POLYPEPTIDE DENDRIMERS AS UNIMOLECULAR CARRIERS OF DIAGNOSTIC IMAGING CONTRAST AGENTS, BIOACTIVE SUBSTANCES AND DRUGS

DENDRIMERES POLYPEPTIDIQUES EN TANT QUE PORTEURS UNIMOLECULAIRES D'AGENTS DE CONTRASTE D'IMAGERIE DIAGNOSTIQUE, DE SUBSTANCES BIOACTIVES ET DE MEDICAMENTS

English Abstract

The invention describes new polypeptide dendrimers and processes for the
synthesis of the same. The polypeptide dendrimers of the invention have a
structure which consists of a multifunctional core moiety from which highly
branched polypeptide chains, formed by short peptide branching units, extend
radially outwards. The outermost branches surround a lower density space with
hollows and channels into which bioactive substances employed in diagnosis and
therapy can be entrapped or covalently linked. For these properties the said
polypeptide dendrimers are particularly useful in a number of areas in biology
and medicine as carriers for the delivery of bioactive substances, including
drugs, or as carriers of bacterial, viral and parasite antigens, gene-therapy
compounds and diagnostic imaging contrast agents.

French Abstract

L'invention concerne de nouveaux dendrimères polypeptidiques ainsi que des procédés pour les synthétiser. Ces dendrimères polypeptidiques ont une structure constituée d'une fraction de noyaux multifonctionnelle à partir de laquelle des chaînes de polypeptides hautement ramifiée, formée de courtes unités de ramification de peptides, s'étendent radialement vers l'extérieur. Les branches le plus à l'extérieur entourent un volume de densité inférieure avec des cavités et des canaux dans lesquels des substances bioactives, utilisées en diagnostic et en thérapie, peuvent être piégées ou liées de manière covalente. Pour ces propriétés, les dendrimères de polypeptidiques sont particulièrement utiles dans de nombreux secteurs en biologie et en médecine en tant que porteurs pour l'administration de substances bioactives, y compris des médicaments, ou en tant que porteurs bactériens, viraux et d'antigènes parasites, de composés de thérapie génique et d'agents de contraste d'imagerie diagnostique.

Claims

26
CLAIMS
1. A polypeptide dendrimer having: i) a multifunctional core moiety; ii) an
exterior
of closely spaced groups constituting the terminals of branched polypeptide
chains
(monodendrons) radially attached to the core that, in turn, form iii) interior
layers
(generations) of short peptide branching units (propagators) with
characteristic
hollows and channels where each propagator contains a trifunctional aminoacid
whose asymmetric carbon (the propagator branching point) is connected to two
equal-length arms bearing identical terminal reactive groups and to a third
arm
(the propagator stem) bearing an activatable functional group,
represented by formula (I):
K(-L)p-M (I) wherein
K is a multifunctional core moiety,
L is a polypeptide monodendron,
p is the number of polypeptide monodendrons irradiating from the core moiety
and
M represents the outermost ramifications of the dendrimer;
2. A polypeptide dendrimer of claim 1 where said K is represented by formula
(II):
X-(CH2)n-X1 (II)
wherein X=X1 or X~X1, and X, X1 are NH or CO or S;
3. A polypeptide dendrimer of claim 1 where said K is represented by formula
(III):
(III)
Y[-(CH2)n-Z]i
wherein Y=C or Y=N; Z is NH or S or Cl or Br or I or a maleimide residue, n=1-
6
and i=3,4;
4. A polypeptide dendrimer of claim 1 where said K is represented by formula
(IV):
X-CH(R)-CO[-NH-CH(R)-CO)n-NH-CH(R)-COO1' (IV)
wherein R is (CH2)m-X1, m=1-5, R1 is methyl or ethyl or butyl or isopropyl,
X=X1 or
X~X1, and X, X1 are NH or CO or S and n=1-6;
5. A polypeptide dendrimer of claim 1 where said L is the single monodendron
whose propagators are represented by formula (V):
-CO-CH(R2)-(CH2)n-NR3- (V)

27
wherein R2=H or the side-chain of natural or synthetic aminoacids, and their
derivatives; R3=H or a linear hydrocarbon radical optionally substituted with
OH or
SH or Cl or Br; R2-CH(CH2)n-NR3 is a 5 or 6 atoms ring, and n=0-6;
6. A polypeptide dendrimer of claim 1 where said L is the single monodendron
whose propagators are represented by formula (VI):
-CO-CH(R2)-CO-N(R3)-(CH2)m-N(R3) (VI)
wherein R2 and R3 have the meaning seen in claim 5 and m=1-6;
7. A polypeptide dendrimer of claim 1 where said L is the single monodendron
whose propagators are represented by one of the residues:
-CO-CH2-NH-NH-; or -CO-CH(R2)-O-; or -CO-CH2-O-N=CH-CO-; or -CO-CH(R2)-
(CH2)n-S-CH2-CO-W; or -CO-NH-CH(CH2-SH)-CO-W or
<IMG>
wherein W=-N(R3)-(CH2)m-NR3, Q=H or -CH3; T is O or S whereas R2, R3 and m
have the meaning seen in claim 5;
8. A polypeptide dendrimer of claim 1 where said L is the single monodendron
whose propagators are represented by one of the residues:
<IMGS>
9. A polypeptide dendrimer of claim 1 where said p is 1 or 2 or 3 or 4;
10. A polypeptide dendrimer of claim 1 where said M is the residue represented
by
formula (VII):
-Aq-B(Ar)-C-Ar[Aq-B(Ar)-C-Ar[Aq-B(Ar-D)-C-Ar-D]2]2 (VII)
wherein A=-CO-CH(R2)-(CH2)n-NR3, R3 and n have the meaning seen in claim 5,
q=1-6, r=1-4 and R2, in addition to the meaning seen in claim 5, is a natural
or

28
synthetic trifunctional aminoacid; B is -CO-CH[-(CH2)n-X1]-X, with X=X1 or
X~X1; X
and X1 are NH or CO or S; n=1-5; C=A or C=-CO(CH2)n-NH- or -(CH2)n-S- with
n=1-6 or C is one of the residues:
<IMGS>
D is a residue represented by formulae (VIII)-(XI):
-Aq-B(Ar-E)-C-Aq-E (VIII)
-Aq-B(Ar)-C-Aq[Aq-B(Ar-E)-C-Aq-E]2 (IX)
-Aq-B(Ar)-C-Aq[Aq-B(Ar)-C-Aq-[Aq-B(Ar-E)-C-Aq-E]2]2 (X)
-Aq-B(Ar)-C-Aq[Aq-B(Ar)-C-Aq-[Aq-B(Ar)-C-Aq[Aq-B(Ar-E)-C-Aq-E]2]2]2 (XI)
wherein A, B, C, q ed r have the meaning seen above , and E is represented by
formulae (XII) and (XIII):
-Aq-B(Ar-P)-C-Aq-P1 (XII)
-Aq-B(Ar)-C-Aq[-Aq-B(Ar-P)-C-Aq-P']2 (XIII)
wherein A, B, C, q and r have the meaning seen above, P=P1 or P~P1, P and P1
being H or a linear hydrocarbon radical optionally substituted with one or
more
linear or branched alkyl groups, acyl, aminoacid, peptide, nucleotide,
oligonucleotide, saccharide, oligosaccharide, protein, monoclonal antibody,
polyethyleneglycol containing 10-400 -CH2-CH2-O- repeats, lipid, enzyme, metal
ligand or their synthetic analogues and derivatives;
11. A polypeptide dendrimer of claims 1-10 wherein the two-dimensional
molecular
diameter of the dendrimers is in the range from about 10 to 100 nm.
12. The dendrimer 2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-
Orn-Gly-Gly-HN-CH2-CH2-N H-Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-
Gly(Gly-Gly-Orn-Gly-H)2)2)2.
13. The dendrimer 2(2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-

29
Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly-HN-CH2-CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-
Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly-H)2)2)2)2.
14. The dendrimer 2(2(2(2(2(H-Gly-Orn-Gly-Gly]Gly-Orn-Gly-Gly)Gly-Orn-Gly-
Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly-HN-CH2-CH2-NH-Gly-Gly-
Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-
Gly-Orn-Gly-H)2)2)2)2)2.
15. The dendrimer 2(2(2(2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-
Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly-HN-CH2-
CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-
Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly-H)2)2)2)2)2)2.
16. The dendrimer 2(2(2(2(2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-
Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-
Gly-Gly-HN-CH2-CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-
Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly-
H)2)2)2)2)2)2)2.
17. The dendrimer N{-CH2-CH2-NH-CO-CH(-CH2-phenyl)-NH-Gly-Gly-Gly-Orn-
Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Orn-Gly-H]2]2]2}3.
18. The dendrimer N{-CH2-CH2-NH-CO-CH(-CH2-phenyl)-NH-Gly-Gly-Gly-Orn-
Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-
Orn-Gly-H]2]2]2]2}3.
19. The dendrimer <IMGS>
20. The polypeptide dendrimers of claims 12-19 wherein the NH2 terminals are
acetylated.
21. A polypeptide dendrimer of claim 1 wherein at least one bioactive or
marker
molecule is covalently linked to the surface of the same.

30
22. A polypeptide dendrimer of claim 21 where the bioactive molecule is
selected
in the group comprising an aminoacid, a peptide, a protein, a nucleotide, an
oligonucleotide, a lipid, a saccharide, an oligosaccharide, and a small
organic
molecule and their synthetic analogues and derivatives.
23. A polypeptide dendrimer of claim 21 where the bioactive molecule is
selected
in the group comprising drugs, cellular receptor ligands, bacterial, viral and
parasite antigens and gene-therapy compounds.
24. A polypeptide dendrimer of claim 21 where the marker molecule is a
diagnostic
imaging contrast agent.
25. A polypeptide dendrimer of claim 1 where the bioactive molecule is
entrapped
in the same.
26. A polypeptide dendrimer of claim 25 where the bioactive molecule is
selected
in the group comprising an aminoacid, a peptide, a protein, a nucleotide, an
oligonucleotide, a lipid, a saccharide, an oligosaccharide, and a small
organic
molecule and their synthetic analogues and derivatives.
27. A polypeptide dendrimer of claim 25 where the bioactive molecule is
selected
in the group comprising drugs, cellular receptor ligands, bacterial, viral and
parasite antigens and gene-therapy compounds.
28. A polypeptide dendrimer of claim 27 where the bioactive molecules are
anticancer drugs.
29. A polypeptide dendrimer of claim 27 where the bioactive molecules are
antibiotics.
30. A polypeptide dendrimer of claim 27 where the bioactive molecules are
antiviral substances.
31. A process for production of the polypeptide dendrimers of claim 1
characterized by the following steps:
i) synthesis of core moieties with at least two reactive functional groups;
ii) divergent synthesis on solid-phase of polypeptide monodendrons with
temporarily or permanently protected terminals;
iii) covalent condensation of polypeptide monodendrons to core moieties;
32. A process for production of polypeptide dendrimers of claim 1
characterized by
the following steps:

31
i) synthesis of core moieties with at least two reactive functional groups;
ii) covalent condensation to the core moieties of polypeptide monodendrons of
generation 1-3 with temporarily protected terminals to obtain the
corresponding
protected dendrimers;
iii) after protecting groups removal, repeated condensations of polypeptide
monodendrons to the dendrimer reactive terminals to obtain the desired final
dendrimers.
33. A process for entrapping into the polypeptide dendrimers of claim 1
bioactive
substances and drugs with molecular weights lower than 1,000 Da, characterized
by the following steps:
(a) adding suitable amounts of polypeptide dendrimers to a concentrated or
saturated solution of said molecules and
(b) precipitating the loaded polypeptide dendrimer after 24 h incubation at
room
temperature in a large volume of a precipitant.
34. A process for entrapping into the polypeptide dendrimers of claim 1
bioactive
substances and drugs with molecular weights higher than 1,000 Da,
characterized
by the selective chemical ligation of polypeptide monodendrons, in aqueous
buffers, to the core moieties in the presence of said molecules.
35. A process for the selective chemical ligation of bioactive substances and
drugs
to the internal functional groups of the polypeptide dendrimers of claim 1, in
aqueous buffers, after loading the dendrimer carrier by diffusion.
36. Use of polypeptide dendrimers of claim 1 as unimolecular carriers of
bioactive
molecules wherein at least one bioactive or marker molecule is covalently
linked to
the surface of the same.
37. Use of polypeptide dendrimers according to claim 36 where the bioactive
molecule is selected in the group comprising an aminoacid, a peptide, a
protein, a
nucleotide, an oligonucleotide, a lipid, a saccharide, an oligosaccharide, and
a
small organic molecule and their synthetic analogues and derivatives.
38. Use of polypeptide dendrimers according to claim 36 where the bioactive
molecule is selected in the group comprising drugs, cellular receptor ligands,
bacterial, viral and parasite antigens and gene-therapy compounds.
39. Use of polypeptide dendrimers according to claim 36 where the marker

32
molecule is a diagnostic imaging contrast agent.
40. Use of polypeptide dendrimers of claim 1 as unimolecular carriers of
bioactive
molecules wherein the bioactive molecule is entrapped into the same.
41. Use of polypeptide dendrimers according to claim 40 where the bioactive
molecule is selected in the group comprising an aminoacid, a peptide, a
protein, a
nucleotide, an oligonucleotide, a lipid, a saccharide, an oligosaccharide, and
a
small organic molecule and their synthetic analogues and derivatives.
42. Use of polypeptide dendrimers according to claim 40 where the bioactive
molecule is selected in the group comprising drugs, cellular receptor ligands,
bacterial, viral and parasite antigens and gene-therapy compounds.
43. Use of polypeptide dendrimers according to claim 40 where the bioactive
molecules are anticancer drugs.
44. Use of polypeptide dendrimers according to claim 40 where the bioactive
molecules are antibiotics.
45. Use of polypeptide dendrimers according to claim 40 where the bioactive
molecules are antiviral substances.
46. Compositions with pharmaceutically acceptable excipients wherein the
polypeptide dendrimers of claim 1 are the unimolecular carriers of bioactive
or
marker molecules covalently linked at the surface of the same.
47. Compositions with pharmaceutically acceptable excipients wherein the
polypeptide dendrimers of claim 1 are the unimolecular carriers of bioactive
molecules entrapped into the same.

Description

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1
POLYPEPTIDE DENDRIMERS AS UNIMOLECULAR CARRIERS OF
DIAGNOSTIC IMAGING CONTRAST AGENTS, BIOACTIVE SUBSTANCES AND
DRUGS
Field of the invention
s The present invention relates to polypeptide dendrimers their processes of
synthesis and their use as carriers for the delivery of bioactive substances,
including drugs, or as carriers of bacterial, viral and parasite antigens,
gene-
therapy compounds and diagnostic imaging contrast agents.
Prior art
Io Dendrimers are highly branched polymers in which a number of primary
branched
chains (monodendrons) irradiating from a multifunctional core moiety
originates
structures and morphologies quite different from classical hyperbranched and
star
polymers. (D. A. Tomalia et al., Angew. Chem. Int. Ed. Engl., 1990, 29, 138-
175;
D. A. Tomalia and H. Dupont Durst, "Topics in Current Chemistry", 1993, 165,
is 193-313). The structural components of dendrimers namely a) a core moiety,
b)
interior layers (generations) composed of branching units forming the
monodendrons radially attached to the core, and c) an exterior of closely
spaced
surface groups generate, as the generations increase, spheroidal structures
with
well-developed internal hollows and channels. The cavities and channels create
a
Zo microenvironment that can be utilized for the entrapment or the covalent
coupling
of guest molecules. The stepwise synthesis of polyamidoamine (PAMAM)
starburst dendrimers with up to 10 generations and their use as host molecules
has been reported in a number of patents and papers. (O. A. Matthews et al.,
Progr. Polym. Sci., 1997, 23, 1-56). Computer modelling of PAMAM dendrimers
2s has shown how the number and dimensions of cavities depend from a) the
number (Nc) of functional groups of the core moiety, b) the number (Nb) of
reactive sites of the branching unit and c) the dimensions and rigidity of the
branching unit. When Nc=3 or 4 and Nb=2, the PAMAM dendrimer series
increases its diameter by approximately 10 A per generation, evolving from a
disk-
30 like shape (generations 0-2) to an oblate spheroid (generations 3,4) to a
nearly
symmetrical spheroid at generations 5 and higher.
Two conceptually different synthetic approaches for the preparation of high-

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2
generation dendrimer exist: the divergent and the convergent approach. Both
approaches are based on a repetition of reaction steps, each repetition
accounting
for the creation of an additional generation. In the divergent synthesis, the
dendrimer is grown stepwise from the core moiety and all reactions are carried
out
s in a single molecule. Since every reaction step occurs incompletely at each
of the
exponentially growing number of terminals (average selectivity lower than
100%),
only limited amounts of defect-free dendrimers are obtained. For instance, an
average selectivity of 99.5% per reaction step leads to only 29% yield of pure
generation 5 poly(propyleneamine) dendrimer. The purification of dendrimers
io obtained by the divergent approach can hardly be achieved as they have very
similar structures to their by-products. In the convergent approach, the
synthesis
of dendrimers begins from the periphery and ends at the core by first
preparing
single monodendrons with the desired number of generations and then joining
them to the core moiety. Dendrimers synthesized by this approach can be
is produced nearly pure since only a constant and low number of reactions are
required for any generation-adding step. Dendrimers can be also obtained in
fewer
steps and higher yields, using pre-branched analogues of both cores
(hypercores)
and branching units (branched monomers) or, alternatively, following "double
exponential" and mixed growth strategies of synthesis.
2o The structural characteristics of dendrimers namely spheroidal surfaces,
internal
voids and nanoscopic dimensions have suggested their use as host molecules
capable of binding guest molecules either at the interior (dendrimers as endo-
receptors) or at the surface (dendrimers as exo-receptors). Various small
molecular weight organic molecules have been entrapped into carboxylate-
Zs terminated hydrocarbon dendrimers. Acetylsalycilic acid and 2,4
chlorophenoxyacetic acid have been encapsulated within, or near the surface
of,
PAMAM dendrimers of generation 4, 5 and 6 and the sequestering of 10-20
molecules of dopamine in the channels of PAMAM dendrimers of generation 6 has
been studied by use of molecular dynamics calculations. (D.A. Tomalia, Angew.
3o Chem. Int. Ed. Engl., 1990, 29, 138-175). Meijer and colleagues have
prepared
the "dendritic box" by building up a shell of Boc-phenylalanine on the surface
of a
poly(propyleneamine) dendrimer of generation 5. (J. F. G. A. Jansen et al.,

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Science, 1994, 266, 1226-1229). When the shell is formed in the presence of
guest molecules, such as Rose Bengal or tetracyanoquinodimethane, those
present in the dendrimer voids are trapped sterically. Liberation of guests is
only
possible after destruction of the shell i. e. by acidolysis of the Boc groups.
The
s number of guest molecules that can be entrapped is dependent upon the guest
size. Only a very limited number of papers dealing with the biocompatibility
and
pharmacokinetics of dendrimers have appeared. PAMAM dendrimers of
generation 3-6 were found to have low toxicity, while the generation 7
dendrimer is
toxic in vivo. A high pancreas uptake and an unexplained high urinary output
for
to the seventh generation dendrimer have been also observed. Haemolysis and
cytotoxicity have been observed for amine-terminated PAMAMs, but not for their
analogues terminating with carboxylate groups. (R. Duncan and N. Malik, Proc.
Int. Symp. Control. Relat. Bioact. Mater., 1996, 23, 105-106). Metal
dendrimeric
chelates have been also studied for diagnostic applications. The Gd (III)
chelate of
is the PAMAM-thiourea-diethylenetriaminepentaacetic acid magnetic resonance
imaging contrast agent (Gd(III)-PAMAM-TU-DTPA) remains circulating in blood
for
longer periods of time than the monomeric chelate, the sixth generation
chelate
being more effective as contrast agent than chelate conjugates based on
polylysine, albumin and dextran supports. By attaching a single monoclonal
2o antibody to a PAMAM dendrimer of generation 2, functionalized at the
surface with
derivatives of tetraacetic or pentaacetic acid for chelation of 9°Y,
"'In, 2'2Bi and
Gd(III), the feasibility of monoclonal antibody guided radiotherapy and
imaging has
been demonstrated. Boronated dendrimer-monoclonal antibody conjugates have
been used successfully as protein probes in electron spectroscopic imaging.
The
Zs transfection of antisense oligonucleotides into a variety of cell lines has
been
carried out in vitro using PAMAM dendrimers. Furthermore, polypeptide
monodendrons of generation 2 and 3, composed of lysyl residues (MAP, multiple
antigen peptides), have been prepared as branched multivalent scaffolds for
peptide conjugation and used as immunogens and immunodiagnostics. (J.P. Tam,
~o J. Immunol. Methods, 1996, 196, 17-32). The author did not however mention
the
possibility to prepare polypeptide dendrimers of globular shape resembling
high
generation spheroidal poly(amidoamines) for the encapsulation of guest
molecules

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in their internal cavities.
The preliminary observations on the in vitro and in vivo properties of PAMAM
dendrimers as well as the harsh conditions that are needed to release guest
molecules from the dendritic boxes, indicate that both microcontainers are not
s suitable as carriers for bioactive substances and drug delivery. Besides
favourable
pharmacokinetic properties, such carriers should have:
1 ) biological stealthiness (biocompatibility).
2) limited and controlled stability towards enzymes. Enzymatic processing is
necessary not only to avoid the chronic toxicity due to non-specific
accumulation in
to the body, but also to obtain the controlled release of guest molecules by
gradual
hydrolysis of the dendrimer structure.
3) high carrying capacity. The internal voids of PAMAMs are not big enough to
encapsulate either a large number of low molecular weight molecules or a
reasonable number of macromolecular guests like, for instance, insulin.
is 4) controlled dimensions, preferably in the 10-100 nm range, to avoid rapid
urinary
clearance and RES (reticuloendothelial system) uptake.
Summary of the invention
The applicant has now surprisingly found that dendrimers with a polypeptide
backbone can have the properties above mentioned and comply with the following
2o aims of the present invention. A first aim of the present invention is that
of
providing water soluble polypeptide carriers with dendrimeric structures,
spheroidal shapes and precisely defined dimensions (unimolecular dendrimeric
carriers), with channels and cavities that can host bioactive substances and
drug
molecules with molecular weights up to 5-7 kDa. A second aim of the present
2s invention is that of providing polypeptide dendrimeric carriers whose
gradual
demolition in vivo, in blood or at the target cellular sites, occurs both by
enzymatic
hydrolysis (which can be controlled and modulated by insertion of D aminoacid
residues into the backbone) and by UV irradiation if the carriers contain
photolabile
bonds. A third aim of the present invention is that of providing loaded
polypeptide
~o dendrimeric carriers whose dimensions and surfaces are tailored to avoid
RES
uptake as well as rapid urinary clearance. An additional aim of the present
invention is the synthesis of polypeptide dendrimeric carriers with antigen
moieties

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s
(peptides, oligonucleotides, saccharides and oligosaccharides deriving from
relevant pathogenic agents) covalently linked to their surface reactive
groups. A
further aim of the present invention is the derivatisation of the surface of
the
polypeptide dendrimeric carriers with biological receptor ligands such as
folic acid,
s sialic acid, mannose, fat acids, vitamins, hormons, oligonucleotides,
monoclonal
antibodies, short peptides, proteins and oligonucleotides for cell targeting.
Then, the object of the present invention are polypeptide dendrimers having:
i. a multifunctional core moiety;
ii. an exterior of closely spaced groups constituting the terminals of
branched
to polypeptide chains (monodendrons) radially attached to the core that, in
turn, form
iii. interior layers (generations) of short peptide branching units
(propagators)
with characteristic hollows and channels, where each propagator contains a
trifunctional aminoacid whose asymmetric carbon (the propagator branching
point)
is connected to two equal-length arms bearing identical terminal reactive
groups
is and to a third arm (the propagator stem) bearing an activatable functional
group,
represented by formula (I):
K(_L)p M (I)
wherein
K is a multifunctional core moiety,
zo L is a polypeptide monodendron,
p is the number of polypeptide monodendrons irradiating from the core moiety
and
M represents the outermost ramifications of the dendrimer.
Further objects of the present invention are the processes for the synthesis
of said
polypeptide dendrimers and the use in biology and medicine of the same as
2s carriers for the delivery of bioactive substances, including drugs, or as
carriers of
bacterial, viral and parasite antigens and gene-therapy compounds and
diagnostic
imaging contrast agents.
Detailed description of the invention
The polypeptide dendrimers, the processes for their synthesis and the use as
3o unimolecular carriers, according to the present invention, will be better
illustrated
in the following description.
The polypeptide dendrimers of this invention consist of highly branched

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polypeptide chains or monodendrons, deriving from repeated condensations of
short peptide branching units or propagators, that irradiate outward from a
multifunctional core moiety, having an exterior of closely spaced groups
constituting the terminals of the monodendrons, and interior layers or
generations
s of propagators with characteristic hollows and channels where each
propagator
contains a trifunctional aminoacid whose asymmetric carbon (the propagator
branching point) is connected to two equal-length arms bearing identical
terminal
reactive groups and to a third arm (the propagator stem) bearing an
activatable
functional group. The polypeptide dendrimers are represented by formula (I):
1 o K(-L)p-M (I )
wherein: K is the multifunctional core moiety and K can be represented by the
formulae:
(I I ) X-(CH2)n-X',
wherein X=X' or X~X', and X, X' are NH or CO or S; or
is (III) Y[-(CH2)n-Z]i~
wherein Y=C or Y=N; Z is NH or S or CI or Br or I or a maleimide residue,
n=1-6 and i=3,4;
or (IV) X-CH(R)-CO[-NH-CH(R)-CO]n-NH-CH(R)-COOR',
wherein R is (CH2)m-X', m=1-5, R' is methyl or ethyl or butyl or isopropyl,
2o X=X' or X~X', and X, X' are NH or CO or S and n=1-fi;
L is the single monodendron whose propagators can be represented by the
formulae: (V) -CO-CH(RZ)-(CH2)~-NR3-
wherein RZ=H or the side-chain of a natural or synthetic aminoacid, and their
derivatives; R3=H or a linear hydrocarbon radical optionally substituted with
OH or
2s SH or CI or Br; RZ-CH(CH2)"-NR3 is a 5 or 6 atoms ring, and n=0-6; and
(V I ) -CO-CH(Rz)-CO-N (R3)-(CH2)m-N (R3)
wherein RZ and R3 have the meaning seen above and m=1-6; or L is the single
monodendron whose propagators can be represented by one of the residues:
-CO-CH2-NH-NH; -CO-CH(R2)-O-; -CO-CH2-O-N=CH-CO-; -CO-CH(R2)-(CH2)~-S
~o CH2-CO-W; -CO-NH-CH(CHZ-SH)-CO-W; -C jN-CH-CO-W ;
HO-CH2-CH-T-CH-Q

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7
-CO ~ ~ CH2-O-; -CO-CH2 ~ ~ CO-CH(CHg)-O-;
N02 N02
-CO-CH2-O ~ ~ CO-CH(CH3)-O-; -CO-(CH2)3 ~ ~ CH(CH3)-O-;
CHgO
s N02 N02
-CO-(CH2)3 ~ ~ CH2-O-; -CO-(CH2)3-O ~ ~ CH(CH3)-NH-
OCH3 OCH3
wherein W=-N(R3)-(CH2)m-NR3, Q=H, -CH3; T is O or S while RZ, R3 and m have
the meaning seen before and p=1-4;
to M is the residue represented by formula (VII):
-Aq-B(Ar)-C-Ar[Aq-B(Ar)-C-Ar[Aq-B(Ar~)-C-Ar~]2l2 (VI I )
wherein A=-CO-CH(R2)-(CH2)n-NR3; R3 and n have the meaning seen before;
q=1-6; r=1-4 and R2, in addition to the meaning seen before, is a natural or
synthetic trifunctional aminoacid; B is -CO-CH[-(CH2)~-X']-X, with X=X' or
X~X'; X
is and X' are NH or CO or S; n=1-5; C=A or -CO(CH2)n-NH; -(CH2)n-S with n=1-6;
or C is one of the residues:
-CO ~ ~ CH2-O-; -CO-CH2 ~ ~ CO-CH(CH3)-O-;
N02
N02
Zo -CO-CH2-O ~ ~ CO-CH(CH3)-O-; -CO-(CH2)3-~ ~ -CH(CHg)-O-;
OCH3
N02 N02
-CO-(CH2)3 ~ ~ CH2-O-; -CO-(CH2)g-O ~ ~ CH(CHg)-NH-;
OCH3 CH3
2s D is a residue represented by formulae (VIII)-(XI):
-Aq-B(A~E)-C-Aq-E (VI I I )
_Aq_B(Ar)-C-Aq[Aq_B(p,~E)_C_Aq_E]2 (IX)
-Aq-B(Ar)-C-Aq[Aq-B(Ar)-C-Aq-[Aq-B(ArE)-C-Aq-E)2]2 (X)
-Aq-B(Ar)-C-AqLAq-B(Ar)-C-Aq-LAq-B(Ar)-C-Aq~Aq-B(ArE)-C-Aq-E~2~2~2 (XI)

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8
wherein A, B, C, q ed r have the meaning seen before, and E is represented by
formulae (X11) and (X111):
-Aq-B(A,-P)-C-Aq-P' (XI I )
-Aq-B(Ar)-C-Aq[-Aq-B(ArP)-C-Aq-P']2 (XI I I )
wherein A, B, C, q and r have the meaning seen before; P=P' or PAP'; P and P'
being H or a linear hydrocarbon radical optionally substituted with one or
more
linear or branched alkyl groups, acyl, aminoacid, peptide, nucleotide,
oligonucleotide, saccharide, oligosaccharide, protein, monoclonal antibody,
polyethylenglycol containing 10-400 -CH2-CH2-O- repeats, lipid, enzyme, metal
io ligand. The terms aminoacid, peptide, nucleotide, oligonucleotide,
saccharide,
oligosaccharide, protein comprise either natural or synthetic analogues and
derivatives.
A characteristic feature of the polypeptide dendrimers of the present
invention is
the limited stability of their backbone to plasma and cellular enzymes and,
more
is important, the possibility of programming the stability towards enzymes in
vivo by
replacing L with D aminoacids. This property distinguishes the polypeptide
dendrimers from PAMAM, polypropylamine, hydrocarbon, polyether, polythioether
and silicon-based dendrimers that, being all stable to enzymatic hydrolysis,
may
accumulate non-specifically in the body creating toxicity problems. By
regulating
2o both the polypeptide dendrimer dimensions (from 10 to 100 nm, to avoid
rapid
urinary excretion and uptake by the RES system) and the liability of the
dendrimer
backbone, it is feasible to balance the retention and the excretion of the
polypeptide dendrimeric carriers in the body. In addition to enzyme
hydrolysis, the
demolition of polypeptide dendrimers with release of guest molecules can be
2s obtained by ultraviolet irradiation of selected bonds when a limited number
of
photolabile residues are inserted in the backbone instead of aminoacid
residues.
As a result, the release of bioactive guest molecules or drugs can be
triggered at
the site of therapeutic utility with generation of fewer systemic side-
effects.
The applicant has surprisingly found that polypeptide dendrimers can be
prepared,
o in accordance with the present invention, by condensing to a core moiety
with 2, 3
or 4 identical functional group, two, three or four polypeptide monodendrons,
previously prepared by stepwise synthesis, using short three-branched peptide

CA 02380178 2002-O1-22
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9
propagators as building blocks. Alternatively, low-generations monodendrons
can
be condensed to a preformed dendrimer (expanded core) to obtain the final
dendrimer. The polypeptide dendrimers of the present invention not only
encapsulate guest molecules of a wide range of molecular weights but,
s surprisingly, show also an extraordinary solubility in water even when
surface
polar groups such as NHZ, OH, and COOH are masked by hydrophobic moieties.
Below are reported methods and examples that demonstrate: 1 ) the feasibility
of
the chemical synthesis of polypeptide dendrimers; 2) the possibility of
entrapment
and encapsulation of guest molecules into the dendrimeric carriers; 3) the
release
io of guest molecules by enzymatic hydrolysis and by ultraviolet irradiation
in vitro
and in vivo; and 4) the non-immunogenicity and adjuvanticity of polypeptide
dendrimers in mice. Numerous embodiments and other features of the present
invention will become better understood with reference to the following
descriptions.
Is General methods of synthesis
According to the present invention, a first general process for the
preparation of
unimolecular polypeptide dendrimers consists in: 1 ) the synthesis of core
moieties
with at least two functional groups; 2) the divergent synthesis of single
polypeptide
monodendrons; 3) the covalent conjugation of the polypeptide monodendrons to
Zo the core moieties. A second general process for the preparation of
polypeptide
dendrimers consists in: 1 ) the synthesis of core moieties with at least two
functional groups; 2) the condensation of monodendrons of generation 1-3,
protected at their termini with removable groups, to the core moieties; 3) the
removal of protecting groups from the low generation dendrimers obtained in
step
2s 2 followed by the reiterated condensation of protected monodendrons to
reach the
target high generation dendrimers; and 4) the removal of protecting groups
from
the final dendrimer followed by surface modification, when necessary.
Protecting
groups, condensing and deblocking agents, solvents and reaction times are
selected considering not only the structure of both core moieties and
propagators,
:o but also the chemical and structural properties of guest molecules.
According to the general formula (I) of the present invention and following
the two
general processes above outlined it is possible, for example, to synthesize

CA 02380178 2002-O1-22
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structurally simple polypeptide dendrimers characterized by a bifunctional
core
such as ethylenediamine to which single monodendrons of generation from 3 to 7
are covalently linked namely 2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn
Gly-Gly)Gly-Orn-Gly-Gly-HN-CH2-CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly
s Gly-Orn-Gly(Gly-Gly-Orn-Gly-H)2)2)2 and 2(2(2(2(2(2(z(H-Gly-Orn-Gly-Gly)Gly-
Orn-
Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-
Orn-Gly-Gly)Gly-Orn-Gly-Gly-HN-CH2-CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orn-
Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-
Orn-Gly(Gly-Gly-Orn-Gly-H)2)2)2)2)2)z)z.
to The objective of entrapping into polypeptide dendrimers molecules with
molecular
weights above 1,000 Da is obtained in two steps: 1 ) assembly of polypeptide
monodendrons on solid supports (Solid-Phase Peptide Synthesis, SPPS), using
short peptide derivatives as building blocks (divergent strategy) and 2)
condensation, in aqueous phase and in the presence of guest molecules, of the
is polypeptide monodendrons to the core moiety by "chemical ligation" methods
as
currently applied for the synthesis of proteins (P.Lloyd-Williams, F.
Albericio and E.
Giralt, "Chemical Approaches to the Synthesis of Peptides and Proteins", 1997,
CRC Press, Boca Raton, 175-200).
The objective of encapsulating into the polypeptide dendrimer molecules with
2o molecular weight below 1,000 Da is obtained both by the above strategy of
trapping guest molecules during dendrimer synthesis and also by first
preparing
"void carriers" that are subsequently filled up by diffusion of small guest
molecules
in their cavities. The objective of preparing polypeptide dendrimers with
photolabile
bonds is obtained following the above methods and using monodendrons with one
2s or more aminoacid residues of the backbone replaced by photolabile
moieties. The
objective of preparing polypeptide carriers with guest molecules covalently
linked
at their interior is obtained by 1 ) preliminary entrapment of guest molecules
into
the dendrimer cavities by diffusion and 2) covalent coupling to the reactive
groups
of the dendrimer carrier. Finally, the objective of conjugating biologically
active
3o molecules to the surface of polypeptide dendrimers for receptor targeting
is
obtained by covalent condensation of a reactive group of the bioactive
molecule
that is not critically important for receptor recognition.

CA 02380178 2002-O1-22
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11
Numerous embodiments and other features of the present invention will become
better understood with reference to the following descriptions. The examples
reported below are not intended to limit the present invention and further
modifications deriving from the natural advancement of the synthetic and
s dendrimer loading protocols are within the spirit and the scope of the
present
invention.
The HPLC analysis was carried out with a Bruker LC21-C apparatus equipped
with the UV Bruker LC313 detector, using Pico Tag Waters columns and
acetonitrile-water buffers A) 10% (v/v) acetonitrile in 0.1 % TFA water and B)
60%
to (v/v) acetonitrile in 0.1 % TFA water; gradient (I) from 0 to 100% B in 25
min and
(II) from 50 to 100% B in 25 min; flow, 1 ml/min, 220 nm detection. Peptide
purification by preparative HPLC was carried out with the Waters Delta Prep
3000
apparatus on a Delta Pack C18-300 (30 mm x 30 cm, 15 ~) column, with the
same eluants and conditions. Flow, 30 ml/min, 220 nm detection. Thin layer
is chromatography was carried out on F 254 silica gel plates (Merck), using as
eluant
1-buthanol/ acetic acid/water (3:1:1 v/v/v). 1% ninhydrin in ethanol and CI2-
Iodine
were used as detecting reagents. 1 H NMR measurements were made with the
200 MHz FT Bruker apparatus. Molecular weights were confirmed by mass
spectrometry on a Voyager-DE apparatus (PerSeptive Biosystems, MA, USA).
zo EXAMPLE 1
This example describes the synthesis of a generation 4 dendrimer by
condensation in liquid phase of a generation 4 monodendron derivative
assembled
on a solid-matrix, to a triamine core.
1. Synthesis of N1CH2-CH2-NH-CO-CH~CH2-phenyl -~213'4HC1
2s 1.91 g of Boc-Phe-OH (7.2 mmole), 150 u1 of N(CH2-CH2-NH2)3 (2.0 mmole),
1.43 g of WSC' HCI (7.5 mmole), 1.15 g of HOBt (7.5 mmole) and 560 ~I of
triethylamine (4.0 mmole) were dissolved in 10 ml of anhydrous DMF at 0
°C and
kept under agitation for 24 h at room temperature. After evaporation of DMF,
the
solid was dissolved in 100 ml of ethyl acetate and extracted with 5% NaHC03
~o (3x20 ml) and brine (3x20 ml). The organic solution was acidified, the
solvent

CA 02380178 2002-O1-22
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12
evaporated and the resulting product, dissolved in 70 ml of ethyl acetate,
further
treated with 4N HCI at 0°C. The mixture was left under agitation at
room
temperature for 30 min. The residue obtained after evaporation of the solvent
was
dissolved in 20 ml of methanol and precipitated with ethyl ether-petroleum
ether
s (1/1, v/v). The solid obtained after filtration was washed repeatedly with
ethyl
ether-petroleum ether (1/1, v/v). M.p.: 167 °C; [a]p22 _1.g (c1, DMF);
R.f.: 0.5;
HPLC: 8,97 min; gradient (I); MS: 589 Da, 611 Da and 627 Da, for M-H+, M-Na+,
M-K+ , respectively.
2. Synthesis of Fmoc-Gly-Orn(Fmoc)-Gly-Gly-OH
io A solution of 14.9 g of Boc-Orn(Fmoc)-OSu (27 mmole) in 30 ml of DMF was
added under agitation at 0°C to a solution of 3. 92 g of H-Gly-Gly-OH
(29.7
mmole) in 45 ml of 5% NaHC03 and 100 ml of DMF. After 1 h at 0°C the
reaction
is continued overnight at room temperature. After DMF evaporation, the residue
was dissolved in 150 ml of 10% citric acid and the product extracted with 200
ml of
is ethyl acetate. The solution was then washed with brine, dried over Na2S04,
filtered and concentrated to a final volume of 50 ml by elimination of the
solvent.
The product was recovered by precipitation with 150 ml of ethyl ether
containing 2
ml of methanol. Yield, 13.9 g. M.p.: 125-128°C; R.f.: 0.7 in 1-butanol/
acetic acid
/water (3:1:1, v/v/v); HPLC: 19.25 min; gradient (I).
20 13.9 g of Boc-Orn(Fmoc)-Gly-Gly-OH were dissolved in 20 ml of TFA and kept
for
1 h at room temperature. After TFA evaporation, the residue was triturated
with
ethyl ether and dried. The salt obtained (14.5 g of TFA~H-Orn(Fmoc)-Gly-Gly-
OH,
24.8 mmole), was dissolved at 0 °C in 50 ml of 5% NaHC03 and 150 ml of
DMF
and left to react with 8.78 g of Fmoc-Gly-OSu (22.3 mmole) for 1 h at 0
°C and
~s overnight at room temperature. After DMF evaporation, the residue was
dissolved
in 10% citric acid, filtered and washed several times with water. The crude
product
was crystallised from ethyl acetate. Yield: 14 g; M.p.: 208-210 °C;
R.f.: 0.63;
HPLC, 23.68 min; gradient (I); [a]p22-20 (c1, DMF).
NMR (DMSO) 8 ppm: 1.32-1.8, m 4H; 2.92-3.06, m 2H; 3.65-3.79, m 6H; 4.18-
~0 4.36, m 7H; 7.31-7.9, m 18H; 7.98, d 1 H; 8.1, t 1 H; 8.25, t 1 H; 12.5, bs
1 H. MS:

CA 02380178 2002-O1-22
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13
748 Da.
3. Synthesis of 2I2I2IAc-Gly-Orn(Ac,-Gly-Gly]Gly-Orn-Gly-Gly]Gly-Orn-Gly_
GIy~lGly-Orn-Gly-GI -OOH.
The synthesis was carried out on a Milligen 9050 machine, using a 0.5 cm
(1.D.)
s column, loaded with 0.5 g of Fmoc-Gly-PEG-PS (Millipore) resin. Loading:
0.18
mmole/g.
1St cycle: a) deprotection: 20% piperidine in DMF, 4 min, flow: 8.1 ml/min; b)
whashing: DMF, 10 min, flow: 4.0 ml/min; c) coupling: 134 mg of Fmoc-Gly-
Orn(Fmoc)-Gly-Gly-OH, 68 mg of HBTU and 28 mg of HOBt were dissolved
Io manually in 0.6 ml of 0.6M N-methylmorpholine (NMM) in DMF and 0.4 ml of
DMF
and then loaded into the column (automatic protocol). Recycle: 5 h, flow: 8.1
ml/min; d) washing: DMF, 15 min, flow: 4.0 ml/min.
2nd cycle: 268 mg of Fmoc-Gly-Orn(Fmoc)-Gly-Gly-OH, 136 mg of HBTU and 56
mg of HOBt dissolved in 1.2 ml of 0.6 M NMM in DMF and 0.3 ml of DMF were
is employed for coupling. A small sample of resin was extracted from the
column,
treated with 20% piperidine in DMF, carefully dried and treated again with
TFA/water (95/5, v/v) for 1 h at room temperature. A single HPLC peak a 2.8
min,
gradient (I), was observed.
3rd cycle: Two couplings were performed. In the first coupling, 400 mg of Fmoc
2o Gly-Orn(Fmoc)-Gly-Gly-OH, 208 mg of HBTU and 80 mg of HOBt dissolved in 1.8
ml of 0.6 M NMM in DMF and 0.2 ml of DMF were employed. Three consecutive
washings with DMF (20 min, flow: 4.0 ml/min), DCM (10 min, flow: 9.0 ml/min)
and
DMF (5 min, flow 4.0 ml/min) were carried out. In the second coupling, 200 mg
of
Fmoc-Gly-Orn(Fmoc)-Gly-Gly-OH, 104 mg of HBTU and 40 mg of HOBt dissolved
2s in 0.9 ml of 0.6 NMM in DMF and 0.1 ml of DMF were employed. Recycle: 3h;
flow: 8.1 ml/min; three washings with DMF and DCM as before. A small sample of
resin, extracted from the column and analysed as before, gave a single HPLC
peak at 6.3 min with gradient (II).
4th cycle: Two couplings were performed. 800 mg of Fmoc-Gly-Orn(Fmoc)-Gly
3o Gly-OH, 416 mg of HBTU, 160 mg of HOBt dissolved in 3.6 ml of 0.6 M NMM in
DMF and 0.4 ml of DMF were employed for the first coupling. Recycle: 3;5 h;
flow:
8.1 ml/min; three washings with DMF, DCM and DMF. In the second coupling, 400

CA 02380178 2002-O1-22
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14
mg of Fmoc-Gly-Orn(Fmoc)-Gly-Gly-OH, 208 mg of HBTU and 80 mg of HOBt
dissolved in 1.8 ml of 0.6 M NMM in DMF were employed. Recycle: 5 h; flow: 8.1
ml/min. The resin was washed and analysed as before. A single, broader HPLC
peak was observed at 8.1 min; gradient (II). The resin was then treated with
20%
s piperidine in DMF for 10 min at a flow of 8.1 ml/min, washed for 15 min with
DMF
at a flow of 4.0 ml/min. and acetylated with 1 M acetic anhydride and 1 M NMM
in
DMF for 1 h, flow: 8.1 ml/min. Finally, the resin was extracted from the
column,
washed with DMF, methanol, DCM and ethyl ether and dried under vacuum
overnight. The peptide monodendron was obtained by suspending the resin in 15
to ml of TFA/water (95/5, v/v) for 1 h at room temperature under stirring.
After
filtration, the resin was washed with 1 ml of TFA and the combined filtrates,
after
partial evaporation of TFA, were added to cold ethyl ether to precipitate the
polypeptide. The mixture was kept at -20 °C for about 3 h. After
filtration, the white
product was dissolved in water and lyophilized three times. Yield: 420 mg. A
is dominating, broad HPLC peak was observed at 8.1 min, gradient (I), together
with
two very small peaks corresponding to products of the second and third cycle.
The
product has been purified by Size Exclusion Chromatography (SEC) on Sephadex
G-50, using 50% acetic acid as eluant. The fractions containing the target
peptide
were lyophilized twice after dilution with water. Yield: 350 mg. MS: 5,021 Da
Zo (Theor. 5.022 Da).
4) Synthesis of N~CH2-CH2-NH-CO-CH(CH2-phenyl)-NH-G~-Gly-Gly-Orn-
GIyjGly-GI~Orn-Gly[Gly-Gly-Orn-GIY[GIG Orn Ac)GIyAc12121213
7.33 mg of N[CH2-CH2-NH-CO-CH(CH2-phenyl)-NH2]3~4HC1 (0.01 mmole),
200.1 mg of the monodendron prepared as reported in 3) (0,04 mmole), 9.6 mg of
Zs WSC ~ HCI (0.5 mmole), 7.7 mg of HOBt (0.5 mmole) and 5.6 p1 of
triethylamine
(TEA) (0.04 mmole) were dissolved in 15 ml of DMF, treated with TEA to reach
an
apparent basic pH, and left to react for 48 h at room temperature under
stirring.
After DMF evaporation, the residue was dissolved in 10 ml of methylethylketone
and the solution extracted with 5% NaHCOg (3x10 ml) and brine (3x10 ml),
~o acidified with 0.1 M HCI and dried over Na2S04. The solid recovered after

CA 02380178 2002-O1-22
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evaporation of the solvent was washed four times with ethyl ether, dried under
vacuum, dissolved again in 50% acetic acid and purified by SEC on Sephadex G-
50 as reported before. Yield: 161 mg; MS: 15,605 Da (Theor: 15,602 Da). The
product has been characterised further by SEC HPLC using a 75HR10/30
s Pharmacia Superdex column (stationary phase: cross-linked agarose-dextran,
13
Vim), using 50 mM NaH2P04 and 100 mM Na2S04 pH 6,5 as eluants; flow: 0.5
ml/min; detection, 220 nm. A single broad peak was observed at 18 min.
Ribonuclease (MW=13,400 Da), Bovine Serum Albumin (BSA) monomer
(MW=66,000 Da) and dimer (MW=112,000 Da) show peaks at 25, 20 and 18 min,
to respectively. These results indicate that the acetylated generation 4
dendrimer
aggregates in the buffer used for SEC HPLC.
EXAMPLE 2
This example describes a three step synthesis of a generation 4 dendrimer
prepared entirely in liquid-phase. In the first step, a generation 2
monodendron
is with NH2 terminals protected by an acid labile group is condensed on a
triamine
core to obtain a generation 2 dendrimer. In the second step, after acidolysis,
the
monodendron is again condensed to the free NH2 terminals of the generation 2
dendrimer to obtain a generation 4 dendrimer. In the third step, after removal
of
the protecting groups, the dendrimer NH2 terminals are acetylated.
1 ) Synthesis of Z-Orn~Bocw-Gly-OCH3
10.44 g of Z-Orn(Boc)-OH (28.5 mmole), 5.75 g of WSC ~ HCI (30 mmole), 4.59 g
of HOBt (30 mmole), 5.47 g of HCI ~ H-Gly-Gly-OCH3 (30 mmole), and 5.6 ml of
TEA (40 mmole) were dissolved in 90 ml of DMF, treated with TEA until basic pH
and then left to react for 12 h at room temperature under stirring. After DMF
Zs evaporation, the residue is dissolved in 300 ml of ethyl acetate and washed
with
0.1 M HCI/brine 1/2 (3 x 40 ml), 5% NaHC03/brine 2/1 (5 x 40 ml) and again
brine
(30 ml). The solution is then acidified with 0.1 M HCI and dried over Na2S04.
The
solvent is then almost completely evaporated and the target product recovered
by
slow crystallization from ethylether/petroleum ether 1/1 v/v. Yield: 13.7 g. A
single
~o HPLC peak was observed at 18.2 min; gradient (I).

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16
2~Synthesis of Boc-GI -~~Boc)-Gly-Gly-OH
13 g of Z-Orn(Boc)-Gly-Gly-OCH3 were dissolved in 170 ml of methanol and
treated with 750 mg of 10% C/Pd. Hydrogenation is continued for 2 h at room
temperature. After elimination of the solid by filtration the resulting
solution is
s concentrated and the product slowly crystallized from ethyl ether petroleum
ether
1 /1. Yield: 9.7 g.
8,83 g of H-Orn(Boc)-Gly-Gly-OCH3 (24.5 mmole), 6.26 g of Boc-Gly-OSu (23
mmole) were dissolved in 30 ml of DMF. 10 mmole of TEA were added to the
solution after 7 h at 0°C. The reaction was continued for 24 h at room
temperature.
to Following DMF evaporation and addition of 300 ml of ethyl acetate, the
organic
solution was extracted 1 M HCI/brine 1/2 (3 x 30 ml), 5% NaHC03/brine 1/1 (3 x
30 ml) and brine ( 2 x 30 ml). After acification with 1 M HCI, and solvent
evaporation, the product was isolated by crystallization from ethyl ether.
Yield:
11.8 g. A single HPLC peak was observed at 15.5 min; gradient (I).
is 5,18 g of Boc-Gly-Orn(Boc)-Gly-Gly-OCH3 (10 mmole) were reacted with 1 M
NaOH in methanol (50 ml) for 15 h at room temperature. After alcohol
evaporation,
the residue was dissolved in 200 ml of ethyl acetate and extracted with 30 ml
of 1
M HCI saturated with NaCI and brine (2 x 20 ml). After acidification and
solvent
elimination, the product was isolated by crystallization from ethyl
ether/petroleum
?o ether 1/1 v/v. Yield: quantitative. A single HPLC peak was observed at
14.48 min;
gradient (I). MS: 527 Da, 543 Da and 565 Da for M-Na+, M-K+ and M-K+-Na+
(Theor: 504 Da).
3~ynthesis of [Boc-Gly-Orn Boc)-Gly-GIyl2-Gly_Orn-Gly-GIL-OH
4,03 g of Boc-Gly-Orn(Boc)-Gly-Gly-OH (8.0 mmole), 1.48 g of HCI ~ H-Gly-
zs Orn(HCI)-Gly-Gly-OCH3 (3.8 mmole), 1.69 g of WSC~HCI (8.8 mmole), 1.35 g of
HOBt (8.8 mmole) and 1.12 ml of TEA (8.0 mmole) were dissolved in 30 ml of
DMF at 0°C. The reaction was kept for 15 h at room temperature under
stirring.
After DMF evaporation, the residue was dissolved in 200 ml of
methylethylketone.
The solution was extracted with 1 M HCI/brine 1/1 (4 x 20 ml), 5% NaHC03 (3 x

CA 02380178 2002-O1-22
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17
20 ml) and brine (3 x 20 ml). After acidification with 1 M HCI, the solvent
was
evaporated and the product crystallized from ethyl acetate/ethyl ether'/< v/v.
Yield:
ca. 4 g of methyl ester were isolated after three further washings with ethyl
ether.
3.97 g were dissolved in 50 ml of warm methanol, the solution left to
equilibrate at
s room temperature and then treated with 4 ml of 1 M NaOH for 16 h. After
evaporation of the solvent, the residue was dissolved in 200 ml of
methylethylketone and 10 ml of 1 M HCI and 20 ml of brine were added to the
solution. The solution was carefully extracted and neutralized with brine (3 x
30
ml). The organic layer was then dried on Na2S04, filtered and the solvent
Io evaporated. The crude product was crystallized from ethyl acetate and the
solid
obtained washed three times with ethyl ether. Yield: 3.7 g. A single HPLC peak
appeared at 19.14 min; gradient (I). MS: 1,298 Da and 1,314 Da for M-Na+ and M-
K+ (Theor.: 1,275 Da).
Synthesis of N~CH2-CH2-NH-CO-CH(CH2-phen~ -fly-Gly-Orn-Gly[GI rL-
is Gly-Orn(Boc)-Gly Boc12~3
510 mg of [Boc-Gly-Orn(Boc)-Gly-Gly]2-Gly-Orn-Gly-Gly-OH (0.4 mmole), 73.3 mg
of N[CH2-CH2-NH-CO-CH(CH2-phenyl)-NH2J3~HCI (0.1 mmole), 96.0 mg of
WSC ~ HCI (0.5 mmole), 77.0 mg of HOBt (0.5 mmole) and 56 ~I of TEA were
dissolved in 20 ml of DMF at room temperature. TEA was added to a basic pH and
2o the mixture left to react for 48 h under stirring. After DMF evaporation,
the residue
is dissolved in 100 ml of methylethylketone and the solution extracted with
0.5%
NaHC03 (3 x 20 ml) and brine (3 x 20 ml). Following acidification with 1 M
HCI,
the organic solution is dried over Na2S04, filtered and evaporated to obtain a
white powdery solid which was repeatedly washed with ethyl ether. Yield: 450
mg.
2s A single HPLC peak was observed at 22.69 min; gradient (I). MS: 4,359 Da
and
4,381 Da for M-H+ and M-Na+ (Theor.: 4,355 Da).
5wnthesis of NfCH2-CH2-NH-CO-CH(CH2-phenyl)-NH-Gly-Gly-Orn-GIyfGly-
Gly-Orn-Gly(Gly-Gly-Orn-GI~GIy-Gly-Orn(Ac)-Gly-Ac]212123
436 mg di N{CH2-CH2-NH-CO-CH(CH2-phenyl)-NH-Gly-Gly-Orn-Gly[Gly-Gly-

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18
Orn(Boc)-Gly-Boc]2}3 (0.1 mmole) were dissolved in 2 ml of warm DMSO. 15 ml
of 4 M HCI in dioxane were then added to the solution left to equilibrate at
room
temperature and the reaction kept for 1 h under stirring. The salt obtained
was
triturated and isolated by centrifugation at 2,000 rpm. After two washings
with ethyl
s acetate, the hygroscopic product was dried under vacuum over P2O5.
363 mg of salt, (0.1 mmole) were dissolved in 2 ml of water, neutralized with
0.1 M
NaOH and added to a 5 ml DMF solution containing 1.53 g of [Boc-Gly-Orn(Boc)-
Gly-Gly]2-Gly-Orn-Gly-Gly-OH (1.2 mmole), 250 mg of WSC ~ HCI (1.3 mmole),
200 mg of HOBt (1.3 mmole) and 210 ~I of TEA (1.5 mmole). The solution was
left
to to react for 48 h at room temperature under stirring. After DMF
evaporation, the
solid was dissolved in 50 ml of methylethylketone and the solution extracted
with
5% NaHC03 (3 x 20 ml) and brine (3 x 20 ml). After acidification with 1 M HCI
and
drying over Na2S04, the organic layer was filtered and evaporated to obtain a
solid residue which was, in turn, repeatedly washed with ethyl ether. The
solid,
is dried under vacuum, was again dissolved in 20 ml of TFA/water 98/2 v/v and
left to
react for 2 h under agitation. The residue obtained after solvent elimination
was
repeatedly washed with ethyl ether and dried under vacuum.
800 mg of trifluoroacetate salt (3.6 mmole) were dissolved in 10 ml of
DMF/water
1 /1 vlv with 905 mg of p-nitrophenylacetate (5.0 mmole) and 700 ~.I of TEA (5
2o mmole). The solution was left to react for 50 h. After evaporation of the
solvent,
the residue was repeatedly washed with ethyl ether and dried under vacuum.
Yield: 1.1 g. The crude was purified by SEC on Sephadex G-50, with 50% acetic
acid as eluant. The fractions containing the target product were pooled and
lyophilized. MS: 15,439 Da (Theor.: 15,431 Da). The MW of the dendrimer has
2s been also determined by SEC HPLC, using a 75HR10/30 Pharmacia Superdex
column, as described in Example 1 ). R.t.: 18 min. The dendrimer is then
identical
to that prepared following the strategy of Example 1 ).
EXAMPLE 3
This example illustrates the synthesis of a generation 7 dendrimer containing
4-[4-
~o (1-(amino)ethyl)-2-methoxy-5-nitrophenoxy]butanoic acid photocleavable
residues.

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NO~
CH30
C~CH3 -Nw-Gly-Orn-Gly[GI~GIy-Orn-Gly[GI~GIy-Orn-Gly[Gly-G~-Orn-
s GIyfGly-G~-Orn-GIY[Gly-Gly-Orn-GIY[Gly-GI -~~Ac)-Gly-Ac121212121212
The synthesis was carried out on a Milligen 9050 apparatus, using a 0.5 cm
(1.D.)
column, loaded with 0.1 g of Fmoc-Cys(Trt)-PEG-PS (Millipore) resin. Loading:
0.16 mmole/g.
In the first cycle of the chain assembly, 4-[4-(1-Fmoc-aminoethyl)-2-methoxy-5-
io nitrophenoxy)butanoic acid was condensed to the cysteine(Trt) residue on
the
resin after Fmoc removal with 20% piperidine in DMF. All subsequent cycles for
the synthesis of the monodendron were conducted following the protocol
described in Example 1 ), using Fmoc-Gly-Orn(Fmoc)-Gly-Gly-OH and the same
solvents and reagents. Yield: 480 mg. After cleavage with TFA/water 95/5 v/v,
the
is crude was purified by SEC on Sephadex G50, using 50% acetic acid as eluant.
The fractions containing the target product were diluted with water and
lyophilized
three times. MS: 41,980 Da (Theor.: 41,972 Da).
/ CO
21 Synthesis of NfCH~-CHI-N---CO-CH-S-CHI-CH(COOH)-NH-
2o N02
N H-Glv-Glv-Orn-GIvfGlv-Glv-Orn-Glvf Glv-Glv-Orn-
CH3
G ly[G ly-G ly-Orn-G Iyf Gly-Gly-Orn-Gly(Gly-GIY-Orn-Gly[G ly-Gly-Orn(Ac)-Gly-
Ac 2121212121213
2s 3.86 mg of Tris(2-maleimidoethyl)amine (Pierce) (0.01 mmole) and 4.0 g of
N02
HOOC-CH(CH2-SH)-NH-CO(CH2)3-O ~ ~ CH(CHg)-NH-Gly-Gly-Orn-
OCH3
3o Gly[Gly-Gly-Orn-Gly[Gly-Gly-Orn-Gly[Gly-Gly-Orn-Gly[Gly-Gly-Orn-Gly[Gly-Gly-

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Orn-Gly[Gly-Gly-Orn(Ac)-Gly-Ac]2]2~2~2~2~2 (0.1 mmole) were dissolved in 5 ml
of
DMF/water 10/90 v/v at room temperature under agitation and left to react for
3 h
at an apparent pH of 7Ø 10 g of thiol-Sepharose 4B resin, preactivated with
2,2'-
dipyridyldisulfide, were then added to the solution to sequester the unreacted
s monodendron by thiol-disulfide exchange in 7 ml of PBS buffer (pH 7.3).
After
elimination of the resin, the solution was evaporated, diluted with water and
lyophilized. The crude was subsequently purified by SEC on Sephadex G50, using
50% acetic acid as eluant. The fraction containing the target product were
diluted
with water and lyophilized. Yield: 751 mg. MS: 126,309 Da (Theor.: 126,299
Da).
io EXAMPLE 4
This example shows the stability of the polypeptide dendrimers described in
Examples 1-3 to enzymatic hydrolysis in vitro.
The degradation in vitro was studied against Leucine-aminopeptidase VI (E.G.
3.4.11.1 ), isolated from pig kidneys, whose activity has been previously
checked
Is with leucine-4-nitroanilide. Dendrimer concentration: 1.10-3 M in 50 mM
Tris.HCl
buffer, pH 8.5, containing 5mM MgCl2. Enzyme concentration: 3 U/ml. The
experiments were performed at 37oC in an oscillating bath. Samples (100 NI
each), withdrawn at fixed time intervals, were blocked with 10% TFA and
centrifuged (10.000 x g , 5 min) before HPLC measurements that were performed
20 on a Waters mod. 660 apparatus equipped with a Lichrosorb RP 18 (10~m)
column. Detection was by a Jasco Uvidec-100-I I detector. Eluant A was 0.1 %
TFA
in water; and eluant B was 0.1 % TFA in acetonitrile; gradient: from 0% B to
21 % B
in 23 min.
The degradation in heparinated human plasma was studied using dendrimer
2s concentrations of ca. 1.0 nmole/ml plasma at 37oC, as described above. The
extent of degradation with time was obtained by comparing the area of the HPLC
signals appearing at a given time to that registered initially. The half-life
of the
generation 4 dendrimer with free amino terminals is ca. 12 h against Leucine-
aminopeptidase VI and ca. 8 h in human plasma. The acetylated generation 4 and
~0 7 dendrimers resulted less labile either to enzymatic degradation by
Leucine-
aminopeptidase VI (half-life, 23 h) or in human plasma (half-life, 16 h).
EXAMPLE 5

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21
This example illustrates the loading by diffusion of the Enkephalinase
inhibitor L-
Trp-L-Ala in a generation 6 polypeptide dendrimer prepared as in Example 1 )
and
its release with time.
30 mg of a generation 6 polypeptide dendrimer with free amino terminals,
s prepared as in Example 1 ), were added to 2 ml of an aqueous solution of 8
mg of
L-Trp-L-Ala and after 24 h the clear solution was precipitated with 15 ml of
ethanol
under stirring. The precipitate was centrifuged, washed with anhydrous ethanol
and dried under vacuum over P205. Yield: 29 mg. 10 mg of the isolated product
were then dissolved in 10 ml of water and the solution injected into a 3-15 ml
to "Slide-A-Lyzer Dialyzer Cassette" (Pierce) ("cut-off', 10,000 Da). The
dialysis was
run against 100 ml of water for 48 h under slow stirring. The absorbance at
280
nm of 200 ~.I solution aliquots, diluted with water to a final volume of 1 ml,
was
determined every 30 min. Increasing absorbance values observed during ca. 12 h
of dialysis indicated a gradual release with time of the dipeptide by slow
diffusion
is from the dendrimeric carrier. A28o of the solution outside the dialysis
cassette
resulted slightly lower (-6%) than that of a reference solution prepared by
dissolving 10 mg of dipeptide in 110 ml of water.
EXAMPLE 6
This example illustrates: a) the entrapment of heparin into a generation 7
2o polypeptide dendrimer containing photolabile bonds during condensation of
the
generation 7 monodendron to a trifunctional core carried out in the presence
of
heparin and b) the release of heparin by photolysis of the loaded dendrimer.
1 ). 1.12 g of sodium heparinate (obtained by depolimerization of ovine
heparin,
MW ca. 2,500 Da; activity, ca.180 IU/mg.) were added to the reagents used in
2s Example 3.2 for the synthesis of the generation 7 dendrimer, at an apparent
pH of
7Ø The monodendron condensation was protracted for 3 h at room temperature.
After elimination of the monodendron in excess with thiol-Sepharose 4B resin,
preactivated with 2,2'-dipyridyldisulfide, and filtration of the resin, the
resulting
clear solution was directly loaded on a. Sephadex G-75 column. The dendrimer
3o was eluted with water at a flow of 0.5 ml/min to separate it from the
excess of
heparin. Yield: 1.04 g.
2). 750 mg of "loaded" dendrimer were dissolved in 6 ml of water and
irradiated at

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22
360 nm for 600 min in a quartz vial. Then, 1 ml of the irradiated solution was
injected intravenously to each of six male rats (ca. 600 g body weight)
deprived of
food for 12 h before the beginning of the experiment (Rats 3-8). The same
procedure was repeated using 1 ml of a non-irradiated solution of 750 mg of
s "loaded" dendrimer dissolved in 6 ml of water for six male rats of similar
weight
(Rats 9-14). Rat 1 is not injected at all, while rat 2 receives an intravenous
injection of 250 mg of heparin dissolved in 1 ml of water. The anticoagulant
effect
of heparin i.e. the time needed to form a fibrin clot for serum samples taken
from
the vein of the tail, was ascertained by the APTT (Activated-Partial
Thromboplastin
io Test) test. The results are reported below
Coagulation time (seconds)
Rat Treatment t=0 1 h 2 h 3 h 4 h 24 h
1 25 26 25 - - -
2 Heparin (iv) 27 54 >300 - - -
3 irradiated dendrimer 25 53 >300 - - -
4 id 26 57 >300 - - -
id 25 54 >300 - - -
6 id 24 56 >300 - - -
7 id 26 55 >300 - - -
8 id 27 54 >300 - - -
9 non-irradiated dendrimer 27 36 85 130 260 28
id 26 39 91 149 252 26
11 id 25 34 90 141 257 28
12 id 26 37 89 153 260 29
13 id 25 40 94 160 268 28
14 id 26 38 89 156 259 25
Within two hours, rats 3-8 showed coagulation times close to those of rat 2,
treated with heparin only. Rats 9-14, treated with the non-irradiated
dendrimer,
showed an increase of coagulation times during four hours. At the first hour,
the
coagulation times are slightly less than that observed for rat 2 after two
hours from
is heparin injection. The coagulation times for rats 9-14 becomes normal after
24 h.
All together, the above results indicate that: a) low MW heparin is entrapped
inside

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23
the dendrimeric carrier; b) photolysis of the photolabile residue incorporated
in the
dendrimer backbone determines the release of heparin from the carrier and c)
the
non-irradiated dendrimer gradually releases heparin in parallel with the slow
enzymatic demolition of its structure in blood.
s EXAMPLE 7
This example reports: a) the absence of immunogenicity in mice of the
generation
4 dendrimer obtained as described in Example 2) and b) its adjuvanticity when
some of the NH2 terminals are covalently linked to the octapeptide antigen Asn
Ala-Asn-Pro-Asn-Ala-Asn-Pro (a short segment of the immunodominant epitope of
to the Plasmodium falciparum Circumsporozoite Protein).
1 ) Immunogenicity Y-of NfCH2-CH2-NH-CO-CH(CH2~henyl~-NH-Gly-GI -y Orn-
Gly[Glv-Glv-Orn-GIy~G~-Glv-Orn-GIvfGlv-Glv-Orn-Glv-H12121213
50 ~g of acetylated dendrimer were dissolved in 50 p,1 of Freund Complete
Adjuvant and injected to 5 groups of C57/8L/6 mice (7-10 mice per group) at
the
is base of the tail. After 3 weeks, 25 ~,g of dendrimer, emulsionated in 25
~.I of
Freund Incomplete Adjuvant, were injected to mice following the same
procedure.
After 10 days, a blood sample was taken from each mice by puncturing the retro-
orbital plexus. Plasma samples were evaluated for the presence of anti-
dendrimer
antibodies by ELISA. Briefly, microtitre 96-well plates (Maxisorp F 96, Nunc,
2o Denmark) were coated overnight in a humid chamber at 4°C with 100 ~I
of a
solution containing 1 ~g/ml of acetylated dendrimer in PBS at pH 7.2. Plates
were
then saturated with PBS and 5% non-fat dry milk for 2 h at room temperature.
After three washings (phosphate buffer, pH 7.4 and 0.05% Tween-20), sera that
were serially diluted in PBS, 2.5% non-fat dry milk and 0.05% Tween 20 were
2s added to the plates for 1 h at room temperature. After washings, rabbit
anti-mice
IgG-specific polyvalent immunoglobulins conjugated to alkaline phosphatase,
diluted in PBS, 2.5% non-fat dry milk and 0.05% Tween 20 were added for 1 h.
Plates were washed and the presence of enzyme evidenced with p-
nitrophenylphosphate substrate. Absorbance at 405 nm was measured with a
3o Dynatech 25000 ELISA reader. Antidendrimer antibodies were not detected. To
avoid the risk of removal of the dendrimer from the wells during reiterated
washings, the experiments were repeated after conjugation (DCI/ HOBt as

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24
coupling reagents, room temperature, 24 h) of the non-acetylated dendrimer to
polyethylene pins, y-irradiated in a 6% v/v aqueous solution of acrylic acid
(M.
Geysen et al., Proc. Natl. Acad. Sci., USA, 1984, 81, 3998-4002). The
antidendrimer antibodies were detected by dipping the polyethylene pins into
the
wells of the microtitration plate, operating as described before. No
antidendrimer
antibodies were again detected in mice sera.
2) Coniugation of Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro to NfCH2-CH2-NH-CO-
CH CH2~henyl -Nw-Gly-Orn-Gl~y-GI -y Orn-G~LGI~y-Orn-Gly[Gly-Gly-
Orn-Gly-H1212121.3
io 400.6 mg of N{CH2-CH2-NH-CO-CH(CH2-phenyl)-NH-Gly-Gly-Orn-Gly[Gly-Gly-
Orn-Gly[Gly-Gly-Orn-Gly[Gly-Gly-Orn-Gly-H]2]2]2}3 (1.8 mmole) were dissolved
in
ml of DMF together with 636 mg of Fmoc-Asn-Ala-Asn-Pro-OH (1.0 mmole),
192 mg of WSC~HCI (1.0 mmole), 154 mg of HOBt (1.0 mmole) and 460 u1 of
TEA. The solution, brought at basic pH with TEA, was stirred for 10 h at room
is temperature and then treated with 218.1 mg of (Boc)20 (1.0 mmole) after
addition
of 500 ~.I of TEA. The mixture was kept under agitation for 10 h, treated with
5 ml
of piperidine, stirred for 2 h and finally precipitated by adding 100 ml of
ethyl ether.
The product was dissolved in 10 ml of water and purified by SEC on Sephadex G-
50 using 50% acetic acid as eluant. The fractions containing the target
product
2o were recovered by lyophilization after dilution with water. 400 mg of the
solid were
again dissolved in 10 ml of DMF and the coupling of Fmoc-Asn-Ala-Asn-Pro-OH to
the dendrimer was repeated once more. After addition of 5 ml of 20% piperidine
in
DMF, and stirring for 3h at room temperature, 100 ml of ethyl ether were added
to
precipitate the product. Yield: 305 mg. The compound was again dissolved in 5
ml
2~ of TFA/water 95/5 v/v and, after one hour at room temperature, 100 ml of
ethyl
ether were added to precipitate a white powdery solid. Following drying over
P2O5
in vacuum, the crude was purified by SEC on Sephadex G-50 Superfine, using
50% acetic acid as the eluant. Yield: 280 mg.
3) Assessment of the adiuvant properties of N~CH2-CH2-NH-CO-CH(CH2-
~o ~henyl)-NH-Gly-Gly-Orn-Gly[Gl~y-Orn-GIyfGly-Gly-Orn-GIyfGly-Gly-Orn(-Pro-

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Asn-Ala-Asn-Pro-Asn-Ala-Asn)-Gly-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Asn121212~3=
Each component of five groups of BALB/c female mice, 7-10 mice per group,
(OLAC, Bicester, Oxon, UK) was injected with 500 ~g of antigen-dendrimer
conjugate dissolved in 50 ml of water as described before. In parallel, the
same
s number of C57/8L/6 mice were injected with 50 ~g of Asn-Ala-Asn-Pro-Asn-Ala-
Asn-Pro dissolved in 50 ~I of water. After three weeks, 25 and 250 p.g of the
same
products were injected again to the two groups of mice. 10 days after, a
sample of
blood was taken from each mice as described before. The sera were tested by an
ELISA test employing (Asn-Ala-Asn-Pro)40 as the antigen. (G. Del Giudice et
al.,
to J. Clin. Microbiol, 1997, 25, 91-96). The antigen-dendrimer conjugate shows
higher anti-Asn-Ala-Asn-Pro antibody titers (as the logarithmic geometric mean
of
antibody titers ~ S.E.M.) at week 45 (4.10~0.01 ) as compared to Asn-Ala-Asn-
Pro-
Asn-Ala-Asn-Pro antigen (2.81~08).
Taking into account all the above results, the polypeptide dendrimers of the
1 s present invention, obtained by chemical synthesis, satisfy the foreseen
objectives.
In particular, unimolecular polypeptide dendrimers can be obtained with the
processes of synthesis described and, furthermore, the practicality of
dendrimer
loading and of controlled release of guest molecules in vivo by enzymatic
hydrolysis and through the application of ultraviolet irradiation has been
2o demonstrated. Applications of the unimolecular carrier polypeptide
dendrimers/guest molecules system in composition with pharmaceutically
acceptable excipients in the medical field are widespread and potentially of
extreme importance namely chemotherapy of cancer, anticoagulant and clot-
dissolving drug therapy, antiviral therapy, vaccines, controlled release of
2s hormones and related bioactive substances. For medical diagnosis, the
controlled
methods of synthesis described above give the possibility to prepare metal
chelates of dendrimeric carriers with precisely defined molecular weights, so
that
the drawbacks due to the presence of imperfect carrier structures are avoided.
Applications to medical diagnosis and therapy are no meant to be restricted to
3o those implementations described, as many other possibilities will be clear
to one
skilled in the medical arts.