ZA200201089B

ZA200201089B - Polypeptide dendrimers as unimolecular carriers of diagnostic imaging contrast agents, bioactive substances and drugs.

Info

Publication number: ZA200201089B
Application number: ZA200201089A
Authority: ZA
Inventors: Antonio Verdini
Original assignee: Servier Lab
Priority date: 1999-07-23
Filing date: 2002-02-07
Publication date: 2003-07-30
Also published as: PL353273A1; WO2001007469A3; JP2003506326A; ITFO990015A1; CA2380178A1; NZ517231A; AU6276600A; CN1364171A; EP1200461A2; NO20020333L; HUP0201975A3; WO2001007469A2; HUP0201975A2; NO20020333D0; AU772167B2

Description

v ( 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

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, 1s 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 ¢) 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 microenvironment that can be utilized for the entrapment or the covalent coupling a 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, 1-66). Computer modelling of PAMAM dendrimers 25s has shown how the number and dimensions of cavities depend from a) the number (Ng) of functional groups of the core moiety, b) the number (Np) of reactive sites of the branching unit and c) the dimensions and rigidity of the branching unit. When Ng=3 or 4 and Np=2, the PAMAM dendrimer series increases its diameter by approximately 10 A per generation, evolving from a disk- 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-

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 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 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 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.

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- terminated hydrocarbon dendrimers. Acetyisalycilic acid and 24 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.

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,

¥ oo A 3

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 5s 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 the seventh generation dendrimer have been also observed. Haemolysis and cytotoxicity have been observed for amine-terminated PAMAMSs, 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 (lll) chelate of the ‘PAMAM-thiourea-diethylenetriaminepentaacetic acid magnetic resonance imaging contrast agent (Gd(l11}-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 : antibody to a PAMAM dendrimer of generation 2, functionalized at the surface with - derivatives of tetraacetic or pentaacetic acid for chelation of *Y, '"'In, 2'?Bi and :

Gd(Ill), the feasibility of monoclonal antibody guided radiotherapy and imaging has been demonstrated. Boronated dendrimer-monocional antibody conjugates have been used successfully as protein probes in electron spectroscopic imaging. The 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,

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 x \ ‘ 4 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 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 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 PAMAMSs 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. 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 © 20 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 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 10 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

I

. , (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, 5 sialic acid, mannose, fat acids, vitamins, hormons, oligonucleotides, monocional 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; il. 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 (1):

K(-L),-M )) wherein

K is a multifunctional core moiety, .

Lis 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 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 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

\ ‘ 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 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 (1):

K(-L),-M " wherein: K is the multifunctional core moiety and K can be represented by the formulae: (in) X~-(CH2)n-X", wherein X=X" or XX", and X, X* are NHor CO or S; or (1) Y[-(cH2)n-Zl;, wherein Y=C or Y=N; Z is NH or S or Cl or Br or | 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 (CHo)m-X", m=1-5, R' is methyl or ethyl or butyl or isopropyl,

X=X'or X«X', and X, X' are NH or CO or S and n=1-6;

L is the single monodendron whose propagators can be represented by the formulae: (V) -CO-CH(R?)-(CH2),-NR?- wherein R2=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

SH or Ci or Br; R%-CH(CH2),-NR? is a 5 or 6 atoms ring, and n=0-6; and (V1) -CO-CH(R?)-CO-N(R3)-(CH2)m-N(R?) wherein R? 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-CH,-NH-NH; -CO-CH(R?)-O-; -CO-CH2-0-N=CH-CO-; -CO-CH(R?)-(CH2),-S-

CH,-CO-W; -CO-NH-CH(CH2-SH)-CO-W, “CON-GH-CO-W X

HO-CHo-CH-T-CH-Q

¥ . , -co-{)-cz-0- -CO-CHa “)~CO-CH(CH3)-0- i

NO2 NO2 -€0-CHp-04_)CO-CH(CH3)-0- coer pencHgo:

CH30

NO2 NO» ‘coor Y-oHz 0: 00-10 )-CH(CHgHH

OCH3 OCH3 wherein W=-N(R?®)-(CH2)m-NR?, Q=H, -CH3; T is O or S while R?, R? and m have the meaning seen before and p=1-4;

Mis the residue represented by formula (Vit): -Aq-B(Ar)-C-A{Aq-B(Ar)-C-AAq-B(A-D)-C-Ar-D]2]2 (Vin) wherein A=-CO-CH(R?)-(CH2)n-NR?; R® and n have the meaning seen before; q=1-6; r=1-4 and R? 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 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: col _)-crzo- -co-cHp{ D-co-cH(cHa)0-

NO2

NO2 ) €0-CHz-04_M)CO-CH(CH3)-0- cor YeHHayo-

OCH3

NO2 NO2 00H )-crz 0: coco ancy:

OCH3 CH3

Dis a residue represented by formulae (VIII)-(XI): -Aq-B(A-E)-C-Aq-E (vir) -Aq-B(Ar)-C-AqlAq-B(ArE)-C-Aq-E]2 (IX) -Aq-B(Ar)-C-AqlAq-B(Ar)-C-Aq-IAq-B(A-E)-C-Ag-E]2]2 (X) -Aq-B(Ar}-C-AqlAq-B(Ar-C-Ag-[Aq-B(Ar)-C-AglAq-B(A-E)-C-Ag-El2]2]2 (XI)

\ ‘ wherein A, B, C, q ed r have the meaning seen before, and E is represented by formulae (X11) and (XII): -Agq-B(A-P)-C-Ag-P" (Xi) -Aq-B(Ar)-C-Aql-Agq-B(A-P)-C-Aq-P'l2 (Xi) s wherein A, B, C, q and r have the meaning seen before; P=P* or P=P*; 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 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 1s 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 } 20 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 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, 10 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

¥ \ , 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, surprisingly, show also an extraordinary solubility in water even when surface polar groups such as NH, 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 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.

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 . 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 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, but also the chemical and structural properties of guest molecules.

According to the general formula (1) of the present invention and following the two general processes above outlined it is possible, for example, to synthesize

KR le oo CT . : 10 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(H-Gly-Orn-Gly-Gly)Gly-Om-Gly-Gly)Gly-Orn-

Gly-Gly)Gly-Orn-Gly-Gly-HN-CH2-CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orm-Gly(Gly-

Gly-Om-Gly(Gly-Gly-Omn-Gly-H)2)2)2 and 2(2(2(2(2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-

Gly-Gly)Gly-Ormn-Gly-Gly)Gly-Ormn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-

Orn-Gly-Gly)Gly-Orn-Gly-Gly-HN-CH2-CH,-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orn-

Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Om-Gly(Gly-Gly-Orn-Gly(Gly-Gly-

Om-Gly(Gly-Gly-Orn-Gly-H)z)2)2)2)2)2)2.

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 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 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 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 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.

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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 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% (v/v) acetonitrile in 0.1% TFA water; gradient (I) from 0 to 100% B in 25 min and (I) from 50 to 100% B in 25 min; flow, 1 mi/min, 220 nm detection. Peptide purification by preparative HPLC was carried out with the Waters Delta Prep 3000 apparatus on a Delta Pack C18-300A (30 mm X 30 cm, 15 p) column, with the same eluants and conditions. Flow, 30 ml/min, 220 nm detection. Thin layer 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 Cly-lodine were used as detecting reagents. TH 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). )

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 N[CHp-CHp-NH-CO-CH(CHp-phenyl NH2]3:4HCI 1.91 g of Boc-Phe-OH (7.2 mmole), 150 ul of N(CHp-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 pul 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% NaHCO3 (3X20 ml) and brine (3X20 ml). The organic solution was acidified, the solvent

Claims

- CLAIMS

1. 1. A polypeptide dendrimer having: i) a multifunctional core moiety; ii) an exterior 2 of closely spaced groups constituting the terminals of branched polypeptide chains 3 (monodendrons) radially attached to the core that, in turn, form iii) interior layers 4 (generations) of short peptide branching units (propagators) with characteristic hollows and channels where each propagator contains a trifunctional aminoacid 6 whose asymmetric carbon (the propagator branching point) is connected to two 7 equal-length arms bearing identical terminal reactive groups and to a third arm 8 (the propagator stem) bearing an activatable functional group, 9 represented by formula (1): K(-L)p-M (I) wherein 11 Kis a multifunctional core moiety, 12 Lis a polypeptide monodendron, 13 pis the number of polypeptide monodendrons irradiating from the core moiety and 14 M represents the outermost ramifications of the dendrimer;

1 2. A polypeptide dendrimer of claim 1 where said K is represented by formula (Il): 2 X-(CH2)p-X? (In ) 3 wherein X=X* or X=X', and X, X' are NH or CO or S;

1 3. A polypeptide dendrimer of claim 1 where said K is represented by formula (lil): © 2 Y[HCH2nZi (Ii) 3 wherein Y=C or Y=N; Z is NH or S or Cl or Br or | or a maleimide residue, n=1-6 4 andi=34,

1 4. A polypeptide dendrimer of claim 1 where said K is represented by formula (IV): 2 3 X-CH(R)-CO[-NH-CH(R)-CO]J,-NH-CH(R)-COOR" (IV) 4 wherein Ris (CH2)m-X', m=1-5, R" is methyl or ethyl or butyl or isopropyl, X=X" or 5 XX, and X, X* are NH or CO or S and n=1-6;

1 5. A polypeptide dendrimer of claim 1 where said L is the single monodendron 2» whose propagators are represented by formula (V): 3 -CO-CH(R?)-(CH2)n-NR3- Vv)

I . R ) 27 4 wherein R*=H or the side-chain of natural or synthetic aminoacids, and their i 5 derivatives; R*=H or a linear hydrocarbon radical optionally substituted with OH or 6 SHor ClorBr; R%-CH(CH2),-NR? is a 5 or 6 atoms ring, and n=0-6;

1 6. A polypeptide dendrimer of claim 1 where said L is the single monodendron 2 whose propagators are represented by formula (V1): 3 -CO-CH(R?)-CO-N(R?*)~(CH2)m-N(R?) (VI) 4 wherein R? and R?® have the meaning seen in claim 5 and m=1-6;

1 7. A polypeptide dendrimer of claim 1 where said L is the single monodendron 2 whose propagators are represented by one of the residues: 3 -CO-CH2-NH-NH-; or -CO-CH(R?)-O-; or -CO-CH5-O-N=CH-CO-; or -CO-CH(R?3)- 4 (CH2)n-S-CHp-CO-W; or -CO-NH-CH(CH2-SH)-CO-W or : 5 -CO-N-GH-co-W 6 HO-CH2-CH-T-CH-Q 7 wherein W=-N(R?)-(CH2)m-NR?, Q=H or -CHa3; T is O or S whereas R?, R® and m } 8 have the meaning seen in claim 5;

1 8. A polypeptide dendrimer of claim 1 where said L is the single monodendron 2 whose propagators are represented by one of the residues: 3 -c0~{_Y-Chz:0- -o-CHp+{_}-CO-CH(CH3)-0- 4 NO2 NO2 -O-CH-04_)-CO-CH(CH3)-O- coer {_orerao: . 6 CH30 7 NO» NO2 8 co-CHpaYerz 0: coro JorCHai: 9 OCH3 OCH3

1 9. A polypeptide dendrimer of claim 1 where said pis 1 or 2 or 3 or 4; 1 10. A polypeptide dendrimer of claim 1 where said M is the residue represented by 2 formula (VII): 3 -Ag-B(Ar)-C-ArjAg-B(Ar)-C-Ar{Ag-B(Ar-D)-C-Ar-D]2lo (VII) 4 wherein A=-CO-CH(R?)-(CH2)n-NR?, R® and n have the meaning seen in claim 5, 5 g=1-6, r=1-4 and R? in addition to the meaning seen in claim 5, is a natural or

LJ -

28 6 synthetic trifunctional aminoacid; B is -CO-CH[-(CH2)n-X"}-X, with X=X' or X=X"; X 7 and X' are NH or CO or 8; n=1-5; C=A or C=-CO(CH2)n-NH- or -(CH2)n-S- with 8 n=1-6 or Cis one of the residues: 9 cO-{_)-Cz 0 -0-CHz~_)-CO-CH(CH3)-0- NO» _ NO2 i -CO-CHp-04_}CO-CH(CH3)-0- cO(cHpI{ yoHCHO: 12 OCH3 13 NO2 NO2 14 ‘co(org{ Y-oHz 0- C0(GHgI0—_)-CH(CHaNH: OCHg3 OCH3 16 Dis a residue represented by formulae (VIII)-(Xl): 17 -Aq-B(Ar-E)-C-Ag-E (vin 18 -Ag-B(Ar)-C-Aq{Ag-B(Ar-E)-C-Ag-E}2 (IX) 19 -Ag-B(Ar)-C-Aq[Ag-B(Ar)-C-Ag-[Ag-B(Ar-E)-C-Ag-EL,). (X) -Ag-B(Ar)-C-Ag[Ag-B(Ar)-C-Ag-[Ag-B(Ar)-C-Aq[Ag-B(Ar-E)-C-Aq-E]2)z)2 (Xn) 21 wherein A, B, C, q ed r have the meaning seen above , and E is represented by 22 formulae (XII) and (XI): © 23 -Ag-B(Ar-P)-C-Ag-P* (XI) 24 -Ag-B(Ar)-C-Aq[-Ag-B(Ar-P)-C-Ag-P]; (Xm) ’ 25 wherein A, B, C, q and r have the meaning seen above, P=P* or P=P?, P and P’ 26 being H or a linear hydrocarbon radical optionally substituted with one or more 27 linear or branched alkyl groups, acyl, aminoacid, peptide, nucleotide, 28 oligonucleotide, saccharide, oligosaccharide, protein, monoclonal antibody, 29 polyethyleneglycol containing 10-400 -CH>-CH>-O- repeats, lipid, enzyme, metal ligand or their synthetic analogues and derivatives; 1 11. A polypeptide dendrimer of claims 1-10 wherein the two-dimensional molecular 2 diameter of the dendrimers is in the range from about 10 to 100 nm. 1 12. The dendrimer 2(z(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly- 2 Om-Gly-Gly-HN-CHz-CHz-NH-Gly-Gly-Orn-Gly(Gly-Gly-Om-Gly(Gly-Gly-Orn- 3 Gly(Gly-Gly-Orn-Gly-H)z)2)2. 1 13. The dendrimer 2(2(2(2(H-Gly-Orn-Gly-Gly)Gly-Om-Gly-Gly)Gly-Orn-Gly-

Ld 2 Gly)Gly-Orn-Gly-Gly)Gly-Omn-Gly-Gly-HN-CHz-CH2-NH-Gly-Gly-Om-Gly(Gly-Gly- 3 Om-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Om-Gly-H):)2)2)». ) 1 14. The dendrimer 2(2(2(2(2(H-Gly-Orn-Gly-Gly]Gly-Orn-Gly-Gly)Gly-Orn-Gly- i 2 Gly)Gly-Om-Gly-Gly)Gly-Omn-Gly-Gly)Gly-Orn-Gly-Gly-HN-CH,-CHy-NH-Gly-Gly- 3 Orn-Gly(Gly-Gly-Ormn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly- 4 Gly-Om-Gly-H)2)2)2)2)z. - t 15. The dendrimer 2(x(2(2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Om-Gly- 2 Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly-HN-CH- 3 CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly- 4 Gly-Omn-Gly(Gly-Gly-Om-Gly(Gly-Gly-Om-Gly-H)2)2)2)2)2)2. 1 16. The dendrimer 2(2(2(2(2(2(2(H-Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly )Gly-Om-Gly- 2 Gly)Gly-Orn-Gly-Gly)Gly-Orn-Gly-Gly)Gly-Om-Gly-Gly)Gly-Om-Gly-Gly)Gly-Ormn- } 3 Gly-Gly-HN-CH2-CH2-NH-Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Orn-Gly(Gly- R 4 Gly-Om-Gly(Gly-Gly-Om-Gly(Gly-Gly-Orn-Gly(Gly-Gly-Om-Gly(Gly-Gly-Om-Gly- Hkh). } 1 17. The dendrimer N{-CHz-CH;-NH-CO-CH(-CHz-phenyl)-NH-Gly-Gly-Gly-Orn- 2 Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Orn-Gly-H}2]2)2}. 1 18. The dendrimer N{-CH2-CH;-NH-CO-CH(-CH.-phenyl)-NH-Gly-Gly-Gly-Orn- 2 Gly[Gly-Gly-Gly-Om-Gly[Gly-Gly-Gly-Ormn-Gly[Gly-Gly-Gly-Om-Gly[Gly-Gly-Gly- 3 Om-Gly-Hlz2]2)2)z2}s.

CO . 1 19. The dendrimer NCH, CH, NZCO— CH-S-CH,-CH(COOH)-NH- 2 NO, 3 :00(CH)0-{_)-CHICH, -NH-Giy-Gly-Gly-On-GHGH-Gl-Gly- Or 4 OCH, 5 Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Orm-Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly- 6 Om-Gly[Gly-Gly-Gly-Orn-Gly[Gly-Gly-Gly-Om-Gly-H] LL L111}, 1 20. The polypeptide dendrimers of claims 12-19 wherein the NH, terminals are 2 acetylated. 1 21. A polypeptide dendrimer of claim 1 wherein at least one bioactive or marker 2 molecule is covalently linked to the surface of the same.

n

1 22. A polypeptide dendrimer of claim 21 where the bioactive molecule is selected 2 in the group comprising an aminoacid, a peptide, a protein, a nucleotide, an 3 oligonucleotide, a lipid, a saccharide, an oligosaccharide, and a small organic 4 molecule and their synthetic analogues and derivatives. 1 23. A polypeptide dendrimer of claim 21 where the bioactive molecule is selected 2 in the group comprising drugs, cellular receptor ligands, bacterial, viral and 3 parasite antigens and gene-therapy compounds. 1 24. A polypeptide dendrimer of claim 21 where the marker molecule is a diagnostic 2 imaging contrast agent. 1 25. A polypeptide dendrimer of claim 1 where the bioactive molecule is entrapped 2 inthe same. 1 26. A polypeptide dendrimer of claim 25 where the bioactive molecule is selected 2 in the group comprising an aminoacid, a peptide, a protein, a nucleotide, an 3 oligonucleotide, a lipid, a saccharide, an oligosaccharide, and a small organic 4 molecule and their synthetic analogues and derivatives. 1 27. A polypeptide dendrimer of claim 25 where the bioactive molecule is selected 2 in the group comprising drugs, cellular receptor ligands, bacterial, viral and 3 parasite antigens and gene-therapy compounds.

) 1 28. A polypeptide dendrimer of claim 27 where the bioactive molecules are 2 anticancer drugs.

; 1 29. A polypeptide dendrimer of claim 27 where the bioactive molecules are 2 antibiotics. 1 30. A polypeptide dendrimer of claim 27 where the bioactive molecules are 2 antiviral substances. 1 31. A process for production of the polypeptide dendrimers of claim 1 2 characterized by the following steps: 3 i) synthesis of core moieties with at least two reactive functional groups; 4 ii) divergent synthesis on solid-phase of polypeptide monodendrons with temporarily or permanently protected terminals; 6 iii) covalent condensation of polypeptide monodendrons to core moieties; 1 32. A process for production of polypeptide dendrimers of claim 1 characterized by 2 the following steps:

x

3 1) synthesis of core moieties with at least two reactive functional groups; 3 4 ii) covalent condensation to the core moieties of polypeptide monodendrons of ) s generation 1-3 with temporarily protected terminals to obtain the corresponding } 6 protected dendrimers; - 7 iii) after protecting groups removal, repeated condensations of polypeptide ) 8 monodendrons to the dendrimer reactive terminals to obtain the desired final ) 9 dendrimers. 1 33. A process for entrapping into the polypeptide dendrimers of claim 1 bioactive 2 substances and drugs with molecular weights lower than 1,000 Da, characterized 3 by the following steps: 4 (a) adding suitable amounts of polypeptide dendrimers to a concentrated or saturated solution of said molecules and N 6 (b) precipitating the loaded polypeptide dendrimer after 24 h incubation at room h 7 temperature in a large volume of a precipitant. 1 34. A process for entrapping into the polypeptide dendrimers of claim 1 bioactive 2 substances and drugs with molecular weights higher than 1,000 Da, characterized 3 by the selective chemical ligation of polypeptide monodendrons, in aqueous 4 buffers, to the core moieties in the presence of said molecules. 1 35. A process for the selective chemical ligation of bioactive substances and drugs : 2 to the internal functional groups of the polypeptide dendrimers of claim 1, in 3 aqueous buffers, after loading the dendrimer carrier by diffusion. . 1 36. Use of polypeptide dendrimers of claim 1 as unimolecular carriers of bioactive 2 molecules wherein at least one bioactive or marker molecule is covalently linked to 3 the surface of the same. 1 37. Use of polypeptide dendrimers according to claim 36 where the bioactive 2 molecule is selected in the group comprising an aminoacid, a peptide, a protein, a 3 nucleotide, an oligonucleotide, a lipid, a saccharide, an oligosaccharide, and a 4 small organic molecule and their synthetic analogues and derivatives. 1 38. Use of polypeptide dendrimers according to claim 36 where the bioactive 2 molecule is selected in the group comprising drugs, cellular receptor ligands, 3 bacterial, viral and parasite antigens and gene-therapy compounds. 1 39. Use of polypeptide dendrimers according to claim 36 where the marker a ) CO oC PCT/EP00/07022 32 molecule is a diagnostic imaging contrast agent.

40. Use of polypeptide dendrimers of clzim 1 as unimolecular carmiers of bioactive mclecules 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 oligonuclectide, a lipid, a saccharide, an oligosaccharide, and a small organic meolecule 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 2nd parasite antigens and gene-therapy compounds.

43. Use of polypeptide dendrimers according to clzim 40 where the bioactive molecules are anticancer drugs.

44. Use of polypeptide dendrimers according to cizim 40 where the bioactive molecules are antibiotics.

45. Use of polypeptide dendrimers accerding 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 unimclecular carriers of bioactive molecules entrapped into the same.

48. Use of polypeptide dendrimers of claim 1 in the manufacture of a preparation for use as unimolecular carriers of bioactive molecules wherein at least one bioactive or marker molecule is covalently linked to the surface of the same.

49. Use according to claim 48 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.

50. Use according to claim 48 where the bioactive molecule is selected in the group comprising drugs, cellular receptor ligands, bacterial, viral and parasite antigens and gene-therapy compounds. - AMENDED SHEET

©, 33 PCT/EP00/07022

51. Use according to claim 48 where the marker molecule is a diagnostic imaging contrast agent.

52. Use of polypeptide dendrimers of claim 1 in the manufacture of a preparation for use as unimolecular carriers of bioactive molecules wherein the bioactive molecule is entrapped into the same.

53. Use according to claim 52 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. 54, Use according to claim 52 where the bioactive molecule is selected in the group comprising drugs, cellular receptor ligands, bacterial, viral and parasite antigens and gene-therapy compounds. —

55. Use according to claim 52 where the bioactive molecules are anticancer drugs.

56. Use according to claim 52 where the bioactive molecules are antibiotics.

57. Use according to claim 52 where the bioactive molecules are antiviral substances.

58. A substance or composition for use in a method as unimolecular carriers of _ bioactive molecules wherein at least one bioactive or marker molecule is covalently linked to the surface of the same, said substance or composition comprising polypeptide dendrimers of claim 1, and said method comprising using said substance or composition as said carrier.

59. A substance or composition for use in a method as unimolecular carriers of bioactive molecules according to claim 58 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.

60. A substance or composition for use in a method as unimolecular carriers of bioactive molecules according to claim 58 where the bioactive molecule is selected in the group comprising drugs, cellular receptor ligands, bacterial, viral and parasite antigens and gene-therapy compounds.

61. A substance or composition for use in a method as unimolecular carriers of AMENDED SHEET

LI 34 PCT/EP00/07022 bioactive molecules according to claim 58 where the marker molecule is a diagnostic imaging contrast agent.

62. A substance or composition for use in a method as unimolecular carriers of bioactive molecules wherein the bioactive molecule is entrapped into the same, said substance or composition comprising polypeptide dendrimers of claim 1, and said method comprising using said substance or composition as said carrier.

63. A substance or composition for use in a method as unimolecular carriers of bioactive molecules according to claim 62 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.

64. A substance or composition for use in a method as unimolecular carriers of bioactive molecules according to claim 62 where the bioactive molecule is selected in the group comprising drugs, cellular receptor ligands, bacterial, viral and parasite antigens and gene-therapy compounds.

65. A substance or composition for use in a method as unimolecular carriers of bioactive molecules according to claim 62 where the bioactive molecules are anticancer drugs. _

66. A substance or composition for use in a method as unimolecular carriers of bioactive molecules according to claim 62 where the bioactive molecules are antibiotics.

67. A substance or composition for use in a method as unimolecular carriers of i bioactive molecules according to claim 62 where the bioactive molecules are antiviral substances.

68. A polypeptide dendrimer as claimed in any one of claims 1 or 12 to 19, substantially as herein described and illustrated.

69. A process as claimed in claim 31 or claim 32, substantially as herein described and illustrated.

70. A process as claimed in claim 33 or claim 34, substantially as herein described and illustrated. AMENDED SHEET a PCT/EP00/07022

71. A process as claimed in claim 35, substantially as herein described and illustrated.

72. Use as claimed in claim 36 or claim 40, substantially as herein described and illustrated.

73. A composition as claimed in claim 46 or claim 47, substantially as herein described and illustrated.

74. Use as claimed in claim 48 or claim 52, substantially as herein described and illustrated.

75. A substance or composition for use in a method as unimolecular carriers of bioactive molecules as claimed in claim 58 or claim 62, substantially as herein described and illustrated.

76. A new polypeptide dendrimer, a new process for producing polypeptide dendrimers, a new process for entrapping bioactive substances and drugs, a new use of polypeptide dendrimers as claimed in claim 1, a new composition, or a substance or composition for a new use as unimolecular carriers of bioactive molecules, substantially as herein described. AMENDED SHEET