
calculated molecular mass = 598.36
observed signal:
599.42 M+1
EXAMPLE 35
SYNTHESIS OF TARGETING UNIT (PEPTIDE) D-ORNLRSGRGEG, CYCLIC
BY VIRTUE OF AN AMIDE BOND BETWEEN THE SIDE CHAIN OF GLU-
TAMIC ACID UNIT AND THE a-AMINO GROUP OF D-ORNITHINE
The functionally protected, resin bound targeting unit (protected
peptide), was synthesized by means of manual synthesis as described in Example
2 above.
The following reagents were employed as starting materials (in this
order):
Fmoc-Gly Resin
Fmoc-L-Glu(OTMSEt)OH
Fmoc-Gly-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-Gly-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-D-Orn(Mtt)-OH
After the last cycle of the coupling process, the still resin-bound targeting
unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 33. Next a sample of peptide was
cleaved from the resin by three hours" treatment with the cleavage mixture de-
scribed in Example 2, and isolated as described in the same example.
Then, the product was identified with the aid of its positive mode
MALDI-TOF mass spectrum by means of the M+1 ion of cyclic D-
OrnLRSGRGEG.
MALDI-TOF data (cyclic D-OrnLRSGRGEG):
calculated molecular mass = 926,50
observed signal:
927.45 M+1
EXAMPLE 36
SYNTHESIS OF TARGETING AGENT D-ORN(DOTA)LRSE-AMIDE (DOTA=
1,4,7,10-TETRAAZACYCLODODECANE-1,4,7,10-TETRAACETlC ACID
COUPLED BY ITS ONE CARBOXYL), CYCLIC BY VIRTUE OF AN AMIDE
BOND BETWEEN THE SIDE CHAIN OF GLUTAMIC ACID UNIT AND THE a-
AMINO GROUP OF D-ORNITHINE
The functionally protected, resin-bound, and cyclized targeting unit,
comprising the targeting motif LRS, was synthesized by means of manual synthesis
as described in Example 34 above. Next, the resin was treated with diluted
TFA (4 % in dichloromethane) in the manner described in example 17
(steps 1-7) to cleave the side chain protecting Mtt-group of omithine. The still
resin-bound unit was then coupled with DOTA-tris-terf-butyl ester by means of
the general method described in Example 2 (steps 12-18) using
HBTU/HOBt/DIPEA activation. Reagent used:
DOTA-tris(tBu ester).
The product was cleaved and isolated as described in Example 2
and identified with the aid of its positive mode MALDI-TOF mass spectrum, in
which the M+1 ion of cyclic D-Orn(Dota)LRSE-amide was clearly predominant.
MALDI-TOF data [cyclic D-Orn(Dota)LRSE-A/H2]:
calculated molecular mass = 984.54
observed signal:
985.52 M+1
EXAMPLE 37
SYNTHESIS OF TARGETING UNIT (PEPTIDE) KLRSD-AMIDE, CYCLIC BY
VIRTUE OF AN AMIDE BOND BETWEEN THE SIDE CHAIN OF ASPARTIC
ACID UNIT AND THE a-AMINO GROUP OF LYSINE
The functionally protected, resin bound targeting unit (protected
peptide), comprising the targeting motif LRS, was synthesized by means of
manual synthesis as described in Example 2 above, in which the the "empty"
resin was deprotected prior to the first coupling in the same manner as described
for the the pre-loaded resins (steps 1-11 in Example 2).
The following reagents were employed as starting materials (in this
order):
Rink amide MBHA Resin
Fmoc-L-Asp(OTMSEt)-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-L-Lys(Mtt)-OH
After the last cycle of the coupling process, the still resin-bound tar-
geting unit was subjected to the cyclization process in which an extra amide
bond is formed as described in Example 33 (as modification which replaces
GIu with Asp and Lys with Orn). Next, a sample of peptide was cleaved from
the resin by three hours" treatment with the cleavage mixture described in Ex-
ample 2, and isolated as described in the same example,
Then, the product was identified with the aid of its positive mode
MALDI-TOF mass spectrum by means of M+1 ion.
MALDI-TOF data (cyclic KLRSD-A/H2):
calculated molecular mass = 598.36
observed signal:
599.21 M+1
EXAMPLE 38
SYNTHESIS OF TARGETING AGENT K(DOTA)LRSD-AMIDE (DOTA=
1,4,7,10-TETRAA2ACYCLODODECANE-1,4,7,10-TETRAACETIC ACID
COUPLED BY ITS ONE CARBOXYL) , CYCLIC BY VIRTUE OF AN AMIDE
BOND BETWEEN THE SIDE CHAIN OF ASPARIC ACID UNIT AND THE a-
AMINO GROUP OF LYSINE
The functionally protected, resin-bound, and cyclized targeting unit,
comprising the targeting motif LRS, was synthesized by means of manual synthesis
as described in Example 37 above. Next, the resin was treated with diluted
TFA (4% in dichloromethane) in the manner described in example 17
(steps 1-7) to cleave the side chain protecting Mtt-group of lysine. The still
resin-bound unit was then coupled with DOTA-tris-ferf-butyl ester by means of
the general method described in Example 2 (steps 12-18) using
HBTU/HOBt/DIPEA activation.
Reagent used:
DOTA-tris(tBu ester).
The product was cleaved and isolated as described in Example 2
and identified with the aid of its positive mode MALDI-TOF mass spectrum by
means of M+1 ion.
MALDI-TOF data [cyclic K(Dota)-LRSE-A/H2 ]:
calculated molecular mass = 984.54
observed signal:
985.52 M+1
EXAMPLE 39
SYNTHESIS OF TARGETING UNIT AC-DLRSK-AHX, CYCLIC VIA SIDE
CHAINS OF ASPARTIC ACID AND LYSINE
The preparation of Ac-DLRSK-Ahx was executed by manual solid
phase peptide synthesis technique that is described in details in Example 2.
The binding of the first structural component (moiety), 6-amino-
hexanoic acid (= Ahx) whose amino function was protected by 9-fluorenyl-
methyloxycarbonyl group (= Fmoc group), to a hydroxyl-functionalized peptide
synthesis resin was carried out by means of dichlorobenzoyl chloride method
(the "equivalents" below are molecular or "mol" amounts relative to the loading
capacity of the resin):
The unloaded ("empty") resin was first washed by shaking with
N.N-dimethylformamide (= DMF) for 20 min and filtered. After addition of five
equivalents of the Fmoc-protected 6-aminohexanoic acid (Fmoc-Ahx-OH) in
DMF (0.2 M solution) and eight equivalents of pyridine onto the resin it was
shaked for 3 min. Next, five equivalents of 2,6-dichlorobenzoylchloride was
added and the mixture was shaken for 18 h (overnight).
After the lengthy treatment the resin was filtered and washed several
times with DMF and dichloromethane in the way described in Example 2
(steps 13 -18). Next, the resin was shaken for 2 hours with a mixture of acetic
anhydride (2M solution, 94 equivalents) and N,N-diisopropylethylamine
(DIPEA, 1.6M solution, 80 equivalents) in /V-methyl pyrrolidinone (NMP) solution,
filtered and washed like earlier ending up in drying at argon gas flow.
The reagents used this far were:
HMP Resin, loading capacity: 1.16 mmol/g, Applied Biosystems Cat. No.
400957.
2,6-dichlorobenzoyl chloride, CAS No. 225-102-4, molecular weight: 209.46
g/mol, Lancaster (Morecambe, England), Cat. No. 8922.
Pyridine, Merck Art. No. 9728.
Fmoc-6-aminohexanoic acid (Fmoc-Ahx-OH), CAS No. 88574-06-5, Novabio-
chem Cat. No. 04-12-1111, Molecular Weight: 353.4 g/mol.
Acetic anhydride, Fuka Cat. No. 45830.
From this on, the synthesis proceeds according to the general
method decribed in Example 2. The stuctural reagents used next in this syn-
thesis, are in sequence as follows:
Fmoc-Lys(Mtt)-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-L-Leu-OH
Fmoc-Asp(2-phenylisopropy! ester)-OH
The still resin-bound product was next cyclized according to Example
17. Finally the sequence was continued with acetic acid (I.e. end-capped at
amino terminal) as follows: Amino protecting Fmoc-group was removed as de-
scribed in Example 2 (steps 1-10). Then the still resin-bound product was
treated with the mixture of acetic anhydride and DIPEA in NMP like was done
after the initial binding of Ahx moiety to the resin. In the end the product was
released from the resin and purified as described in Example 2. Identification
was based on M+1 ion of MALDI mass spectrum.
MALDI-TOF data ( cyclic Ac-DLRSK-Ahx)
calculated molecular mass = 754.43
observed signal: 755.60
EXAMPLE 40
SYNTHESIS OF TARGETING AGENT AC-DLRSK-AHX-DOX (DOX =
DOXORUBICIN COUPLED VIA ITS AMINO GROUP) COMPRICING
DOXORUBICIN LINKED VIA AN AMIDE BOND TO THE CARBOXYL GROUP
OF C-TERMINAL SPACER MIOETY (AHX = 6-AMINOHEXANOYL) OF THE
N-CAPPED (AC = ACETYL) CYCLIC TARGETING UNIT AC-DLRSK-AHX
COMPRICING TARGETING MOTIF LRS
The "targeting unit" compound ( a peptide derivative) Ac-DLRSK-
Ahx was prepared as described in Example 39. Doxorubicine was linked to pu-
rified Ac-DLRSK-Ahx in N,A/-dimethylformamide (= DMF) solution by means of
PyAOP/DIPEA activation as follows:
Equimolar amounts of Ac-DLRSK-Ahx and PyAOP were combined
in DMF as 0.05 M solution, two molar equivalents of DIPEA (2 M solution in
NMP) was mixed in and after five minutes equimolar (in respect to Ac-DLRSK-
Ahx) amount of doxorubicin hydrochloride (0.05 M solution in DMF) was
added. After the reaction was allowed to proceed one hour at dark (protected
from light) the mixture was diluted with diethyl ether. The centrifugued solid
precipitate was purified by reverse phase HPLC chromatography and identified
; by positive mode MALDI mass spectrum as described in Example 2.
Material used:
Doxorubicin hydrochloride, CAS No. 25316-40-9, molecular weight: 580.0
g/mol, Sigma Cat. No. D-1515.
MALDI-TOF data (Ac-DLRSK-Ahx-Dox, -DLRSK- moiety cyclic):
calculated molecular mass = 1279.60
observed signal:
1280.29 M+1
EXAMPLE 41
SYNTHESIS OF TARGETING AGENT AMF-AHXDLRSK [AMF = 4-AMINO-
10-METHYLFOLIC ACYL], COMPRISING THE EFFECTOR UNIT 4-AMINO-
10-METHYLFOLIC ACID COUPLED VIA ITS CARBOXYL GROUP TO THE N-
TERMINAL AMINO GROUP OF THE PEPTIDE AHXDLRSK BY VIRTUE OF
AN AMIDE BOND, AND ALSO COMPRISING THE TARGETING UNIT
DLRSK, THAT IS CYCLIC BY VIRTUE OF LACTAM BRIDGE BETWEEN THE
SIDE CHAINS OF THE OUTERMOST MEMBERS OF THE SEQUENCE
The resin-bound, a spacer (= Ahx) comprising targeting unit
(AhxDLRSK) was prepared as described in Example 9 above, including cycli-
zation (according to Example 21). Next, the sequence AhxDLRSK was contin-
ued on resin with glutamic acid by means of the general coupling technique
described in Example 2. As final coupling the sequence (now E-AhxDLRSK,
where "E" will be a part of the "Amf moiety) was ended with "Amf minus E
acid", i.e. 4-[/V-(2,4-diamino-6-pteridinyl-methyl)-W-methylamino]-benzoic acid
hemihydrochloride dihydrate, by means of the general coupling techniques
with the exceptions of PyAOP as activation reagent (instead of HBTU and
HOBt), reaction time 5 hours, and nealy equimolar ratio of reagents to resin-
bound peptide in the treatment step 12 of Example 2.
The stoichiometric reagent ratios in that step were:
"resin-bound peptide" / "Amf minus E acid" / PyAOP / DIPEA = 1 : 1.2 : 1.2 :
2.4, (time 5 h).
After isolation and purification, according to Example 2, the product
was identified on the basis of M+1 ion in positive mode MALDI mass spectrum.
Reagents used:
Fmoc-L-Glu(OtBu)-OH, CAS No. 71989-18-9, Applied Biosystems Cat. No.
GEN911036, Molecular Weight: 425.5 g/mol.
4-[N-(2,4-diamino-6-pteridinyl-methyl)-/V-methylamino]-benzoic acid hemihy-
drochloride dihydrate, CAS No. 19741-14-1, Aldrich Cat No. 86,155-3, molecu-
lar weight 379.59 g/mol, designated as "Amf minus E acid".
MALDI-TOF data (Amf-AhxDLRSK, cyclic):
calculated molecular mass = 1148.58
observed signals:
1149.62 M+H
EXAMPLE 42
SYNTHESIS OF TARGETING AGENT PTXSUC-AHXDLRSK (PTXSUC =
PACUTAXEL MONOSUCCINATE), COMPRISING THE EFFECTOR UNIT
PACLITAXEL AS MONOSUCCINATE COUPLED VIA ITS SUCCINYL (SUC-
CINIC CARBOXYL) GROUP TO THE AMINO GROUP AT 6-AMINO-
HEXANOYL (= AHX) MOIETY OF THE PEPTIDE AHXDLRSK BY VIRTUE OF
AN AMIDE BOND (OR TARGETING AGENT WHERE EFFECTOR UNIT PA-
CLITAXEL IS LINKED VIA THE SPACER UNIT 6-(SUCCINYLAMINO)-
HEXANOYL TO THE TARGETING UNIT DLRSK), AND ALSO COMPRISING
THE TARGETING UNIT DLRSK, THAT IS CYCLIC BY VIRTUE OF AN AM-
IDE BOND BETWEEN THE SIDE CHAINS OF THE OUTERMOST MEMBERS
OF THE SEQUENCE
Paclitaxel succinate, described in the end of this example, was dis-
solved as 0.012 M solution in DMF and equimoiar amount of 0.05 M PyAOP in
DMF was added, followed by double molar amount of 2.0 M DIPEA in NMP.
After 2 minutes equimolar amount (per paclitaxel succinate) of side-chain-to-
side-chain cyclic targeting compound AhxDLRSK, described in Example 9
above, was added as 0.015 M solution in DMF. After staying overnight the mix-
ture was diluted with diethyl ether. The centrifuged solid precipitate was puri-
fied by reverse phase HPLC chromatography as described in Example2, in-
cluding the identification of the product based on its M+1 ion in the positive
mode MALDI-TOF mass spectrum.
MALDI-TOF data (PtxSuc-AhxDLRSK, cyclic):
calculated molecular mass = 1647.76
observed signal:
1648.57 M+1
Packlitaxel succinate was synthesized by following the procedure
described in the article: Chun-Ming Huang, Ying-Ta Wu and Shui-Tein Chen
(2000). Targeting delivery of paclitaxel into tumor cells via somatostatin receptor
endocytosis. Chemistry & Biology 2000, Vol 7 No 7. 453-461.
Herewith 0.05 M paclitaxel in pyridine was stirred with 12-fold excess
of succinic anhydride for 3 hours. After evaporation of the solvent in reduced
pressure, the residue was dissolved in water and freeze-dryed (lyophi-
lized).
Materials used in the synthesis of paclitaxel succinate:
Paclitaxel (from Taxus yannesis), CAS No. 33069-62-4, molecular weight:
853.9 g/mol, Sigma product No. T-1912.
Succinic anhydride, CAS No. 108-30-5, molecular weight: 100.08 g/mol, Fuka
product No. 14089.
EXAMPLE 43
SYNTHESIS OF TARGETING AGENT CPTC-AHXDLRSK [CPTC = (S)-(+)
CAMPTOTHECIN LINKED AS ESTER AT ITS HYDROXYL GROUP VIA
CARBONIC ACYL, I.E. (S)-(+)-CAMPTOTHECIN CARBONYL MOIETY],
COMPRISING THE EFFECTOR UNIT CAMPTOTHECIN CARBONATE COU-
PLED TO THE AMINO GROUP AT 6-AMINOHEXANOYL (= AHX) MOIETY
OF THE PEPTIDE AHXDLRSK BY VIRTUE OF AN AMIDE BOND (OR TAR-
GETING AGENT WHERE EFFECTOR UNIT (S)-(+)-CAMPTOTHECIN IS
LINKED VIA THE SPACER UNIT 6-(CARBONYLAMINO)-HEXANOYL TO
THE TARGETING UNIT DLRSK), AND ALSO COMPRISING THE TARGET-
ING UNIT DLRSK, THAT IS CYCLIC BY VIRTUE OF AN AMIDE BOND BE-
TWEEN THE SIDE CHAINS OF THE OUTERMOST MEMBERS OF THE SE-
QUENCE
Camptothecin p-nitrophenylcarbonate, described in the end of this
example, was dissolved as 0.02 M solution in DMF and combined with 0.04 M
solution of equimolar amount of cyclic targeting compound AhxDLRSK, de-
scribed in Example 9 above, in the same solvent. After staying overnight, 2 M
DIPEA in NMP was added in 10% excess (I.e. equimolar amount multiplied by
1.1). After being stirred overnight the mixture was diluted with diethyl ether and
the centrifuged solid precipitate was purified by reverse phase HPLC chroma-
tography as described in Example2, including the identification of the product
based on its M+1 ion in the positive mode MALDI-TOF mass spectrum.
MALDI-TOF data (Cptc-AhxDLRSK, cyclic):
Calculated molecular mass = 1086.51
Observed signal:
1087.26 M+1
The synthesis of camptothecin p-nitrophenylcarbonate: 0.29 mmol
of 4-nitrophenyl chloroformate and 0.10 mmol of (S)-(+)-camptothecin were
dissolved in 12 mL of dichloromethane (DCM). Next, 1.71 mmol of 4-
(dimethylamino)-pyridine (DMAP) was added to the DCM solution on cooling
water bath. The mixture was then shaken for two hours followed by dilution
with 30 mL of DCM. After washings: twice with 0.1% hydrochloric acid and
once with saturated aqueous sodium chloride solution, the DCM solution was
dried with disodium sulfate, filtered, and concentrated to small volume. The
product was precipitated by addition of diethyl ether and gathered after cen-
trifugation.
Materials used in the synthesis of camptothecin p-nitrophenyl-
carbonate:
4-nitrophenyl chloroformate, CAS No. 7693-46-1, molecular weight: 201.57
g/mol, Fluka product No. 23240.
(S)-(+)-camptothecin, CAS No. 7689-03-4, molecular weight: 348.36 g/mol, Al-
drich product No. 36,563-7.
DMAP; 4-(dimethyIamino)-pyridine, CAS No. 1122-58-3, molecular weight
122.17, Fluka product No. 29224.
EXAMPLE 44
SYNTHESIS OF TARGETING AGENT DOTA-AHXDLRSGRGK-AMIDE
(DOTA = 1,4,7,10-TETRAAZACYCLODODECANE-1,4,7,10-TETRAACETIC
ACID COUPLED BY ITS ONE CARBOXYL), COMPRISING POTENTIAL
METAL CHELATOR GROUP "DOTA"AS EFFECTOR LINKED VIA A SPACER
UNIT, 6-AMlNOHEXANOYL (= AHX), TO THE TARGETING UNIT
"DLRSGRGK", THAT IN TURN, IS CYCLIC VIA THE SIDE CHAINS OF AS-
PARTIC ACID MOIETY ("D") AND LYSINE ("K"). ALL THE BONDS NEEDED
FOR MENTIONED COMBINATION ARE AMIDE BONDS
The functionally protected, resin bound targeting unit (protected
peptide), comprising the targeting motif LRS, was synthesized by means of
manual synthesis as described in Example 2 above, in which the the "empty"
resin was deprotected prior to the first coupling in the same manner as de-
scribed for the the pre-loaded resins (steps 1-11 in Example 2).
The following reagents were employed as starting materials (in this
order):
Rink amide MBHA Resin
Fmoc-L-Lys(Mtt)-OH
Fmoc-Gly-OH
Fmoc-L-Arg(Pbf)-OH
Fmoc-Gly-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Arg(Pbf>OH
Fmoc-L-Leu-OH
Fmoc-L-Asp(2-Phenylisopropyl ester)-OH
After these coupling cycles the still resin-bound targeting unit was
subjected to the cyclization process in which an extra amide bond is formed as
described in Example 17. Next, still two couping cycles were carried out as de-
scribed in Example 2 above employing following starting materials:
Fmoc-6-aminohexanoic acid
DOTA-tris-(tBu ester).
Finally the the resin was cleaved and the product purified as de-
scribed in Example 2, and identified by means of its M+1 ion in positive mode
MALDI-TOF mass spectrum.
MALDI-TOF data (Dota-AhxDLRSGRGK-amide):
calculated molecular mass = 1367.76
observed signal:
1368.70 M+1
EXAMPLE 45
SYNTHESIS OF TARGETING AGENT GD-DOTA-AHXDLRSGRGK-AM1DE
(GD = CHELATED GADOLINIUM1", DOTA = 1,4,7,10-
TETRAAZACYCLODODECANE-IA^IO-TETRAACETIC ACID MINUS
THREE HYDROGENS COUPLED BY ITS ONE ACYL TO FORM AN AMIDE
BOND), COMPRISING GADOLINIUM ATOM AS DETECTABLE EFFECTOR
LINKED BY CHELATOR "DOTA"VIA A SPACER UNIT, 6-AMINOHEXANOYL
(= AHX), TO THE TARGETING UNIT "DLRSGRGK", THAT IN TURN, IS CY-
CLIC VIA THE SIDE CHAINS OF ASPARTIC ACID MOIETY ("D") AND LY-
SINE ("K")
14.9 mg of the chelator-peptide compound described in Example 44
and 24 mg of Gadolinium trichloride hydrate (five-fold excess) was dissolved in
1 mL of aqueous 0.05 M ammonium acetate. Next day the solution was sub-
jected to reverse phase HPLC purification as described in Example 2 with the
exception of 0.05 M ammonium acetate as aqueous buffer (instead of aqueous
TFA). Identification was positive mode MALDI-TOF mass spectrum at neutral
matrix (no TFA). The purified yield was 9.4 mg.
MALDI-TOF data:
Calculated molecular mass: 1522.66 based on the most abundant isotopes
Observed signal M+1:1523.66 as typical isotopic pattern
LIST OF REAGENTS
4-Amino-10 methylfolic acid; (+)amethopterin; methotrexate
hydrate; Formula weight: 454.4 g/mol, CAS No. 59-05-2, Sigma A-6770
Boc-amino-oxyacetic acid; Boc-NH-OCH2-COOH, Molecular weight: 191.2
g/mol, CAS No., Novabiochem Cat. No. 01-63-0060
Boc-Cys (Trt)-OH, CAS No: 21947-98-8, Novabiochem, cat. no 04-12-0020
D-Biotin (Vitamin H), CAS No. 58-85-5, molecular weight: 244.3, Sigma B-
4501,99%
DL~2,3-diaminopropionic acid monohydrochloride, CAS No. 54897-59-5
C3H8N2O2.HCI, Acros Organics, New Jersey USA; Ceel Belgium, Cat. No.
204670050
Diethylenetriaminepentaacetic dianhydride, CAS No. 23911-26-4
molecular weight: 357.32, Aldrich cat. no. 28,402-5
Fmoc-6-aminohexanoic acid (Fmoc-6-Ahx-OH), CAS No. 88574-06-5
Fmoc-Asp(2-phenylisopropyl ester)-OH, Molecular weight: 473.53 g/mol
Bachem Cat. No. B-2475.0005
Fmoc-L-Asn-OH, CAS No. 71089-16-7, Applied Biosystems, cat. no: GEN
911018
Fmoc-Gly Resin, Applied Biosystems Cat. No. 401421,0.65 mmol/g
Fmoc-GIy-OH, CAS No. 29022-11-5, Novabiochem Cat. No. 04-12-1001
Molecular Weight: 297.3 g/mol
Fmoc-L-Asn-OH, Applied Biosystems Cat. No. Gen 911018, Molecular weight:
354.40
Fmoc-L-Arg(Pbf)-OH, CAS No. 154445-77-9, Applied Biosystems Cat. No.
GEN911097, Molecular Weight: 648.8 g/mol
Fmoc-L-Cys(Trt)-OH, CAS No. 103213-32-7, Applied Biosystems Cat. No.
GEN911027, Molecular Weight: 585.7 g/mol
Fmoc-L-Leu-OH, CAS No. 35661-60-0, Applied Biosystems Cat. No.
GEN911048, Molecular Weight: 353.4 g/mol
Fmoc-L-Lys(Fmoc)-OH, CAS No. 78081-87-5, Molecular weight: 590.7 g/mol
PerSeptive Biosystems Cat. No. GEN911095, Hamburg, Germany
Fmoc-Lys(Mtt) Resin, 0.68 mmol/g, Bachem Cat. No. D-2565.0005
Fmoc-L-Lys(tBoc)-OH, CAS No. 71989-26-9, Molecular Weight: 468.6 g/mol
Applied Biosystems Cat. No. GEN911051
Fmoc-Ser(tBu) Resin, Applied Biosystems Cat. No. 401429, 0.64 mmol/g
Fmoc-L-Ser(tBu)-OH, CAS No. 71989-33-8, Perseptive Biosystems Cat. No.
GEN911062, Molecular Weight: 383.4 g/mol
Fmoc-L-Tyr(tBu)-OH, Applied Biosystems Cat. No. GEN911068
CAS No. 71989-38-3, M.W. 459.5
LIST OF SUPPLIERS
Applied Biosystems, Warrington, WA1 4SR,United Kingdom
Bachem AG, Hauptstrasse 144, CH-4416 Bubendorf, Switzerland
Calbiochem-Novabiochem, CH-4448 Laufelfingen, Switzerland
Fluka Chemie GmbH, Buchs, Switzerland
Merck KGaA, Darmstadt, Germany
PE Biosystems, Warrington, United Kingdom
Perseptive Biosystems, Warrington, United Kingdom/HamburgGermany
Sigma Aldrich Chemie, Steinheim Germany
(also Riedel-deHae"n)
Sigma-Genosys LTD, Pampisford, Cambridge, UK
Bio-Whittaker, Verviers, Belgium
Harlan Laboratories, Horst, The Netherlands
Genset SA, Paris, France
AmershamPharmacia Biotech, Uppsala, Sweden
Qiagen, Hilden, Germany
Terumo, Leuven, Belgium
Vector Laboratories, Burlingame, USA
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WE CLAIM:
1. A tumor targeting unit comprising a peptide sequence:
Cy-Rrn-Dd-Ee-Ff-Rrm-Cyy
or a pharmaceutically or physiologically acceptable salt thereof, wherein,
Dd-Ee-Ff is Aa-Bb-Cc, wherein
Aa, is isoleucine, leucine or tert-leucine, or a structural or functional
analogue thereof;
Bb is arginine, homoarginine or canavanine, or a structural or func-
tional analogue thereof;
Cc is serine or homoserine, or a structural or functional analogue
thereof;
Rr are each, independently, any amino acid residue or structural or
functional analogues thereof;
n and m, independently, are integers 0-4 and the sum of n and m
does not exceed four, and,
Cy and Cyy are entities capable of forming a cyclic structure by
means of an amide or ester bond,
wherein the peptide is cyclic or forms part of a cyclic structure.
2. The tumor targeting unit as claimed in claim 1, wherein a cyclic
structure is formed by a lactam or lactone bond.
3. The tumor targeting unit as claimed in claim 2, wherein one of Cy
and Cyy is aspartic acid, glutamic acid or a structural or functional analogue
thereof, and the other is lysine, ornithine or a structural or functional analogue
thereof.
4. The tumor targeting unit as claimed in any one of claims 1 to 3,
wherein the sum of n and m is 2 - 4.
5. The tumor targeting unit as claimed in any one of claims 1 to 3,
wherein either n or m or both are zero.
6. The tumor targeting unit as claimed in any one of claims 1 to 5,
wherein Rr is any amino acid residue, except histidine or lysine.
7. The tumor targeting unit as claimed in claim 6, wherein Rr is selected
from the group consisting of glycine, arginine and structural or functional
analogues thereof.
8. The tumor targeting unit as claimed in any one of claims 1 to 7,
wherein Dd-Ee-Ff is LRS.
9. The tumor targeting unit as claimed in claim 8, having the formula
DLRSK (SEQ ID NO. 1), DGRGLRSK (SEQ ID NO. 2), DRGLRSK (SEQ ID
NO. 3), DRYYNLRSK (SEQ ID NO. 4), DSRYNLRSK (SEQ ID NO. 5), ,
DLRSGRK (SEQ ID NO. 6) DLRSGRGK (SEQ ID NO. 7), OLRSE (SEQ ID
NO. 8), OLRSGRGE (SEQ ID NO. 9) or KLRSD (SEQ ID NO. 10).
10. The tumor targeting unit as claimed in any one of the previous
claims, wherein the unit is derivatized, activated, protected, resin bound or oth-
er support bound.
11. A tumor targeting agent comprising at least one targeting unit as
claimed in any one of claims 1 to 10, coupled to at least one effector unit.
12. The tumor targeting agent as claimed in claim 11, wherein the
effector unit is a directly or indirectly detectable agent or a therapeutic agent.
13. The tumor targeting agent as claimed in claim 12, wherein the .
detectable agent comprises an affinity label, a fluorescent or luminescent la-
bel, a chelator, a metal complex, an enriched isotope, radioactive material or a
paramagnetic substance.
14. The tumor targeting agent as claimed in claim 13, wherein the
detectable agent is a rare earth metal.
15. The tumor targeting agent as claimed in claim 12, wherein the
therapeutic agent is selected from the group consisting of cytotoxic and cyto-
static substances and radiation emitting substances.
16. The tumor targeting agent as claimed in claim 12, wherein the
detectable agent is gadolinium.
17. The tumor targeting agent as claimed in claim 15, wherein the
therapeutic agent comprises doxorubicin, daunorubicin, methotrexate or
boron.
18. The tumor targeting agent as claimed in any of claims 11-17,
comprising an optional unit.
19. A diagnostic or pharmaceutical composition comprising at least
one targeting unit as claimed in any one of claims 1 to 10, or at least one tar-
geting agent as claimed in any one of claims 11 to 18.
20. The composition as claimed claim 19 for the preparation of a
medicament for the treatment of cancer or cancer related diseases.
21. The composition as claimed in claim 20, wherein said cancer or
cancer related disease is a solid tumor.
22. The composition as claimed in claim 21, wherein said cancer is
selected from the group consisting of carcinoma, sarcoma, melanoma or
metastases.
This invention relates to novel tumor targeting motifs, units and agents, as well as tumor targeting peptides and
analogues thereof. The targeting agents typically comprise at least one targeting motif, Aa-Bb-Cc, and at least one effector unic. The
invention further relates to specific tumor targeting peptides, pharmaceutical and diagnostic composisitons comprising such peptides.
Disclosed are also methods for diagnosing or treating cancer.

Documents:

778-kolnp-2005-granted-abstract.pdf

778-kolnp-2005-granted-assignment.pdf

778-kolnp-2005-granted-claims.pdf

778-kolnp-2005-granted-correspondence.pdf

778-kolnp-2005-granted-description (complete).pdf

778-kolnp-2005-granted-drawings.pdf

778-kolnp-2005-granted-examination report.pdf

778-kolnp-2005-granted-form 1.pdf

778-kolnp-2005-granted-form 18.pdf

778-kolnp-2005-granted-form 3.pdf

778-kolnp-2005-granted-form 5.pdf

778-kolnp-2005-granted-gpa.pdf

778-kolnp-2005-granted-letter patent.pdf

778-kolnp-2005-granted-reply to examination report.pdf

778-kolnp-2005-granted-specification.pdf

778-kolnp-2005-granted-translated copy of priority document.pdf


Patent Number 216890
Indian Patent Application Number 778/KOLNP/2005
PG Journal Number 12/2008
Publication Date 21-Mar-2008
Grant Date 19-Mar-2008
Date of Filing 02-May-2005
Name of Patentee KARYON OY.
Applicant Address VIIKINKAARI 4, FIN-00790, HELSINKI
Inventors:
# Inventor's Name Inventor's Address
1 ELO HANNU KAUPPANEUVOKSENTIE 12, FIN-00200, HELSINKI
2 BERGMAN MATHIAS SKUTHOLMINKAARI 16, FIN-01100, OSTERSUNDOM.
3 AUVINEN MERJA WESTERDIN PUISTOTIE 10 A,FIN-02160 ESPOO
PCT International Classification Number C07K 7/06
PCT International Application Number PCT/FI2003/000723
PCT International Filing date 2003-10-03
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 20021763 2002-10-03 Finland