CA1316456C - Starburst conjugates - Google Patents

Abstract

ABSTRACT

Starburst conjugates which are composed of at least one starburst polymer in association with at least one unit of a carried pharmaceutical material have been prepared. These conjugates have particularly advantageous properties due to the unique characteristics of the starburst polymer.

35,444-F"A"

Description

131645~

STARBURST CONJUGATES
The present invention concerns the use of dense star polymers as carriers for pharmaceutical materials (the "carried"
material). In recent years polymers referred to as dense star polymers or starburst polymers have been developed. It has been found that the size, shape and properties of these dense star polymers of Starburst* polymers can be molecularly ~ailored to meet speciali~ed end uses. Starburst polymers have significant advantages which can provide a means for the delivery of high concentrations of carried material per unit of polymer, controlled delivery, targeted delivery and/or multiple species delivery or use.
This application is one of three closely related paten~
applications serial numbers 544,73~, 544,735 and 544,736 all filed on August 18, 1987. The present application deals with all cases in which the carried material is a pharmaceutical material, application serial no. 544,736 deals with all cases in which the carried material is an agricultural material and application serial no. 544,735 deals with all the remainin~ cases in which the carried material is neither a pharmaceutical nor an agricultural material.
In its broadest aspect, the present invention is directed to polymer conjugate materials comprising dense star polymers or starburst polymers associated with pharmaceutical materials (hereinafter these polymer co~jugates will frequently be referred to as "starburst conjugates" or "conjugates", process for preparing ~hese conjugates, compositions containing the * Trademark

-2- 131~6 conjugates, and methods of using the conjugates and compositions.
The conjugates of the present invention are suitable for use in a variety of applications where specific delivery is desired, and are particularly suited for the delivery of biologically active agents.
In a preferred embodiment of the present invention, the starburst conjugates are comprised of one or more starburst polymers associated with one or more bioactive agents.
The starburqt conjugates offer significant benefits over other carriers known in the art due to ,r 15 the advantageous properties of the ~ starburst polymers. Starburst polymers exhibit molecular architecture characterized by regular dendritic branching with radial symmetry. These radially symmetrical molecules are referred to as possessing "starburst topology". These polymers are made in a manner which can provide concentric dendritic tiers around an initiator core. The starburst topology is achieved by the ordered assembly of organic repeating units in concentric, dendritic tiers around an initiator core; this is accomplished by introducing multiplicity and self-replication (within each tier) in a geometrically progressive fashion through a number of molecular generations. The resulting highly functionalized molecule~ have been termed "dendrimers"
in deference to their branched (tree-like) structure as well aq their oligomeric nature. Thus ? the terms starburst oligomer and starburst dendrimer are encompassed within the term starburst polymer.
Topological polymers, with size and shape controlled domainq 9 are dendrimers that are covalently bridged 35,444-F"A" -2~

~3~ ~31~56 through their reactive terminal groups, which are referred to as starburst "bridged dendrimers." The term bridged dendrimer is also encompassed within the term "starburst polymer".

The following description of the figures aid in understanding the present invention.
Figure 1 depicts various generations of starburst dendrimers Fi~ure 2A depicts a dendrimer having unsymmetrical (unequal) branch junctures.
Fi~ure 2B depict~ a dendrimer having symmetrical (equal) branch juncturesO
Fi~ure 3 depicts dendrimer sizes relative to antibody dimensions.
Figure 4 shows carbon-13 spin lattice relaxation times (T1) for aspirin incorporated into variou~ dendrimer generations. (Example 1) Fi~ure 5 shows the re3ults of the dynamic analysis of Example 2.
Fi~ure 6 shows the influence of generation 6.5 dendrimer on the dialysis rate of pseudoephedrine at pH
9.5 from Example 2.
Fi~ure 7 shows the effect of dendrimer hydrolysis on the permeability of pseudoephedrine of Example 3.
Fi~ure 8 Comparison of Percent Salicylic Acid released into the receptor compartment in the presence of 35,444-F"A" -3-~ ~ 31~45~

starburst polymer (Gen - 4.0) at pH 5.0 and 6.65 with salicyclic acid control, Example 4.
Fi~ure 9 Comparison of percent salicyclic acid lost from donor compartment with starburst polymer (Gen =
4.0) in receptor compartment at pH 8.0 to salicyclic acid content, Example 4.
Fi~ure 10 Comparison of percent salicyclic acid lost from donor compartment in presence of starburst polymer (Gen =4.5) to salicyclic acid control, Example 4.
The tarbur~t polymers are illustrated by Figure 1 wherein ~ represents an initiator core (in ~- 15 this figure a tri-functional initiator core shown by the far left drawing,); Z represents a terminal group, shown in the fir~t instance by the second drawing from the left, referred to as a starbranched oligomer; A, B, C, D, and E represent particular molecular generations of starburst dendrimers; and (A)n~ (B)n~ (C)n, (D)n~
and (E)n repre3ent starburst bridged dendrimers.
The starburst dendrimers are unimolecular assemblages that possess three distinguishing architectural features, namely, (a) an initiator core, (b) interior layers (generations, G) composed of repeating units, radially attached to the initiator core, and (c) an exterior surface of kerminal functionality (i.e., terminal functional groups)

3 attached to the outermost generation. The size and shape of the starburst dendrimer molecule and the functional groups present in the dendrimer molecule can be controlled by the choice of the initiator core, the number of generations (i.e., tiers) employed in creating the dendrimer, and the choice of the repeating 35,444-F"A" _4_ ~ 316~
64~g3-410 unlts employed at each generation. Since the dendrimers can ~e lsolated at any partlcular generatlon, a means ls provlded for obtainlng dendrl~ers having deslred properties.
The choice of the starburst dendrimer components affects the propertles of the dendrlmers. The lnltlator core type can affect the dendrimer shape, produclng (dependlng on the choice of inltlator core)l for example, spheroid-shaped dendrimers, cylin-drlcal or rod-shaped dendrimers, elllpsoid-shaped dendrlmers, or mus~lroom-shaped dendrimers. Sequential building of generations (l.e., generatlon number and the size and nature of the repeating units) determlnes ~he dlmensions of the dendrimers and the nature of their interior.
Because starburst dendrimers are branched polymers containlng dendrltic ~ranches havlng functlonal groups dlstribute on the periphery of the branches, they can be prepared with a varlety of propertles. For example, starburst dendrlmers, such as those deplcted in Figure 2A and Figure 2B can have dlstinct pro-pertles due to the branch length. The dendrlmer type shown in Figure 2A (such as Denkwalter, U.S. Patent 4,289,872) possesses unsymrnetrical (unequal segment) branch ~unctures, exterlor ~l.e., surface~ groups ~represented by Z'), interior moietles ~represent-ed by Z) but much less lnternal void space. The dendrimer type shown in Figure 2B possesses syrnmetrical (equal segment) branch ~unctures with surface groups ~represented by Z'), two dlfferent interlor moietles ~represented respectlvely by X and Z) with interior vold space which varies as a ~unctlon of the generation (G~. The dendrimers such as those depicted ln Flgure 2B can be advanced through enough generations to totally enclose and contain void space, to give an ent;ty with a predominantly hollow interior and a highly congested surface. Also, starburst dendrimers, when advanced through sufficient generations exhibit "starburst dense packing" where the surface of the dendrimer contains sufficient terminal moietieq such that the dendrimer surface becomeq congested and encloses void spaces within the interior of the dendrimer. This congestion can provide a molecular level barrier which can be used to control diffusion of materials intG or out of the interior of the dendrimer.
Sur~ace chemi~try of the dendrimers can be controlled in a predetermined fashion by selecting a repeating unit which contains the desired chemical functionality or by chemically modifying all or a portion of the surface functionalities to create new surface functionalities. These surface~ may either be targeted toward specific sites or made to resist uptake by particular organs or cells e.g. by reticuloendo-thelial cells.

In an alternative use of the starburst dendrimers, the dendrimer~ can themselves be linked together to create polydendric moieties (starburst "bridged dendrimers") which are also suitable a~
carriers.
In addition, the dendrimers can be prepared so as to have deviations from uniform branching in particular generations, thus providing a means of adding discontinuities (i.e., deviations from uniform 35 9 444-F"A" -6- _ 131~5~
branching at particular locations within the dendrimer) and different properties to the dendrimer.
The starburst polymers employed in the starburst conjugates of the present invention can be prepared according to methods known in the art, for example, U. S. Patent 4,587,329.
Dendrimer~ can be prepared having highly uniform size and shape and most importantly allow for a greater number of functional groups per unit of surface area of the dendrimer, and can have a greater number of functional groups per unit of molecular volume as compared to other polymers which have the same molecular weight, same core and monomeric components and same number of core brancheq as the starburst polymers. The increaqed ~unctional group denqity of the dense starburst polymers may allow a greater quantity of material to be carried per dendrimer.
Since the number of functional groups on the dendrimers can be controlled on the surface and within the interior, it also provides a means for controlling the amount of bioactive agent to be delivered per den-drimer. In a particularly preferred embodiment of thepresent invention the starburst polymers? particularly the starburst dendrimers, are targeted carriers of bioactive agents capable of delivering the bioactive agents to â particular target organism or to a particular determinant or locus in a target organism.
An analogy can be made between early generation starburst dendrimers (i.e. generation = 1-7) and classical spherical micelles. The dendrimer-micelles analogy wa~ derived by comparing features which they had in common such as shape, size and surface D.

35,444-F'IA'' -7-13164~6 Table I

Parameter Regular Classical Sta~burst Dendrimers --- Mlcelles Shape Spherical Spherical Size 20-60~ 17-4 (dia~eter) Surface 4-202 ~=6-192 (Z is the 0 aggregation number number of surface groups)(generation =
2-7) area2/surface group 130-80A2 127-75A2 tA ~
0~l nm lA2 = 10-2 nm2) ' 15 In Table I, the shape was verified by scanning transmission electron micrographs (STEM) microscopy and intrinsic viscosity (~) measurements. The size was verified by intrinsic viscosity [~] and size exclusion chromatography (SEC) measurements. The surface aggregation numbers were verified by ~ ~me~ and high field NMR. The area/surface group was calculated from SEC hydrodynamic measurements.
The first five generations of starburst polyamidoamine (PAMAM) dendrimers are microdomains which very closely mimic classical spherical micelles in nearly every respect (i.e. shape, size, number of sur~ace groups, and area/surface groups). A major 3 difference, however, i~ that they are covalently fixed and robust compared to the dynamic equilibrating nature of micelles. This difference is a significant advantage when using these microdomains as encap~ulation devices.

35,444-F"A" -8- _ -9- ~31~6 As further concentric generations are added beyond five, congestion of the surface occurs. This congestion can lead to increased barrier characteristics at the surface and manifests itself as a smaller surface area per head (surface) group as shown in Table II.

3o 35,444~F"A" -9-13:L6~

r "~ N 3~ 1 r~
r~
N ~S
CD ~ 0 r~
~¦ N ~ 'S ~ t`l 'S J~
~r ~ .,1 ". o ~ ~ N oc~

I o 'r O O _I ~ C

~1 ¦ N ~ ~ o N N ~ 0 I i N ~ ~ C

C ¦ N N "~ N N 1~7 ~1 E
a~ ,~ x Ul O O
~ . o~l1~ N ~1~1 N 1~
U7CO 7~IIS ~ ' ~ a- Il C ~ ~ N 1'` '~
~ ~ 1 ~I:U C ~1 rL. e ~ 1 ~ 5 o NE
o~ ~ N ~ CS
D ~) ' I~ . U~ ~ ~ -- ~
_I N O ~0 N N _I Ul O O

~;1 E 31 N
u a~ ~
3 e 0~ _1 o ~1 ~ 0 ~a ~1 ,~ ~1 C ~ U ~ O ~ 0 11 ~ ~ 2 ~ a. rd o ~ c o o ~ ~ o U~ 3 ~ --o ~ o -35, 444-F`'A" -10-1 3 ~ 6 6~3-4104 For example, amine ~ermin~ted generations 5.0, 6.0, 7.0, 8.0 and 9.0 have decreased surface areas of 104, 9Z, 73, 47 and 32A~ per Z group, respectlvely. This character~stic corresponds to a transltlon from a less congested mlcelle-llke surface to a more congested bl-layer~monolayer barrler-like surface normally associated wlth vesicles (liposomes) or Langmuir-Blodgett type membranes.
If this surface congestlon ls occurrlng, the change ln physlcal characterlstlcs and morphology should be observed as the generatlons lncrease from the lntermedlate generation (6-8) to the more advanced generations (g or lO). The scannlng transmission electron micrographs (STEM) for generations = 7.0, 8.0 and 9.0 were obtained after remov~ng the methanol solvent from each of the samples to provide colorless, light yellow solid fllms and stain-ing wlth osmium tetraoxlde. The morphological change predlcted occurred at the generation G=9.0 stage. The microdomains at gen-eration = 9.0 measure about 33A in diameter and are surrounded by a colorless rim whlch ls about 25A thlck. Apparently methanolic solvent has been entrapped wlthln the 25A outer membrane-like barrler to provlde the dark stalned lnterior. Thus, at generatlon = 9.0, the starburst PAMAM is behaving topologlcally llke a vesi-cle (llposome). However, this starburst is an order of magnltude smaller and very monodlspersed compared to a llposome. ~onse-quently, the present dendrlmers can be used to molecularly encap-sulate solvent fllled void spaces of as much dlameter as about 33A
(volume about 18,000~3) or more. These mlcelle sized prototypes appear to behave llke a covalently fixed liposome in thls advanced generatlon stage. This ~ehavlor enables these prototypes to serve ~12- ~31~4~

as drug delivery agents or as carriers for non-chelating radionuclides in starburst antibody conjugates for the treatment of various mammalian diseases.

Dendrimers suitable for use in the conjugates of the present invention include the dense ~tar polymers or starburst polymers described in U. S.
Patents 4,507,466, 4,558,120, 4,568,737 and 4 7 587,329.
In particular, the present invention concerns a starburst conjugate which comprises at least one starburst polymer associated with at least one carried pharmaceutical material. Starburst conjugates included within the scope of the pre~ent invention include those represented by the formula:

(P)x * (M)y (I) wherein each P represents a dendrimer;
x represents an integer of 1 or greater;
each M represents a unit (for example9 a molecule, atom, ion, and/or other basic unit) of a carried pharmaceutical material, said carried pharmaceutical material can be the same carried pharmaceutical material or a different carried pharmaceutical material, preferably the carried pharmaceutical material is a bioactive agent;
y represents an integer of 1 or greater; and * irdicate~ that the carried pharmaceutical material is associated with the dendrimer.

35,444-F"A" -12~ _ _13_ ~316~S

Preferred ~tarburst conjugate~ of formula (I) ~ are those in which M is a drug, radionuclide, ~he~
`~ ~ chelated metal, toxin, antibody, antibody fragment, antigen, signal generator, signal reflector, or signal absorber; particularly preferred are those in which x-1, and y-2 or more.
Also included are starburst conjugate3 of formula (I) wherein the starburst dendrimers are covalently linked together, starburst bridged dendrimer~, optionally via linking groups, so as to form polydendric assemblages (i.e., where x>1). Uses of these starburst bridged dendrimers include topical controlled release agents, radiation synovectomy, and Other~-As used herein, "associated with" means thatthe carried material(s) can be encapsulated or entrapped within the core of the dendrimer, dispersed partially or fully throughout the dendrimer, or attached or linked to the dendrimer, or any combination thereof. The association of the carried material(s) and the dendrimer(s) may optionally employ connectors and/or spacers to facilitate the preparation or use of the starburst conjugates. Suitable connecting groups are groups which link a targeting director (i.e., T) to the dendrimer (i.e., P) without significantly impairing the effectiveness of the director or the effectiveness of any other carried material(s) (i.e., M) present in the qtarburst con~ugate. These connecting groups may be cleavable or non-cleavable and are typically used in 5 f~ric order to avoid ~AUU:}I hindrance between the target director and the dendrimer, preferably the connecting group3 are stable (i.e., non-cleavable). Since the ~ize, shape and functional group density of the 35,444-F"A" ~13-_14_ ~3~

starburst dendrimers can be rigorously controlled, there are many ways in which the carried material can be associated with the dendrlmer. For example, (a) there can be covalent, coulombic, hydrophobic, or chelation type association between the carried material(s) and entities, typically functional groups, located at or near the surface of the dendrimer; (b) there can be covalent, coulombic, hydrophobic, or chelation type association between the carried material(s) and moieties located within the interior of the dendrimer, (c) the dendrimer can be prepared to have an interior which is predominantly hollow allowing for physical entrapment of the carried materials within the interior (void volume), wherein the release of the carried material can optionally be controlled by congesting the surface of the dendrimer ~ith diffusion controlling moieties; or (d) various combinations of the aforementioned phenomena can be employed.
ZO
Dendrimers, herein represented by ~P~s include the dense star polymers described in U. S. Patent

4,507,466, 4,558,120, 4,568,737 or 4,587,329.

Carried pharmaceutical materials, herein represented by "M", which are suitable for use in the starburst conjugates include any materials in vivo or in vitro use for diagnostic or therapeutic treatment which can be associated with the dense star dendrimer 30. without appreciably disturbing the physical integrity of the dendrimer, for example, drugs such as antibiotics, analgesics, hypertensives, cardiotonics, and the like such as acetaminaphen, acyclovir, alkeran, amikacin, ampicillin, aspirin, bisantrene, bleomycin, neocardiostatin, chloroambucil, chloramphenicol, cytarabine, daunomycin, doxorubicin, fluorouracil, 35,444-F"A" -14_ -15- l 3 l ~ 5 gentamycin, ibuprofen, kanamycin, meprobamate, methotrexate, novantrone, nystatin, oncovin, phenobarbital, polymyxin, probucol, procarbabizine, rifampin, streptomycin, spectinomycin, symmetrel, thioguanine, tobramycin, trimethoprim, valban; and toxins such as diphtheria toxin, gelonin, exotoxin A, abrin, modeccin, ricin, or toxic fragments thereof;
metal ions such as the alkali and alkaline-earth metals; radionuclides such as those generated from actinides or lanthanides or other similar transition elements or from other elements, such as 67cu~ 90y, 111In, 131I, 186Re, 105RhS 99mTe, 67Ga~ 153sm~ 159Gd, 175y~, 177LU, 88y, 166Ho~ 115mIn~ 109pd~ 82Rb~ 194Ir, 140ga~ 149pm~ 199AU, 140La, and 188Re; signal generators such as fluorescing entities; signal reflectors such as paramagnetic entities for example Fe~ Gd, ~Mn; chelated metal such as any of the metals given, whether or not they are radioactive when associated with a chelant; signal absorber~ such as an electron beam opacifiers; antibodies including monoclonal antibodies and anti-idiotype antibodies;
antibody fragments; hormones; biological response modifiers such as interleukins, interferons, viruses and viral fragments; diagnostic opacifiers; and fluorescent moieties. Carr.ied pharmaceutical materials include scaveng.ing agents such as chelants, antigens, antibodie~ or any moieties capable of selectively scavenging therapeutic or diagnostic agents.
Preferably the carried pharmaceutical materials are bioactive agents. As used herein, "bioactive".
referY to an active entity such as a molecule, atom, ion and/or other entity which is capable of detecting, identifying, inhibiting, treating, catalyzing, 35,444-F"A" -15- _ -16- ~316~

controlling, killing, enhancing or modifying a targeted entity such as a protein, glycoprotein, lipoprotein, llpid, a targeted cell, a targeted organ, a targeted organism [for example, a microorganism or animal (including mammals such as humans)] or other targeted moiety.
The starburst conjugates of formula (I) are prepared by reacting P with M, usually in a suitable solvent, at a temperature which facilitate~ the association of the carried material (M) with the starburst dendrimer (P).
Suitable solvents are solvents in which P and M
- 15 are at least partially miscible and inert to the formation of the conjugate. If P and M are at lea~t partially miscible with each other, no solvent may be required. When desired, mixtures of suitable solvents can be utili~ed. Examples of such suitable solvents are water, methanol, ethanol, chloroform, acetonitrile, toluene, dimethylsulfoxide and dimethylformamide.
The reaction condition~ for the formation of the starburst conjugate of formula (I) depend upon the particular dendrimer (P), the carried pharmaceutical material (M), and the nature of the bond (*) formed.
For example if P is the PEI (polyethyleneimine) starburst dendrimer with a methrlene carboxylate surface, M is a radionuclide, e.g. yttrium, then the reaction is conducted at room temperature in water.
~~ ~ f'o~ car~ 5 ~:~ However, if P is an ester terminated~(PAMAM)starburst dendrimer, M is aspirin, then the reaction is conducted at room temperature in chloroform. Typically, the temperature can range from room temperature to reflux.

35,444-F"A" -16-_17_ l 31 6~ ~6 The qelection of the particular solvent and temperature will be apparent to one qkilled in the art.
The ratio of M:P will depend on the size of the dendrimer and the amount of carried material. For example, the ~ ratio (ratio of moles) of any ionic M
,.~
to P usually is 0.1-1,000:1, preferably 1-50:1, and more preferably 2-6:1. The weight ratio of any drug or toxin M to P usually is 0.1-5:1, and preferably 0.5-3:1~
When M is a radionuc]ide, there are three waysthe starbur~t conjugate can be prepared, namely: (1) P
can be u-qed as a chelantO For example a ~- 15 methylenecarboxylate surface PEI or PAMAM will chelate a metal such as yttrium or indium. (2~ A chelate can be covalently bonded to P. For example, an amine terminated PEI starburst dendrimer can be reacted with 1-tp-isothiocyanatobenzyl)diethylenetriaminepenta-acetic acid and then chelated, or a complex such asrhodium chloride chelated with isothiocyanatobenzyl-2,3,2-tet can be reacted. (3) A prechelated radionuclide can be associated with Pby hydrophobic or ionic interaction.
Particularly preferred starburst conjugates are those conjugates which contain a target director (herein designated a~ "T'~) and which are represented by the formula (T)e * (P)x * (M)y (II~

wherein 35,444-F"A" -17-1316~
64~93-410 each T represents a targe~ dlrector;
e represents an integer of 1 or greater; and P, x, , M, and y are as prevlously defined !lereln.
Preferred among the starburst con~ugates of formu~a (II) are those in whlch M is a drug, radlonucllde, chelator, chelated metal, toxin, slgnal generator, signal reflector, or slgnal absorber.
Also preferred coniugates are those con~ugates in which e=l or 2;
an~ those in which ~=1 and y=2 or more. Particularly preferred conjugates are those in whlch x=l, e=l, y=2 or more and M and T
are assoclated wlth the polymer via the same or different con-nectors.
The starburst con~ugates of formula (II) are prepared either by formlng T P and then addlng M or by formlng P M and then addlng T. Elther reaction scheme is conducted at temperatures which are not detr~mental to the particular con~ugate component and ln the presence of a sultable solvent when requlred. To control pH, buffers or addltlon of sultable acid or base i5 used.
The reaction conditlons are dependent on the type of assoclatlon formed( ), the starburst dendrimer used (P), the carrled pharma-ceutical material (M), and the target dlrector (T). For example,when T is a monoclonal antlbody and M is a radionuclide, th~ T P
assoclatlon ls done through a functlonal group such as an lsothio-cyanate in water or ln water wlth an organic modlfier such as acetonitrile or dimethylformamide. Usually, the con~ugatlon is done in a buffer at pH 7-10, preferably pH 8.5-9.5. The formed con~ugate is then chelated with a radionuclide such as yttrlum acetate, preferably at room temperature.

J ;~.

Alternati~ely, P and M can be chelated, usually in water, before conjugation to T. The conjugation with T
is carried out in a suitable buffer.
The ratio of T:~ i5 preferably 1:1, especially when T is an antibody or fragmentO The ratio of M:P
will be as before.
Target directors capable of targeting the starbur~t conjugates are entities which when used in the starburst conjugates of the present invention result in at least a portion of the starburst conjugates being delivered to a desired target (for example, a protein, glycoprotein, lipoprotein, lipid, a - 15 targeted cell, a targeted organ, a targeted organism or other targeted moiety) and include antibodies, preferably monoclonal antibodies, antibody fragments such as Fab, Fab', F(ab')2 fragments or any other antibody fragments having the requisite target specificity, hormones, biological response modifiers;
epitopes; chemical functionalities exhibiting target specificity; and the like.
The antibodies or antibody fragments which may be used in preferred starburst conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art, see, for example, Kohler and Milstein (1975, Nature 256:495-497; and 1976, Eur. J. Immunol. 6:511-519). Such antibodies normally have a h~ghly specific reactivity.
In the antibody targeted starbur~t conjugates, antibodies directed against any antigen or hapten may 35,444-F'IA" -19- _ -20- 13~ 6 be used. Although conventional polyclonal antibodies may be used, monoclonal antibodies offer several advanta~es. Each monoclonal antibody is highly specific for a single epitope. In addition, large amounts of each monoclonal antibody can be produced.
Antibodies used in the present invention may be directed against, for example, tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules. For a more complete list of antigens see U. S. Patent 4,193,983.
It may be desirable to connect more antibodies or fragments to the dendrimer, and in particular instances to connect antibodies of different specificities. For example, a bifunctional conjugate which has the ability to localize and bind to a tumor and then scavenge circulating cytotoxic, diagno3tic, or biostatic compounds can be designed.
In the absence of a target director (or in the presence of a target director if desired), due to the ~. ~u~b~
i~ 25 ~ eP of functional groups which can be located at or near the surface of the dendrimer, all or a sub~tantial portion of such functional groups can be made anionic, cationic, hydrophobic or hydrophilic to effectively aid delivery of the sta-rburst conjugate to a desired target of the oppoqite charge or to a hydrophobic or hydrophilic compatible target.
Preparation of the conjugates of formula (II) using a P with a protected handle (S) is also intended 35,444-F"A" -20- _ -21- 1316~

as a proceqs to prepare the conjugates of formula (II).
The reaction scheme is shown below:

S*P loading S*P*M deprotection P*M

T*P*M linking _¦ ~

where S*P represents the protected dendrimer;
S*P*M represents the protected dendrimer conjugated with M;
P*M represents the dendrimer conjugated with M (starburst conjugate~;
T*P*M represents the starburst conjugate linked to the target director.
Suitable solvents can be employed which do not b~f~ rb~,~7 yf 4L~ effect P*M. For example when S is t- _ , S
can be removed by aqueous acid.

Also preferred are starburst conjugates in which the polymer is associated directly, or via connectors; these starburst conjugates are represented by the formula:

[(T)e - (C')f]g * (P)x * [(C )h ~ (M)y]k (III) wherein each C' represents the same or different connecting 3S group;

35,444-F"A" -21 -22~ 131~6 each C " represents the same or di~ferent connecting group;
g, and k each individually represent an integer of 1 or greater;
f and h each individually represent an integer of 0 or greater;
- indicate~ a covalent bond in lnstances where a connecting group is present; and P, x, *, M, y, T, and e are as previously defined herein.
~- 15 Preferred among the starburst conjugates of formula (III) are thoqe in which M is a radionuclide, drug, toxin, qignal generator, signal reflector or signal absorber. Also preferred are those conjugates in which x_1. Particularly preferred conjugates are those in which x, e, f, h, and y are each 1, and g is one or more and k is each individually 2 or more. Most preferred are those conjugates in which x, e, ~, h, y7 and g are each 1, and k is 2 or more. Also particularly preferred are those starburst conjugates in which M represents a bioactive agent such as a radionuclide, drug, or toxin.
Suitable connecting groups which are represented by C " are groups which link the carried pharmaceutical material to the dendrimer without significantly impairing the effectiveness of the carried pharmaceutical material or the effectiveness of the target director(s) present in the starburst conju-gate. These connectors must be stable ~i.e., non-clea-vable) or cleavable depending upon the mode of activity 35,444-F"A" -22--23- 1316~56 of the carried pharmaceutical material and are c.
typically used in order to avoid -~t~e hindrance between the carried pharmaceutical material and the polymer.

Most preferred are conjugates in which the dendrimer is associated directly, or via connecting group(s), to one antibody or antibody fragment. The polymer in these preferred conjugates may, in addition, be optionally a~sociated either directly, or via connecting group(s), to one or more other carried materials, preferably a radioisotope. Such starburst conjugates are repre~ented by the formula:

[~Antibo~Y)e - (C )~lg * (P)x * [(C )h ( )y k (IV) wherein each Antibody represents an antibody or antibody fragment capable of interacting with a desired epitope;
- indicates a covalent or coulombic bond in instances where a connecting group is present; and p, x, *, M, T, e, y, C', C " , g, k, f, and h are as previously deflned herein.
For the above synthesis of starburst dendrimers (P) which have ~ functional group available for linking 3 (C' or C") with a targeting director (T), the preferred proceqs requireq that the reactive functionality be protected as a synthetic precursor. This protection is preferred because it enables the synthesi~ of dendrimer or conjugates of very high quality. This process allow~ for the chemical binding of a unit of carried 35,444-F"A" -23- _ 1 3 ~ 6 -21~-pharmaceutical material (M) to the terminal functional groups of the starburst dendrimer (P) in ways which would otherwise result also in reaction with a linking functional group, thus making it impossible to attach to the targetting director ~T). Subsequent deprotection or synthetic conversion into the desired linking functional group thus enables the starburst conjugate to be linked to the targetting director.
One of the preferred "functional groups for linking" (hereafter referred to as a "handle") is an aniline moiety. This group is preferred because it can be used directly for linking to the targetting director, or it can be readily modified to other functional groups suitable for reaction with the targetting director, e.g. isothiocyanate, isocyanate, emithiocarbazide, semicarbazide, bromoacetamide~
iodoacetamide, and maleimide. The aniline moiety is also preferred as a handle for linking with the targetting directors because it can be readily protected for use in starburst dendrimer synthesis, or the nitro group can be used as a precursor which can be converted into the desired amino function at the end of the synthesis.
There are a number of protecting groups which are suitable for protecting the anilino amino functionality during starburst dendrimer synthesis.
(See Theodora W. Green, Protective Groups In Organic Synthesis., Pub. John Wiley & Son, New York, 1981). A
preferred class of protecting groups are the carbamates shown below.

35,441~-F"A" -24- _ -25~ ~ 3~

~ H2 ~ - ~ NHCOR

Many carbamates have been used for protection of amines. The most preferred carbamates for starburst dendrimer synthesis is the t-butoxycarbamate, R =
-C(CH3)3. Deprotection is achieved by mild acid hydrolysi~. Also preferred is the benzylcarbamate f 15 protecting group, R_-CH2- ~ / , which is preferred when the dendrimer is susc~ible to acid hydrolysis.
Deprotection is achieved by catalytic hydrogenation.
Also preferred is the 9-fluorenylmethylcarbamate, R- -CH2 ~

The phthalimide protecting group is also a preferred example, R ~ ~NH2 ~ R~ ~ N

35 9 444-F"A" 25- _~

1 3~ Sfi 64693-4]04 Other protecting groups used for amines which are well known in the literature could also be used in this synthetic scheme.
The above preferences are given as illustrative examp]es only but are not the only protecting groups such as carbamates which can be used. Any protecting group which is stable under the reaction conditions and can be removed wi-thout altering the integrity of the starburst dendrimer can be employed.

An alternate process involves the reaction of an activated aryl halide, e.g. 4-nitrofluorobenzene, with an amino-function; on the agent for conjugation, e.g. starburstpolyethyleneimines (PEI), and subsequent catalytic hydrogenation of the nitro group to the aniline functionali-ty for subsequent conjugation. It is particularly useful for agents, e.g. polyamines, which need further modification prior to use, due to the relative chemical inertness of the nitrophenyl functionality to all non-reducing reaction conditions. The more common bifunctional linking agents, e.g. active esters or diisocyanates, which are reactive under a large number of reaction conditions and which would render them unusuable for conjugation include:

.~
h 27 ~31~;6 o o ~ i ~ Cc~2cH2 ~ N

ICH2CNH ~ -C2-N ) ; and The invention also include~ the use of ..
nitro~substituted arylsulphonyl halides to give sulphonamides~

e.g. 02N- ~ S2X

The advantage of this proce~s over known proce~ses of introducing an aminophenyl group for conjugation is that it takes place at a late stage of the synthe~iq. Gansow et al., U.S. Patent 4,472,509, in hi~ process introduced the nitrophenyl group at the 35,444-F"A" -27-first step of a long synthe-tic procedure, -thereby having limitations on the chemistry available.

This process also introduces a handle which is clearly differentiable from the remainder of the molecule. Manabe et al.
disclosed that the ring opening of succinic anhydride by residual amines gave a coupling group through which conjugation to an anti-body was possible. This method however gave no means of differentiating between any unchelated sites on the ~olymer, since the chelating groups were the same as the linking group.

The above process can introduce an aminophenyl functionality into any agent containing an amino group which is then conjugated with some bioactive agent, e.g. monoclonal anti-body or enzyme. The agent can be conjugated by oxidative coupling to carbohydrates on the bioactive agent, e.g. antibody. The aminophenyl group also can be converted into an isothiocyanate or isocyanate for subsequent reaction with the pendant amino groups of lysine residues on the bioactive agent.
The present process also provides for direct chelation of lanthanides with starburst dendrimers, preferably by PEI
acetate dendrimer. In contrast, Denkwalter, U.S. Patent 4,289,872 states that just putting acetates on the surface works. However, the present reaction shows that PEI acetate, works much better than PAMA~l, i.e~ surface of iminodiacetates is only part of the story, the nature of the backbone, and branching is important also. The PEI acetate has better chelating properties than the PAMAM acetate.

`:

2,~- ~ 3 ~ s s rco2~

~ N
N ~ L Co2e N ~ \ ~ ¦ C023 L e PEI

~~ 15 rco2 ,. ~ I
~ CNH ~ C029 N r Co2e CNH ~ L
co2e PAMAM

Preferred among the starburst conjugates of formula ~IV) are those in which M is a radionuclide, drug~ toxin, signal generator, signal reflector or signal absorber. Also preferred are those conjugates 3o in which x-1. Particularly preferred are those conjugates in which x, et f9 h, and y are each 1, and g is one or more and k is each individually 2 or more.
Most preferred are those conjugates in which x, e7 f, h9 y, and g are each 1, and k is 2 or more. Also particularly preferred are those starburst conjugates in which 1'Antibody" represents a monoclonal antibody or 359444-F"A" -29- _ ~30- 13~6~

an epitope binding fragment thereof; and especially preferred are those in which M represents a bioactive agent such as a radionuclide, drug, or toxin.
The starburst conjugates can be used for a variety of in vitro or in vivo diagno~stic applications such as radioimmunoassays, electron microscopy, enzyme linked immunosorbent assays, nuclear magnetic resonance spectroscopy, contrast imaging, and immunoscintography, 1Q in analytical applications, in therapeutic applications a~ a carrier of antibiotics, radionuclides, drugs or other agents suitable for use in the treatment of disease states such as cancer, autoimmune diseases, central nervous sy~tem disorders, infectious disease~, and cardiac disorders, or used as starting materials ~or making other useful agents.
The present i.nvention is also directed to starburst conjugate compositions in which the starburst conjugates are formulated with other suitable vehicles~
The starburst conjugate compositions may optionally contain other active ingredients, additives and/or diluents.
The pre~erred starburst polymer for use in the starburst conjugates of the present invention is a polymer that can be described as a starburst polymer having at least one branch (hereinafter called a core branch), preferably two or more branches, emanating ~rom a core, said branch having at least one terminal group provided that (l) the ratio of terminal gr~ups to the core branches i~ more than one, preferably two or greater, (2) the density o~ terminal groups per unit volume in the polymer is at least 1.5 times that of an extended conventional star polymer having similar core 35,444-F"A" -30--31-- ~3t~

and monomeric moieties and a comparable molecular ~eight and nu~ber of core branches, each of such branches of the extended conventional star polymer bearing only one terminal group, and (3~ a molecular volume that is no more than about 80 percent of the molecular volume of said extended conventional ~tar polymer as determined by dimensional studies using scaled Corey-Pauling molecular models. As used herein, the term "den~e" as it modifies "star polymer" or "dendrimer" means that it has a smaller mol*cular volume than an extended conventional star polymer having the same molecular weight. The extended conventional star polymer which is used as the base for comparison with the dense star polymer is one that has the same molecular weight, same core and monomeric components and same number of core branches aq the dense star polymer. By "extended" it is meant that the individual branches of the conventional star polymer are extended or stretched to their maximum length, e.g~ 9 as ~quch branches exist when the star polymer i~
completely solvated in an ideal solvent for the star polymer. In addition while the number of terminal groups is greater for the dense star polymer molecule than in the conventional star polymer molecule, the chemical structure of the terminal groups is the same.
Dendrimers used in the conjugates of the present invention can be prepared by processes known in 3 the art. The above dendrimers, the various coreactants and core compounds, and process for their preparation can be a~ defined in U. S. Patent 4,587,329.
The dendrimers~ for use in the conjugates of JJ the present invention, can have terminal groups which are sufficiently reactive to undergo addition or 35,444-F"A" -31--32- ~31~

substitution reaction~. Examples of such terminal group~ include amino, hydroxy, mercapto, carboxy, alkenyl, allyl, vinyl, amido, halo, urea, oxiranyl, aziridinyl, oxazolinyl, imidazolinyl, sulfonato, phosphonato, isocyanato and isothiocyanato. The terminal groups can be modified to make them biologically inert, for example, to make them non-immunogenic or to avoid non-specifc uptake in the liver, spleen or other organ. The dendrimers differ from conventional star or star-branched polymers in that the de~drimers have a greater concentration of terminal groups per unit of molecular volume than do conventional extended star polymers having an f 15 equivalent number of core branches and an equivalent core branch length. Thus, the density of terminal groups per unit volume in the dendrimer uYually is at least 1.5 times the density of terminal groups in the conventional extended star polymer, preferably at least

5 times, more preferably at least 10 times, most preferably from 15 to 50 times. The ratio of terminal groups per core branch in the dense polymer is preferably at least 2, more preferably at least 3, most preferably from 4 to 1024. Preferably9 for a given polymer molecular weight, the molecular volume of the dense star polymer is less than 70 volume percent, more preferably from 16 to 60, most preferably from 7 to 50 volume percent of the molecular volume of the 3~ conventional extended star polymer.
Preferred dendrimer~ for use in the conjugates of the present invention are characterized as having a univalent or polyvalent core that is covalently bonded to dendritic branches. Such ordered branching can be 35,444-F"A" -32- _ -33~

illustrated by the following sequence wherein G
indicates the number of generations:

G = 1 G = 2 ~ N-~ N~
~ ' ~
N

/ \ ~ N
H H N / \
H H H H

~ -N-'---' ~ N

f ~ ' f ~
N N N N
H H H H H H H H

Mathematically, the relationship between the number (#) of terminal groups on a dendritic branch and the number o~ generations of the branch can be represented a~ follows:

35,444-F"A" -33- _ -3~- ~31~45~

NrG
# of terminal groups per dendritic branch = -wherein G is the number of generations and Nr is the repeating unit multiplicity which is at least 2 as in the case of amines. The total number of terminal groups in the dendrimer is determined by the following:

G
NcNr # of terminal groups per dendrimer =

wherein G and Nr are as defined before and Nc represents the valency (often called core functionality) of the core compound. Accordingly, the dendrimers of this invention can be represented in its component parts as follows:

( Te~minal (Core)~(Repeat Unit~ G Moiety ~ Nc Nr_l ~

wherein the Core, Terminal Moiety, G and Nc are as defined before and the Repeat Unit has a valency or functionality of Nr ~ 1 wherein Nr is as defined beforeO
A copolymeric dendrimer which is a preferred dendrimer for the purposes of this invention is a unique compound constructed of polyfunctional monomer 35,444-F"A" -34- _ 3 13~5~

units in a highly branched (dendritic) array. The dendrimer molecule is prepared from a polyfunctional initiator unit (core compound), polyfunctional repeating unit~ and terminal units which may be the same or different from the repeating units. The core compound is represented by the formula ~ (ZC)Nc wherein ~ represents the core, zc represents the functional groups bonded to ~ and Nc represents the core functionality which is prefera~ly 2 or more, mo~t preferably 3 or more. Thus, the dendrimer molecule comprises a polyfunctional core, ~ bonded to a number (Nc) of functional groups, zc, each of which is connected to the monofunctional tail of a repeating unit, X1Y1(Z1)N1~ of the first generation and each of the Z groups of the repeating unit of one generation is bonded to a monofunctional tail of a repeating unit of the next generation until the terminal generation is reached.
In the dendrimer molecule, the repeating units are the same within a single generation, but may differ from generation to generation. In the repeating unit, X1Y1(Z1)N1, X1 represents the monofunctional tail of the first generation repeating unit, y1 represents the moiety constituting the first generation, z1 represents the functional group of the polyfunctional head of the repeating unit of the first generation and may be the same as or different from the functional groups of the 3 core compound, ~ (ZC)Nc, or other generations; and is a number of 2 or more, most preferably 2? 3 or 4, which represents the multiplicity of the polyfunctional head of the repeating unit in the fir~t generation.
Generically, the repeating unit is represented by the formula XiYi(Zi)Ni wherein "i" represents the 35,444-F"A" -35--36~ 1316~

particular ~eneration from the firqt to the t-l generation. Thus, in the preferred dendrimer molecule, each z1 of the first generation repeating unit is connected to an x2 of a repeating unit of the second generation and so on through the generations such thak each zi group for a repeating unit XiYi(Zi)Ni in generation number "i" is connected to the tail (Xi+1) of the repeating unit of the generation number "i+1".
The final or terminal of a preferred dendrimer molecule comprises terminal units, XtYt(Zt)Nt wherein t represents terminal generation and xt, yt~ zt and Nt may be the same as or different from xi, yi~ zi and Ni except that there is no succeeding generation connected to the zt groups and Nt may be less than two, e.g., zero or one. Therefore the preferred dendrimer ha~ a molecular formula represented by i(~(ZC) )~i yi (Z)i~ n~xtYt(z )N~)NcnNn where i is 1 to t-1 wherein the symbols are as previously defined. The 3 function is the product of all the values between its defined limits. Thu~

~ Nn = (N1)(N2)(N3)~-(Ni-2)(Ni-l) n-1 35,444-F"A" -36- _ -37- ~3~4~

which is the number of repeat units, XiYi(Zi)Ni, comprising the ith generation of one dendritic branch and when i is 1, then 11 = 1 n=1 In copolymeric dendrimers, the repeat unit for one generation differs from the repeat unit in at least one other generation. The preferred dendrimers are very symmetrical as illustrated in structural formulas deqcribed hereinafter. Preferred dendrimers may be converted to functionalized dendrimers by contact with another reagent. For example, conversion of hydroxyl in the terminal generation to ester by reaction with an acid chloride gives an ester terminally functionalized dendrimer. This functionalization need not be carried out to the theoretical maximum as defined by the number of available functional groups and, thus, a functionalized dendrimer may not have high symmetry or a precisely defined molecular formula as is the case with the preferred dendrimer.
In a homopolymeric dendrimer, all of the repeat units, XiYi(Zi)Ni, are identical. Since the values of A 3 all Ni are equal (defined as Nr)7 the product function representing the number of repeat un ts reduces to a simple exponential form. Therefore, the molecular formula may be expressed in simpler form as:

35,444-F"A" -37- _ -38~ ~3~4~6 ~ ~ (Zc)N ~ ~(X Y (~ )N3 NCNri-l ~ (XtYt(Z )N~ NcNr(t-1 where i = 1 to t~1 This form still shows the distinction between the different generations i, which each consist of NCNr(i-1) repeating units, XiYi(Zi)Ni. Combining the generations into one term gives:

(Zc)N ~ (X Y (Z )N~ ~xtYt(zt)Nt) Nc Nr(t-1)_ CNr(t-1) Nr-l or (~ ~Zc)N ) ~ (xrY (Z )Nr) N (t~ N ) (t 1 Nr-1 Nc Core Repeat Unit` Terminal Unit 35,444-F"A" -38- _ -39~ 131~

wherein XrYr(Zr)Nr is the repeating unit which is used in all generations i.
Consequently, if a polymer compound will fit into these above formulae, then the polymer is a starburst polymer. Conversely, if a polymer compound will not fit into these above formulae, then the polymer is not a starburst polymer. A1SQ, to determine whether a polymer is a starburst polymer, it is not necessary to know the process by which it was prepared, but only whether it fits the formulae. The formulae also demonstrate the generations (G) or tiering of dendrimers.
~~ 15 Clearly, there are several ways to determine the ratio of agent (M) to dendrimer (P) which depend upon how and where the association of P*M occurs. When there is interior encapsulation, the weight ratio of M:P usually is 10:1, preferably 8:1, more preferably 5 1, most preferably 3:1. The ratio can be as low as 0.5:1 to 0.1:1. When interior stoichiometry is used, the weight ratio of M:P is the same as for interior encapsulation. When exterior stoichiometry is determined, the mole~mole ratio of M:P is given by the following formulae:

M : P
3 (A) 5 NCNtNrG I 1 (B) 3 NCNtNr~
~C3 1 NCNtNrG-1 1 where Nc means the core multiplicity, Nt means the terminal group multiplicity, and Nr means branch 35,444-F"A"-39- _ ~4693-~104 ~ullcture multlpllcity. The ~C.NtNr~-l term will result in the num-ber of Z groups. Thus, lor e~ample, ~A) above will result when protelns, en~ymes or hlghly charged molecules are on the surface;
(~) above when it ls aspirin or octanoic acld; ~C) above when it i5 carboxylate lons or groups.
Of course other structures of various dimensions can be readily prepared by one skllled in the art by approprlately vary-ing the dendrimer components and num~er of generations employed.
A roughly scaled comparlson of three dlfferent dendrlmer serles relatlve to an IgG antibody is seen in F'lgure 3. The series of drawlngs indlcated by Figure 3~B) I shows the starburst polyaml-doamines (PAMAM); by II shows the starburst polyethers (PE); and by ~II shows the starburst polyethyleneimlnes (PEI). In a manner similar to that of Flgure 1, all three series (I, II and III) have thelr far left ~rawlng showing the lnltlator core, the next draw-ing from the left showlng the starhranch ollgomer, and the remain-ing drawings showlng the starburst ollgomers and respectlve star-burst bridged dendrimers. It can be seen that ln these series of scale drawlngs that the dendrlmer dlmenslons are close to and in fact smaller than those noted for the IgG antlbody Flgure 3~A).
The IgG antlbody ls shown to the far left ln Flgure 3. The scale is 1 mm = 3.5A. In Figure 3~A~ the varlable reglon ls shown by (A); the constant reglon by (~); and the carbohydrate attachment sites by (C). Approximate measurements shown on Figure 3 are (1) is 35-40A; (2) ls 70A; and (3) ls 60A. These dlmenslonal proper-tles are preferred for lnstance where targetlng lnvolves exlting from the vascular system. T~lerefore dendrlmer dlameters of 125 Angstrom ~41- 131~

unit~ or less are particularly preferred in that they may allow exiting from the vascular system to targeted organs serviced by continuous or fenestrated capillaries. These dimensions are significant in that they are small compared to the size of a potential targeting component such as an antibody (see Figure 3).
A linear polymer of comparable molecular weight would have a radius of gyration, (in its fully extended form), that would be much larger than the same molecular weight dendrimer. A linear polymer of this type would be expected to adversely affect the molecular recognition properties of many accapted targeting components. It is also de~irable that the conjugates be of minimum molecular volume so as not to discourage, extravasation, e.g., by coupling Fab, Fab' or other appropriate antibody fragment to low molecular volume dendrimers.
2~ Dendrimers are deslrable for the delivery of radionuclides or strongly paramagnetic metal ions to tumor sites because of their ability to chelate a number of metal ions in a small volume of space.
Coupling to antibodies or antibody fragments which are specific for tumors may deliver a number of metals per antibody, with only a single modification to the antibody.
Linking target directors to dendrimers is another aspect of the present invention. In preferred embodiments of the present invention, particularly where it is desired to use an antibody as a target director, a reactive functional group such a~ a carboxyl, sulfhydryl, reactive aldehyde, reactive olefinic derivative, isothiocyanato, isocyanato, amino, reactive aryl halide, or reactive alkyl halide can 35,444-F"A" -41_ !1 31~3~
~9~-4104 convenlently be employed on the dendrimer. The reactive func-tlonal groups can be lntroduced to the dendrimer using known technlques, for example:
(1) Use o~ a heterofunct~onal inl~iAtor (as a starting materlal for synthesizing the dendrlmer) which has incorporated lnto it functlonal groups o~ di~ferent reactivity. In such heterofunctlonal initiator at least one o~ the functlonal groups will serve as an lnitiatlon slte for dendrimer formatlon and at least one of the other functlonal groups will be available for llnking to a target dlrector but unable to lnitiate dendrlmer synthesls. For e~ample, use of protected aniline allows further modlficatlon of NH2 groups wlthln the molecule, wlthout reactlng the NH2 of the anlllne.
The functlonal group whlch wlll be avallable for linklng to a target dlrector may be part of the lnltlator molecule ln any one of three forms; namely:
(a) In the form in whlch lt wlll be used for linking with the target dlrector. This ls possible when none of the synthetlc steps lnvolved in the den-drimer synthesls can result ln reactlon at thls center.
(b) When the functlonal group used for linking to the targeting dlrector ls reactlve ln the synthetlc steps involved ln the dendrimer synthesls, lt can be protected by use of a protectlng group, whlch renders the group unreactlve to the synthetlc procedures lnvolved, but can ltself be readl.ly removed ln a manner _4~_ ~3~5~

which does not alter the integrity of the remainder of the macromolecule.
(c) In the event that no simple protecting group can be formed for the reactive functionality to be used for linking with the targeting director, a synthetic precursor can be used which is unreactive in all the synthetic procedures used in the dendrimer synthesis. On completion of the synthesis, this functional group must be readily convertible into the desired linking group in a manner which does not alter the integrity of the remainder of the molecule.
(2) Coupling ~covalently) the desired reactive functional group onto a preformed dendrimer, the reagent used must contain a functionality which is readily reacted with the terminal functional groups of the dendrimer. The functional group to be ultimately used to link with the targetting agent can be in its final form, as a protected functionality, or as a synthetic precursor. The form in which this linking functionality is used depends on its integrity during the synthetic procedure to be utilized, and the ability of the final macromolecule to withstand any conditions nece~sary to make this group available for linking.
For example, the preferred route for PEI uses F ~ N02 35,444-F"A" ~43 _44 13~

Example~ of heterofunctional initiator~ for use in (1) above, include the following illustrative examples:

H2N ~ ~ 2CH

~CNHCH2GH2NH2 //

H2N ~ CH2C~H

3o 35,444-F"A" -44- _ -45~

\ /
( CH3 ~ 3COCN~C~CH2(~ ;

,CNHCH2~H2NH2 r- 1 5 ( CH3 ) 3COCNH~ CH2 H2N ~CH2CH2NH2 35, 444-F"A" -45- _ 13~6~6 H2N ~ CH2CH

02N ~3 CH2CH2NH2 2C ÇH2NH2 02N ~~>--CH2CH ; and 2~

35, 444-F"A" -46-~CH2CH2NH2 fH2NCH2CH2NH2 02N --CH2 lH

CH21fCH2CH2NH2 1 c CH2CH2NH2 C
There are several chemistries of particular importance:
1) Starburst Polyamidoamides ("PAMAM") Chemistry;
2) Starburst Polyethyleneimines ("PEI") Chemistry;
3) Starburst PEI compound with a surface of PAMAM;
4) Starbur~t polyether ("PE") chemistry.

3o 35,444-F"A" -47- _ -48- ~31~56 Modifications of the dendrimer surface functionalities may provide other useful functional groups such as the following:
-OP03H2, -P03H2, -P03H(-1), _po3(-2), -C02( 1), -S02H, -S02( 1), -S03H, -S03(~ NRlR2, -R5, -OH, -ORl, -NH2, perfluorinated alkyl, -CNHR1, -COH, ,9 ,.
O O

ln -(CH2)n ~ ' N=C ~

-NHCH2~ ' -(CH2)n ~
R3 R4 N -(CH2)n wherein R represents alkyl, aryl or hydrogen;
R1 represents alkyl, aryl, hydrogen, or (CH2)n -N X

~ (CH2)n 35,444-F"A" -48-_49_ 131~4~6 R2 represents alkyl, aryl, or ~ (CH2)n ~ (CH2)n ~
R3 represents -OH, -SH, -C02H, -S02H, or -S03H;
R4 represents alkyl, aryl, alkoxy, hydroxyl, mercapto, carboxyl, nitro, hydrogen, bromo, chloro, iodo, or fluoro;
R5 represents alkyl;
X represents NR, O or S; and n represents the integer 1, 2 or 3;
polyethers; or other immuno insensitive moieties.
20The choice of functional group depends upon the particular end use for which the dendrimer is designed.
Linking of antibodies to dendrimers is another aspect of the present invention. Typically, the antibodies or antibody fragments are linked to the dendrimer by techniques well known in the art such as attachment between a functional group on the dendrimer and moieties such as carbohydrate, amino, carboxyl, or sulfhydryl functionalities present in the antibody or antibody fragment. In some instances connecting groups may be used as connectors or spacers between the dendrimer and antibody or antibody fragment. The attachment of the dendrimer to the antibody or antibody fragment should be performed in a manner which does not significantly interfere with the immunoreactivity of 35,444-F"A" -49_ -50- ~316~

the antibody or antibody fragment, that is, by binding the antibody or antibody fragment via a functionality in the antibody or antibody fragment which is not a part of the antigen recognition and binding site.

The following examples further illustrate the invention but are not to be construed as a limitation on the scope of the invention. The lettered examples concern the preparation of starting materials; the numbered examples concern the preparation of products~

Exam~le A: Preparation of 2-Carboxamido-3-(4'-nitro-phenyl)-propanamide.
p-Nitrobenzyl malonate diethylester (2.4 grams (g), 8.13 mmole) was dissolved in 35 ml of methanol.
The solution was heated to 50-55C with stirring and a stream of anhydrous ammonia was bubbled through the solution for 64 hours. The solution was cooled and the white, flocculant product was filtered and recrystallized from 225 milliliters (ml) of boiling methanol to afford 1.85 g (7.80 mmole) of bis amide in 96% yield (mp = 235.6C(d)).
The structure was confirmed by MS,1H and 13C NMR
spectroscopy.
Anal: Calc. for Cl oH 1 1 04N3 C H N
Theo: 50063 4.69 17.72 Found: 50.75 4.81 17.94 35,444-F"A" -50- _ Example 8: Preparation of l-Amino-2-(aminomethyl)-3-(4'-nitrophenyl)propane.
2-Carboxamido-3-(4'nitrophenyl)propan^mide (2.0 g, 8.43 mmole~ was slurried in 35 ml of dry tetrahydro-furan under a nitrogen atmosphere with stirring. Tothis mixture was added borane/tetrahydrofuran complex (106 ml, 106 mmole) via syringe. The reaction mixture was then heated to reflux for 48 hours during which time the suspended amide dissolved. The solution was cooled and the tetrahydrofuran was removed in vacuo using a rotary evaporator. The crude product and borane residue was dissolved in 50 ml of ethanol and thiq solution wa~ purged with anhydrous hydrogen chloride gas. The solution wa~ refluxed for 1 hour and the solvent removed in vacuo. The crude hydrochloride salt was dissolved in 15 ml of deionized water and extracted with two 50 ml portions of methylene chloride. The aqueous layer was cooled in an ice bath under an argon blanket and 50% sodium hydroxide was 510wly added until basic pH=11.7. The basic aqueous layer was extracted with four 25 ml portions of methylene chloride and these combined extracts were evaporated (rotary) to give 1.45 g of amber colored oil. This oil was triturated with diethyl ether (50 ml) and filtered under pressure through a short silica gel (grade 62 Aldrich) column. The column was washed with 100 ml of ether and the combined filtrates were vacuum evaporated giving 1.05 g (5.02 mmole) of the titled diamine as a clear oil (mp = 275-278C(dj bis HCl salt).
The structure was confirmed by MS, lH and 13C NMR
spectroscopy.

35,444-F"A" -51--52- ~ 31 6~5 6 A_al: Calc. for C1oHl7N3o2cl2 C H N
Theo: 42.57 6.07 14.89 Found: 43.00 6.14 15.31 Example_C: Preparation of l-Amino-2-(aminomethyl)-3-(4'-aminophenyl)propane.
Borane/tetrahydrofuran solution (7Q ml, 70 mmole) was added under nitrogen via a cannula needle to a flask containing 4-amino-benzyl malonamide (1.5 g, 7.24 mmole) with stirring. The solution was brought to reflux for 40 hour~. The colorless solution was cooled and excess tetrahydrofuran was removed by rotary evaporation leaving a clear gelatinous oil. Methanol (50 ml) was cautiously added to the oil with notable gas evolution. Dry hydrogen chloride was bubbled through the suspension to effect dissolution and the solution was then refluxed for 1 minute. The methanol/HCl was rotary evaporated and the resulting hydrochloride salt was carried through the same dissolution/reflux procedure again. The hydrochloride salt obtained was dissolved in 10 ml oY water and cooled in an ice bath under argon. Concentrated sodium hydroxide (50%) was added slowly with stirring to pH=11. The aqueous portion was then extracted with 2 X
100 ml portions of chloroform which were combined and filtered through a short silica gel plug without drying. The solvent was removed in vacuo (rotary) 3 affording the title compound (0.90 g, 5.02 mmole) in 70% yield (Rf=0.65 ~ CHCl3/MeOH/NH40H conc - 2~2/1).
The structure was confirmed by 1H and 13C NMR and used without further purification.

35,444-F'~A" -52- _ -53_ 1316~5~

Example D: Preparation of 6-(4-Aminobenzyl~-1,4,8,11-tetraaza-5,7-dioxoundecane.
4-Aminobenzyl malonate dimethylester (2.03 g, 8.43 mmole) was dissolved in lO ml of methanol. This solution was added dropwise to a stirred solution of freshly distilled ethylene diamine ~6.00 g, 103.4 mmole) in 10 ml of methanol under nitrogen over a 2 hour period. The clear solution was stirred for 4 days and Thin Layer Chromotography (TLC) analysis indicated total conversion of diester (R~ = 0.91) to the bis amide (Rf = 0.42 - 20% conc NH40H/80% ethanol). This material was strongly ninhydrin positive. The methanol and excess diamine were removed on a rotary evaporator and the resulting white solid was vacuum dried (10~1 - mm, 50C) overnight to afford crude product (2.45g, 8.36 mmole) in 99% yield. An analytical sample was recrystallized from chloroform/hexane, MP = 160-161C.
The mass spectral, 1H and 13C NMR data were consistent with the proposed structure.

Example E: Reaction of Mesyl Aziridine with 1-Amino-2-(aminomethyl)-3 (4-nitrophenyl)propane.

l-Amino-2-(aminomethyl)-3-(4-nitrophenyl)-propane (400 mg, 1.91 mmole, ~96% pure) was dissolved in 10.5 ml of absolute ethanol under nitrogen. Mesyl aziridine (950 mg, 7.85 mmole) was added to the stirred diamine solution as a solid. The reaction was stirred at 25C for 14 hours using a magnetic stirrer and during this period a white, gummy residue formed on the sides of the flask. The ethanol was decanted and the residue was triturated with another 15 ml portion of ethanol to remove any unreacted aziridine. The gummy product was 35,444-Ft'A" ~53_ ~54~ ~ 316~6 vacuum dried (lO1mm, 25C) to afford the tetrakis methyl sulfonamide (1.0 g, 1.44 mmole) in 75% yield (Rf - 0.74 - NH40H/ethanol - 20/80). The structure was confirmed by lH and 13C nuclear magnetic resonance (NMR) spectroscopy.

Example F: Preparation of 2-(4-Nitrobenzyl)-1,3-(bis-N,N-2-aminoethyl)diaminopropane.

The crude methylsulfonamide (650 mg, 0.94 mmole) was dissolved in 5 ml of nitrogen purged, concentrated sulfuric acid (98%). This solution was maintained under nitrogen and heated to 143-146C for 27 minute~ with vigorous stirring. A slight darkening was - 15 noted and the cooled ~olution waq poured into a stirred solution of ether (60 ml). The precipitated white salt cake was filtered and immediately dissolved in lO ml of deionized water. The pH of the solution was adjusted to pH=11 with 50% NaOH under argon with cooling. The resulting solution was mixed with 90 ml of ethanol and the precipitated inorganic salts were filtered. The solvent was removed from the crude amine under reduced pressure and to the resulting light brown oil was added 190 ml of toluene under nitrogen. The mixture was stirred vigorously and water was removed through azeotropic distillation (Dean-Stark trap) until the remaining toluene acquired a light yellow color (30-40 ml remaining in pot). The toluene was cooled and decanted from the dark, intractable residues and salt.
Thi~ solution was stripped of solvent in vacuo and the resulting light yellow oil waq vacuum dried (0.2 mm, 35~) overnight affording 210 mg of the product (60%) which was characterized by MS, lH and 13C NMR.

35,444-F"A" -5~- _ 13164~6

6~693-41~4 E~xample ~: Preparatlon of a starburst polymer (containing an anlline derlvative) of one half generatlon represented by the followlng scheme:

~ 11 H2C=CHCOCH3 H2N--~O ~ CH2CH(CNE~CH2CH2NH2)2 + - >`
\___J CH30H
Compound ~1 f o O

2 ~ CH2CH(CNHCH2CH2N(CH2CH2CoCH3)2)2 Compound #2 Methyl acrylate (2.09 g, 24 mmole) was dissolved in methanol (15 ml). The compound 6-(4-amlnobenzyl)-1,4,8,11-tetra-aza-5,7-dloxoundec,ane (1.1 g, 3.8 mmole) (i.e., Compound #1, pre-paratlon described ln Example D) was dlssolved ln methanol (10 ml) and was added slowly over 2 hours with ri.gorous stirring to the methyl acrylate solutlon. The reaction ml~ture was stlrred for 48 hours at amblent temperatures. The solvent was removed on the rotary evaporator malntainlng the temperature below 40C. The ester (Compound ~2) was obtalned as a yellow oil (2.6 g). No carboxyethylation of the anlline ~unction was observed.

--c~i, ~j -56- 131~4~6 Example H: Preparation of a starburst polymer (containing an aniline moiety) of one generation;
represented by the following scheme:

Compound #2 ~ H2NcH2cH2NE~2 H2N ~ ~H2CH(CNHCH2CH2N(CH2CH2CNHCH2CH2NH2)2 Compound ~3 r l5 The ester (Compound #2) (2.6 g, 3.7 mmole) was dissolved in methanol (lO0 ml). this was carefully added to a vigorously stirring solution of ethylene diamine (250 g, 4.18 mole) and methanol (100 ml) at such a rate that the temperature did not rise above 40C. After complete addition the reaction mixture was stirred for 28 hours at 35-40C (heating mantle). After 28 hours no ester groups were detectable by infrared spectroscopy. The solvent was removed on the rotary evaporator at 60C. The excess ethylene diamine was removed using a ternary azeotrope of toluene-methanol-ethylene diamine. Finally all remaining toluene was azeotroped with methanolO Removal of all the methanol yielded 3.01 g of the product (Compound #3) as an orange glassy solid.

35,444-F"A" -56- _ ~57~ 13~64~6 Example I: Preparation of a starburst polymer ~containing an aniline moiety~ of one and one half generations represented by the ~ollowing scheme:

Compound #3 + H2C=CHCOCH3 H2N_ ~ -cH2cH(c~cH2cH2N(cH2c82cNHcH2cH2N(c~2c~2c~H3)2~2)2 Compound #4 , 15 The amine (Compound #3) (2.7 g, 3.6 mmole) was dissolved in methanol (7 ml) and was added slowly over one hour to a stirred solution of methyl acrylate (3.8 g, 44 mmole) in methanol (15 ml~ at ambient temperatures. A slight warming of the solution was observed during the addition. The solution was allowed to stir at ambient temperatures for 16 hours. The solvent was removed on the rotary evaporator at 40C.
After removal of all the solvent and excess methyl acrylate the ester (Compound #4) was obtained in 4.7 g yield as an orange oil.

3o 35,444-F"A'~ -57- _ -58~ 6 ExamDle J: Preparation oP a starburst polymer (containing an aniline moiety) of one half generation represented by the following scheme:

H2N ~ CH2cH~cH2NH2)2 + H2C=CHCOCH3 Compound #5 CH30H

H2N- ~ -CH2CH(CH2N(CH2CH2COCH3)2)2 Compound #6 The triamine (Compound #5, the preparation of this compound is shown in Example C) (0.42 g, 2.3 mmole) was dissolved in methanol (10 ml) and was added dropwise over one hour to methyl acrylate (1.98 g, 23 mmole) in methanol (10 ml). The mixture was allowed to stir at ambient temperatures for 48 hours. The solvent was removed on the rotary evaporator, maintaining the temperature at no higher than 40C. The excess methyl acrylate was removed by repeated azeotroping with methanol. The ester (Compound #6) was isolated as an orange oil (1.24 g).

35,444-F"A" -58- _ ~59 1316~

Example K: Preparation of a starburst polymer (containing an aniline moiety) of one generation;
represented by the following scheme:

Compound #6 + H2NCH2CH2NH2 o ~(cH2~(cH2cH2cNHcHzcH2N~2)2)2 Compound #7 The ester (Compound #6) (1.24 g7 2~3 mmole) was dissolved in methanol (50 ml) and was added dropwise over two hours to ethylenediamine (73.4 g, 1.22 mole) in methanol (100 ml). A small exotherm was noted, vigorous stirring was maintained~ The solution was left to stir at ambient temperatures for 72 hours. The solvent was removed on the rotary evaporator at 60C.
The exces~ ethylene diamine was removed using a ternary azeotrope of toluene-methanol-ethylenediamine. Finally all remaining toluene was removed with methanol and then pumping down with a vacuum pump for 48 hours gave the amine (Compound #7) (1.86 g) as a yellow/orange oil.

35,444-F''AI' -59- _ -60- ~31~

Example L: Preparation of a starburst polymer (containing an aniline moiety) of one and one hal~
generations; represent by the following scheme:

Compound #7 ~ H2C=CHCOCH3 O O
E~ 2N ~ ~ 2C~ ( CEI 2N ( CH 2CM 2 CNHCE; 2CE1 2N ~ CH 2C~ 2COC1~ 3 ) 2 ) 2 ) 2 Compound #8 The amine (Compound #7) (1.45 g, trace of methanol remained) was dissolved in methanol (100 ml) and was added slowly over 1~ hours to a stirred solution of methyl acrylate (5.80 g) in methanol (20 mll. The solution was allowed to stir for 24 hours at room temperature. Removal of the solvent followed by repeated azeotroping with methanol enabled the removal Of all the excess methyl acrylate. After pumping down on a vacuum pump for 48 hours the ester (Compound #8) was isolated a~ an orange oil (2.50 g, 1.8 mmole).

Example M: Hydrolysis of 4.5 generation dendrimer and preparation of calcium salt.
4.5 Generation PAMAM (ester terminated, initiated off NH3) (2.11 g, 10.92 meq) was dissolved in 25 ml of methanol and to it was added 10~ NaQH (4.37 ml, 10.92 meq) (pH - 11.5-12). After 24 hours at room temperature, the pH was about 9.5. After an additional 35,444-F"A'i -60-~31~

20 hours, the solution was rotovaped (rotary evaporated), 50 ml of toluene added, and rotovaped again.
The resulting oil was dissolved in 25 ml of methanol and precipitated as a white gum upon addition of 75 ml of diethyl ether. The liquid was decanted, and the gum was rotovaped to give a very fine off-white powder which upon further drying gives 2.16 g of product (98% yield). No ester groups were found upon NMR and infrared analysi The sodium salt of 4.5 Generation PAMAM (e~ter terminated, initiated from NH3) was replaced by the calcium ~alt via dialy~iY. The sodium salt (1.03 g) was dissolved in lO0 ml of water and passed through hollow fiber dialysis tubing (cut off = 5000) at 3 ml/minute. The exterior of the tubing was bathed in 5%
CaCl2 solution. This procedure was then repeated.
The resulting solution was again dialyzed, this time against water, then repeated two additional times.
Evaporation provided 0.6 g of wet solid, which was taken up in methanol (not totally soluble) and is dried to give 0.45 g of off-white crystals.
C369H5920141N9lCa24 Calc. - 10.10% Ca++
0 M Wt. - 9526.3 Calc. - C-4432.1, H-601.8, 0-2255.9, N-1274.6, Ca-961.9) Theo: C-46.5, H-6.32, N-13.38, Ca-10.10 Found: C-47.34, H-7.00, N-13.55, Ca~8.83 35,444-F''A'I -61-13~64~

Example N- Preparation of dendrimers with terminal carboxylate groups.
Half-generation starburst polyamidoamines were hydrolyzed to convert their terminal methyl ester groups to carboxylates. This generated spheroidal molecules with negative charges dispersed on the periphery. The dendrimers hydrolyzed ranged from 0.5 generation (three carboxylates) to 6.5 generation (192 carboxylates).
The products could be generated as Na~, K', Cs+
or Rb+ salts.

Example 0: N-t-butoxycarbonyl-4-aminobenzyl malonate ~ 15 dimethylester 4-Aminobenzyl malonate dimethylester (11.62 g, 49 mmol) waq dissolved in 50 ml of t-butanol:water (60:40 with stirring~ Di-t-butoxydicarbonate (19.79 g, 90 mmol) was added and the reaction mixture stirred overnight~ The butanol was removed on the rotary evaporator, resulting in a yellow suspension of the product in water. Extraction into methylene chloride, drying (MgS04) and evaporation gave a yellow oil (21~05 g, contaminated by di~t-butoxydicarbonate).
Recrystallization from 2-propanol:water (75:25) yield pale yellow crystals (11.1 g, 33 mmol, 67%). The structure was confirmed by 13C NMR and purity checked by hplc analysis (spherisorb O~S-1, 0.05M H3P04 pH 3:
CH3CN 55:45). The material was used without further purification.

35,444-F"A" -62-13~

64693-~10 E am~_QP. N-t--butoxycar~onyl--6--~4-amlnoben~yl~-1,4,8,1:L-tetraaza-5,7-dioxoundecane N-t-butoxycarbonyl-4-amlnobenzy:L malonate dlmethylester ~8.82 g 26 mmol), prepared ln Example O, was dissolved in 50 ml of methanol. Thls solutlon was added dropwise l2 hours) to a solu-tion of freshly dlstllled ethylenediamine (188 g 3.13 mole) and 20 ml of me~hanol, under a nltrogen atmosphere. The solutlon was allowed to stlr for 2~ hours. The ethylene dlamlne/methanol solu-tlon was removed on the rotary evaporator. The product was dls-solved in methanol and toluene added. Solvent removal on therotary evaporator gave the crude product as a whlte solld (10.70 g contamlnated with ethylenediamine). The sample was divided lnto two samples for purlfication. Azeotropic removal of ethylene-dlamine wlth toluene, using a soxhlet extractor with sulphonated ion exchange beads in the thimble to trap the ethylenedlamine, resulted ln partial decomposltlon of the product, giving a brown oil. The remaining product was lsolated as a whlte solid from the toluene on cooling (2.3 g approximately 50 percent). Analysis of a 10 percent solutlon ln methanol by gas chromatography ~Column, Tenax 60~80) showed no ethylenedlamine detectable ln the sample (<0.1 percent). The second fractlon was dissolved in methanol to glve a 10 percent solution (by weight) and purified from the ethylenedlamlne by reverse osmosis, using methanol as the solvent.
~The membrane used was a Filmtec FT-30, ln an Amicon TClR thin channel separator, the ethylenedlamine crosslng the membrane.1 The product was lsolated as a whlte solid ~2.7 g), ln which no detectable amounts o~ ethylenediamine could be found by gas chromatography. The 13~ NMR data and HPLC analysis ~Spherisorb ODS-l, -64- 131~4~

0.05M H3POI~ pH 3:CH3CN 55.45) were consistent with the proposed structure. The product was used with no further purification.

Example Q: Preparation of a starbur~t dendrimer of one half generation from N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane (5.0 g 13 mmol~, prepared in Examp ? P, was dissolved in 100 ml of methanol. Methyl acryla e (6.12 g, 68 mmol) was added and the solution stirred at ambient temperatures for 72 hours. The reaction was monitored by HPLC (Spherisorb ODS1, Acetonitrile: o.O4M Ammonium acetate 40:50) to optimize conversion to the desired product. The solution was concentrated to 30 percent solids, and methyl acrylate (3.0 g 32 mmol~ was added. The reaction mixture ~as stirred at ambient temperature~ until no partially alkylated products were detectable by HPLC (24 hours).
Removal of the solvent at 30C by rotary evaporation, and pumping down at 1 mm Hg for 24 hours gave the product as yellow viscous oil, yield 7.81 g. The 13C
NM~ data wa~ consistent with the proposed structure.
The product was used without further purification.

Example R: Preparation of a starburst dendrimer of one full generation from N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane The half generation product (Example Q) (7.70 g, 10.45 mmol) was dis~olved in 75 ml of methanol and added dropwise over 2 hours to a stirred solution of ethylenediamine (400 ml, 7.41 mol) and methanol (50 ml). The reaction mixture wa~ stirred at ambient temperatures for 48 hours. The ethylenediamine and 35,444-F"A" -64 -65- 1 3 1 6 ~ 5 6 methanol were removed by rotary evaporation to give a yellow oil (11.8 g contaminated with ethylenediamine).
The product was dissolved in 90 ml of methanol, and purified from the ethylenediamine by reverse osmosis B rj (Filmtec FT-30 membrane and Amicon TC1R thin channel separator, methanol as solvent). After 48 hours, no ethylenediamine could be detected by gas chromatography (Column, Tenax 60/80). Removal of the solvent on the rotary evaporator, followed by pumping down on a vacuum line f~r 24 hours gave the product as a yellow glassy solid ~6.72 g). Analysis by HPLC, PLRP-S column, acetonitrile:O.015M NaOH, 10-20 percent gradient in 20 min.) and 13C NMR analysis was consistent with the r~ 15 proposed structure.

ExamPle S: Preparation of a starburst polymer of one and one half generation from N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7 dioxoundecane The one generation product (Example R) (2.14 g, 25 mmol) wa~ dissolved in 12.5 ml of methanol, and methyl acrylate (3.5 g, 39 mmol) in 5 ml of methanol was added. The solution was stirred at ambient temperatures for 48 hours, monitoring the progress of the reaction by HP~C (Spherisorb ODS-1, acetonitrile:
0.04M ammonium acetate, 60:40). A second aliquot of methyl acrylate was added (3.5 g 39 mmol) and the reaction mixture stirred at ambient temperature~ for a further 72 hours. Remo~al of the solvent on the rotary evaporator gave the product as a yellow oil (3.9 g) after pumping down overnight with a vacuum pump. The product was used with no further purification.
~
r/~de~ r(~

35,444-F"A" -65- _ -66- 13164~

Example T: Preparation of a starburst polymer of two full generations from N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza 5,7-dioxoundecane The one and one half generation product (Example S) (3.9 gJ 2.5 mmol) was dissolved in 50 ml of methanol, and was added dropwise over 2 hours to a stirred solution of ethylenediamine (600 g, lO mol) and methanol (50 ml). The solution was stirred at ambient temperatures under an atmosphere of nitrogen for 96 hours. The ethylenediamine/methanol was removed on the rotary evaporator to give a yellow glassy solid (4.4 g contaminated with ethylenediamine). A 10 percent solution of the product wa~ made in methanol, and purified from the ethylenediamine by reverse osmosis (membrane used as a Filmtec ~T-30, in an Amicon TClR
thin channel separator) until no ethylenediamine could be detected by gas chromatography (Column, Tenax 60/80.
Removal of the solvent gave the product as a yellow glassy solid (3.52 g). The 13C NMR data and HPLC
analysis (PLRP-S column, acetonitrile:O.015 M NaOH, 10 to 20 percent gradient in 20 minutes, were consistent with the proposed structure.

Example U: Reaction of the two generation starburst with Bromoacetic Acid to give a methylene carboxylate terminated starburst dendrimer The second generation product (Example T) (0.22 g, 0.13 mmol) was dissolved in 15 ml of deionized water 3 and the temperature equilibrated at 40.5C. Bromoacetic acid (0.48 g, 3.5 mmol) and lithium hydroxide (0.13 g, 3.3 mmol) were dissolved in 5 ml of deionized water, and added to the reaction mixture. The reaction pH was carefully maintained at 9 7 Wi th the use of a pH stat (titrating with 0.1N NaOH~, at 40.5C overnight.

35,444-F"A" -66- _ -67- 13164~6 Monitoring by reverse phase HPLC, (Spherisorb ODS-1 column, eluent 0.25 M H3P04 p~l 3 [NaOH]; acetonitrile ~5:15) confirmed the synthesis of predominantly a single component.

Example V: Preparation of Isothiocyanato functionalized second generation methylene-carboxylate termin3ted starburst dendrimer Five ml of a 2.8 mM solution of the second generation methylenecarboxylate terminated starburst dendrimer (Example U) was diluted with 20 ml water and the pH adjusted to 0.5 with concentrated hydrochloric acid. After one hour at room temperature the mixture was analyzed by HPLC to verify the removal of the butoxycarbonyl group and then treated ~ith 50 percent A sodium hydroxide to ~ ~ the pH to 7. A pH stat (titrating with 0.1 N NaOH) was used to maintain the pH
at 7 and 225 ~l thiophosgene was added. After 15 minutes at room temperature the pH of the mixture was adjusted to 5 with 1N HCl. The mixture washed with chloroform (20 ml x 2) then concentrated on ~ rotary evaporator at reduced pressure. The residue recovered 0.91 g is a mixture of the isothiocyanate and salts.

Example W: Preparation of second generation starburst polyethyleneimine-methane sulfonamide To a solution of 125 g N-methanesulfonyl-aziridine in 50 ml ethanol was added 25.0 g tris(2-amînoethyl)amine. The solution was stirred at room temperature for 4 days. Water was added to the reaction mixture as needed to maintain the homogeneity of the solution. The solvent was removed by distillation in vacuo to give the 2nd generation 35,444-F"A" -67~ _ -68~
1 3 ~
starburst PEI-methane sulfonamide as a yellow glass (161 g)-Example X: Cleavage of methane sulfonamides to form second generation starb~rst polyethyleneimine A solution of 5.0 g of seccnd generationstarburst PEI-methane sulfonamide, from Example W in 20 ml of 38 percent HCL was sealed in a glass ampoule.
The ampoule was heated at 160C for 16 hours, then cooled in an ice bath and opened. The solvent was removed by distillation in vacuo and the residue dissolved in water. After adjusting the pH of the solution to greater than or equal to 10 with 50 percent ~- 15 NaOH, the solvent was removed by distillation ln vacuo.
Toluene (150 ml) was added to the residue and the mixture heated at reflux under a Dean-Stark trap until no more water could be removed. The solution was filtered to remove salts and the filtrate concentrated in vacuo to give 1.9 g second generation starburst PEI
as a yellow oil.

Example Y Preparation of third generation starburst polyethyleneimine-methane sulfonamide To a solution of 10.1 g second generation starburst PEI 9 from Example X, in 100 ml ethanol was added 36.6 g N methanesulfonylaziridine. The solution was stirred at room temperature for 1 week. ~ater was added as needed to maintain the homogeneity of the solution. The solvent was removed by distillation in vacuo to give third generation starburst PEI-methane sulfonamide as a yellow glass (45.3 g).

35,444-F"A" -68- _ -69- 131 64~

Example Z: Cleavage of methane sulfonamides to form 3rd generation st;arburst polyethyleneimine The methane sulfonamide groups of third generation starburst PEI-methane sulfonamide (5.0 g), from Example Y, were removed by the same procedure as described for the ~econd generation material in Example X to give 2.3 g third generation starburst PEI as a yellow oil.
Example AA: Reaction of a third generation starburst polyethyleneimine with 4-fluoro-nitrobenzene The third generation starburst polyethyleneinine (Example Z) (1.06 g, 1 o2 mmol) was dissolved in 12 ml of absolute ethanol. (4-Fluoro)-nitrobenzene (120 ~l, 1.2 mmol~ was added and the reaction mixture refluxed overnight. The solvent was removed on the rotary evaporator~ and the bright yellow oil dissolved in water. The aqueous solution was washed with chloroform to remo~e any unreacted (4-fluoro)-nitrobenzene. Removal of the water gave the product as a deep yellow oil (0.80 g). The 13C NMR
spectrum was consistent with the proposed structure.
(No attempt was made to determine the nature of the statistical distribution). The product was used without further purification.

3 Example B8: Reaction of the nitrophenyl derivative of the third generation starburst pol~ethyleneimine with glycolonitrile.
The nitrophenyl derivative of the third generation starburst polyethyleneimine (Example AA) (0.80 g) was dissolved in 20 ml of deionizea water.

35,444-F"A" -69--70- 1316~6 Sodium hydroxide (2.80 g~ 50 percent w/w) was added to the stirred solution, and the solution purged with nitrogen, venting through a sodium hydroxide scrubber.
Glycolonitrile (2.85 ml of a 70 percent aqueous solution) was added at ambient temperatures. A yellow precipitate was observed to form after a few minutes.
After two hours, the temperature was slowly raised to a reflux, and the solution maintained at a reflux with a nitrogen purge for 24 hours. Removal of the water gave the product as a yellow solid contaminated with glycolic acid and sodium hydroxide. The 13C NMR
spectrum was consistent with the proposed structure.
The product was used without further purification.

Example CC: Hydrogenation of the nitrophenyl derivative to the aminophenyl methylenecarboxylate terminated third generation starburst polyethyleneimine.
The yellow solid from Example BB (1.70 g) was dissolved in lO ml of deionized water, the resulting pH
of the solution was approximately 11. Palladium on charcoal (200 mg of 5 percent Pd/C) was added to the reaction mixture in a glass Parr shaker bottle. The reaction mixture was placed under a pressure of 40 psi (275 kPa) of hydrogen, and shaken at ambient temperature in a Parr hydrogenation apparatus, for 6 hours. The reaction mixture was then filtered through a 0.5 m Millipore filter to remove the Pd/C and the solvent removed in vacuo and was gel filtered through a Biogel P2 resin (25 g swollen with water).
Acidification with HCl resulted in an orange brown solution, which was purged with nitrogen overnight.
Removal of the solvent in vacuo gave the product as the hydrochloride salt which was a pale brown solid (3.98 g~ contaminated with NaCl and glycolic acid, maximum 35,444-F"A" -7--71- 1 3 1 fi~ ~6 theoretical amount of product 1.15g). The product ~as used with no further purificate.

Example DD: Preparation of 4-isothiocyanatophenyl methylenecarboxylate terminated third generation starburst polyethyleneimine The product ~rom Example CC (3.98 g) was dissolved in 15 ml of deionized water and an aliquot (2.5 ml) of this solution was diluted with 10 ml water.
the pH of the solution was adjusted to 7 with sodium hydroxide. A pH stat (titrating with 1N NaOH) was used to maintain the pH and 200 ~l thiophosgene was added.
t- 15 After 10 minutes the pH of the mixture was adjusted to 4 with hydrochloric acid. Water was removed on a rotary evaporator at reduced pressure (a small amount of n-butanol wa~ added to prevent foaming) . The residue was washed with methylene chloride and then dried. The crude product (0.95 g) a mixture of isothiocyanate (0.14 g) and salts was used without further purificationO

Example EE: Preparation of a methylenecarboxylate-terminated second generation starburst polyamidoamine (initiated from ammonia) The second generation starburst polyamidoamine (2.71 g, 2.6 mmol) and bromoacetic acid (4.39 g, 31.6 3 mmol) were dissolved in 30 ml of deionized water and the pH adjusted to 9.7 with 5N NaOH using a pH stat.
The reaction was maintained at this pH for a half hour, and the temperature was slowly raised to 60C and was maintained at 60C for three hours at constant pH. The pH was raised to 10.3, and the reaction mixture 35,444-F"A" ~71--72- 1316~56 remained under control of the pH stat at ambient temperatures overnight. The reaction mixture was refluxed for a further four hours prior to work up.
Removal of the solvent, and azeotroping the final traces of water with methanol gave the product as a pale yellow powder (8.7 g, contaminated with sodium bromide). The 13C NMR spectrum was consistent with the propose structure ~with some contamination due to a small amount of defected material as a result of some monoalkylation).

~xample FF: Preparation of a methylenecarboxylate terminated second generation starburst polyethyleneimine (initiated from ammonia) r l5 The second generation ~tarburst polyethyleneimine ~2.73 g, 6.7 mmol), from Example X, and bromoacetic acid (11.29 g 81 mmol) were dissolved in 30 ml of deionized water. The pH was slowly raised to pH 9.5 maintaining the temperature below 30C. The temperature was raised slowly to 55C, and the reaction pH maintained at 9.5 for 6 hours with the aid of a pH
stat (titrating with 5N NaOH). The pH was raised to 10.2, and maintained at that pH overnight. Removal of the solvent on the rotary evaporator, and azeotroping the fihal traces of water using methanol 7 gave the product as a yellow powder (17.9 g, contaminated with sodium bromide). The 13C NMR spectrum was consistent with the proposed structure (with some contamination due to a small amount of defected material as a result of some monoalkylation).

35,444-F"A" 72--73- 13~6~

Exam~le GG: Preparation of a 3 l/2, 4 1/2, 5 1/2 and 6 1/2 generation starburst PAMAM
To a lO wt%,1solution of 2.46 g 3 generation PAMAM starburst was added 2.32 g of methyl acrylate.
This mixture was allowed to sit at room temeprature f'or 64 hr. After solvent and excess methyl acrylate removal, 4.82 g of product was recovered (105% o~
theore,tical).

Preparation of higher 1/2 generation starburst PAMAM'S:

Generations 4 1/2, 5 1/2 and 6 1/2 were prepared as described above with no significant differences in reactant~concentrations, reactant ratios or reaction times.

Example HH: Preparation of a 4~ 5 and 6 generation starburst PAMAM:
To 2000 g of predistilled ethylenediamine was added 5.4 g of 4 1/2 generation starburst PAMAM as a 15 wt% solution in methanol. This was allowed to sit at room temperature for 48 hrs. The methanol and most of the excess ethylenediamine were removed by rotary evaporation under water aspirator vacuum at temperature less than 60C. The total wt of product recovered was 8.07 g. Gas chromatography indicated that the product still contained 34 wt% ethylenediamine at this point.
A 5.94 g portion of this product was dissolved in lO0 ml methanol and ultrafiltered to remove the residual ethylenediamine. The filtration wa~ run using an Amicon TC1R thin channel recirculating separator equipped with an Amicon YM2 membrane. An in-line pressure relief valve was used to maintain 55 psig (380 kPa) pressure 35,444-F"A" -73-_7l~_ ~3~4~6 across the membrane. The 100 ml was first concentrated to 15 ml by forcing solvent flow exclusively through the membrane. After this initial concentration, the flow was converted to a constant volume retentate recycle mode for 18 hrs. After this time, 60 ml of methanol was passed over the membrane to recover product still in the module and associated tubing. The product was stripped of solvent and 2O53 g of 5 generation starburst PAMAM was recovered. Analysis by gas chromatography indicated 0.3~ residual ethylenediamine remained in the product.

Preparation of generation 4 and 6 proceeded as r 15 above with the only difference being the weight ratio of ethylenediamine to starting material. To prepare 4th generation thiq ratio was 200:1 and for 6th generation this ratio was 730:1.

Example 1: Incorporation of 2-(acetyloxy)benzoic acid (aspirin) into starburst dendrimers.
A widely accepted method for ascertaining whether a "probe molecule" is included in the interior of a micelle is to compare its carbon-13-spin lattice relaxation times (T1) in a non-micellized versus micelliæed medium. A substantial decrease in T1 for the micellized medium is indicative of "probe molecule"
inclusion in the micelle. Since starburst dendrimers are "covalently fixed" analogs of micelles, this T1 relaxation time technique was used to ascertain the degree/extent to which various pharmaceutical type molecules were associated with starburst polyamidoamines~ In the following examples, T1 values for (acetyloxy)benzoic acid (I~ (aspirin) were 35,444-F"A" -74--75~ ~ 3 1 6? 4 5 6 determined in solvent (CDCl3) and then compared to T1 values in CDCl3 at various [I:dendrimer] molar ratios.
Inclusion of aspirin (I) into various starburst polyamidoamine dendrimers as a function of generation.
Various half generation (ester terminated~
initiated from NH3) starburst polyamidoamine dendrimers ~G = 0.5 ~ 5.5) were combined with 2-(acetyloxy)benzoic acid in CDCl3 to give acid:tertiary amine ratios of =
1Ø A plot of T1 values for 2-(acetyloxy)benzoic acid versus generation of starburst dendrimer added (see Figure 4 where represent C-4, n represent C-6, and o represent C-5) show~ that T1 reaches a minimum over the ,- 15 generation range of 2.5 ~ 5.5 for carbons 4, 5 and 6 in 2-(acetyloxy)benzoic acid. This demonstrates association of 2-(acetyloxy)benzoic acid in the dendrimers (G = 2.5 ~ 5.5) and further confirms that polyamidoamine dendrimer~ (Gen = ~.5 or greater) can function as carrier molecules.

Example 2 Release of pseudoephedrine from starburst dendrimer - PA~AM
Pseudoephedrine (o~83 mg/ml) and starburst PAMAM dendrimer [1.0 mg/ml; G= 6.5; terminal group (Z) =
192 (methyl ester)] were dissolved in deionized distilled water and the pH of the donor phase was adjusted to 9.5, with sodium hydroxide solution? and stored at room temperature for about 12 hours.
Solution of pseudoephedrine alone was treated in the same way (control). The drug dendrimer solution was stored at 40C for 8 hours after the first experiment and dynamlc dialysis performed. Dialysis membrane used was a spectra/Por 7, MWC0 1,000 28.6 mm in diameter in 35,444-F"A" -75--76- 131~4~

spectrum separation cells (half cell volume 5 and lO
ml, cell dimensions: 38 mm diameter for both the cells and the cell depth of 10 and 20 mm for 5 and lO ml cells, respectively).

Samples were analyzed by a HPLC procedure developed for pseudoephrine conditions for which are as follows:

Column: uBondapak C-18 Mobile phase: pH 3.2 phosophate buffer plus acetonitrile (80:20) Flow rate : 0.3 ml/min Detection: UV at 210 nm Retention time: 13.3 min The dialysis membrane was washed with deionized water and was kept soaking in the receptor phase for at least 12 hours prior to use. The dialysis membrane was placed in between the donor and the receptor compartment wa~ stirred with a small magnetic spin bar.
Known volumes of donor and receptor solutions were introduced into the respective compartments and transfer of pseudoephedrine to the receptor compartment was followed as a function of time. To maintain sink conditions the entire receptor phase was removed periodically (every 30 minutes) and replaced with fresh receptor phase. The amount of pseudoephedrine was assayed in the sampled receptor phaseO Experiments were conducted at room temperature (22C). The receptor phase was plain deionized distilled water.

35,444-F''AI' -76- _~

1 3 1 6 ~

The results of dynamic analysis are shown in Figure 5. In Figure 5, the ~represents pseudoephedrine only (control), the ~ represents pseudoephedrine plus the dendrimer, and the ~ represents pseudoephedrine plus the dendrimer at 40C, 8 hours before dialysis. It is apparent that in presence of ~ = 6.5 dendrimer in the donor compartment the rate of dialysis of pseudoephedrine is reduced. Storing the donor solution at 40C, appears to further reduce the rate of dialysis.

The experiment was repeated at lower concentrations (the ratio of number of drug molecules to the number of terminal groups was kept the same).
~- 15 G - 6.5 dendrimer 120 ~/ml pseudoephedrine 100 ~iml (122 ~/ml salt).
Dynamic dialysis of pseudoephedrine (alone) at this lower concentration was almost identical to that at higher concentration. Figure 6 summarizes the results of this experiment where represents pseudoephedrine only (control), and o represents pseudoephedrine plus dendrimer.

Example 3 The procedure of Example 2 was repeated using the modifications given below.
Receptor phase: pH 7.4 phosphate buffer Donor phase: pH 7.4 phosphate buffer plus drug and dendrimer in the following ratios:

35,444-F"A" -77-~316~

1. G 6.5 : Drug :: 1 : 192 2. G 5.5 : Drug :: 1 : 96 3. G 4.5 : Drug ~ 48 4. G 6.5H: Drug :: 1 : 192 5. G 5.5H: Drug ~96 6. G 4.5H: Drug :: 1 : 48 The above donor phase compositions plu~
pseudoephedrine alone were subjected to dynamic dialysis. The letter ~H~t after the dendrimer generation number stands for hydrolyzed dendrimer.
Hydrolysis was accomplished by the procedure described in Examples M and N.

The results of these experiments are summarized in Figure 7 where the donor and receptor compartment contained pH 7.4 phosphate buffer. For pseduoephedrine alone (P) the mean curve of three experiments is plotted (shown by the solid line), and one typical run from the other experiments are shown. In Figure 7, the following symbols represent the dendrimer of the indicated generation.

35,444-F"A" -78-:~ 31 ~6 Table III
Symbol Dendrimer Generation 5.5 6.5 ~ 4.5 ~ 5.5H
0 6.5H
~ 4.5H

,- 15 Pseudoephedrine appears not to associate with the dendrimer (unhydrolized) at pH 7.4. Hydrolysis of the terminal functional groups into carboxylate form, has a dramatic effect on the ~ialysis rate (reduction). The generation number appears not to influence the dialysis rate.

Exampl~_4: Interaction studies of salicylic acid with PAMAM starburst dendrimers 2~
This example evaluated interaction characteristic~ of salicyclic acid with PAMAM starburst dendrimer~. These dendrimers consisted of an ammonia initiated core with repeating units derived from N-(2-aminoethyl) acrylamide. Both full (amine terminatedfunctions) and half ~ester terminal groups) generation polymers were included in the studies. The ratio of salicyclic acid to starburst dendrimers utilized in the experiments re~ulted in approximately one salicyclic acid molecule to one terminal amine functional group for full generation polymers. In the half-generation 35,444-F"A" ~79--80- ~316~6 polymer study, the same ratio was employed with adjustments made for the higher molecular weight polymer.
The experiments were conducted at room temperature using an equilibrium static cell dialysis methadology. Spectrum static dialysis cells (half cell volume, 10 ml) separated by SpectraPor 6 membranes (molecular weight cutoff = 1000) were utilized for all experiments. Transport of salicyclic acid was monitored as a function of time by removing aliquots from appropriate cell compartments and assayed by HPLC
analysi~ using a U.V. detector at 296 nm (Bondupak C-18 ~olumn, eluting mobile phase of acetonitrile/0.1M
phosphate buffer (pH 3.2) at a ratio of 20:80 (~/V), set at a flow rate of 30 ml/hour).
Ten ml of a solution containing 1 mg/ml salicyclic acid and 2.5 mg/ml starburst polymer (Gen 4.0) adjusted to pH 6.65 and 5.0 with HCL solution were placed in the donor compartment of the dialysis cell and an equal volume of purified water adjusted to the same pH's placed in the receptor compartment.
Transport of salicyclic acid into the receptor compartment was monitored. The results are given in Figure 8. In Figure 8, the free acid is represented by , the acid plus generation 4.0 dendrimer, pH 6.65 is . represented by 09 and the acid plus generation 4.0 dendrimer, pH 5.00 is represented by O.
Due to the lower percent ionization of the amine groups on the polymer at pH 6, a greater extend of interaction with salicylic may be expected at pH 5, resulting in less compound transported at the lower pH.
The results given in Figure 8 indicate a much lower 35,444-F"A'i -80-~l ~316~6 percentage of salicylic acid transported in the presence of polymer at both pH's studied compared to the salicyclic acid control study. It is also observed that more salicyclic acid is transported at pH 6.65 than at pH 5.0 as predicted. The data demonstrates an interaction of the starburst polymer with salicylic acid that can be controlled by pH~ Sustained release characteristics are also implied by the data since the salicyclic acid levels in the presence of polymer continue to rise past the approximate 12-hour equilibrium point observed in the control study.
To further investigate the interaction characteristics of salicylic acid with starbur~t polymers (Gen = ~.0) an experiment was designed at pH
8Ø The design of the study differed from that previously described in that only the salicylic acid solution (1 mg/ml), adjusted to pH 8.a, was placed in the donor compartment and the polymer solution (2.5 mg/ml) placed in the receptor compartment. Loss of salicylic acid from the donor compartment was monitored as previously described. The results of the experiment are given in Figure 9. In Fig 9, the free acid is represented by --- , and the acid plus generation 4.0 dendrimer at pH 8.0 is represented by --~

As indicated in Figure 9, the equilibrium characteristics of sa~icylic acid in the donorcompartment with starburst polymers in the receptor compartment differs from the salicylic acid control study. Based on the ionization characteristics of the molecules at pH 8, approximately 6-7~ interaction is expected. The observed extent of interaction is indicated to be on the order of 4-5%. The lower 35,444-F"A" 81--82- 13164~

association observed may be due to experimental variability or to an ionization constant of less than one.
This experiment indicates an uptake or removal of free salicylic acid from the contlnuous phase of the system by the polymer. This type of action could result in suppression of reactivity of molecules suggesting a possible chelating type of property associated with the polymers.
The interaction characteristics of salicylic acid at pH 6.65 with a half generation starburst polymer (Gen = 4.5) haYing ester terminated functional groups were evaluated. Salicylic acid (1 mg/ml) was combined with starburst polymer (Gen = 4.5~ 3.6 mg/ml at pH 6.65. Ten ml of the solution was placed in the donor compartment and transport from the donor compartment was monitored as previously described. The results are given in Figure 10. In Figure 10, the free acid is represented by ---, and the acid plus polymer is represented by ---o---.
Under these experimental conditions no charge interaction is predicted to occur since the tertiary amine groups are non~ionized at pH 6.65. As is indicated in Figure 10, the loss of salicylic acid in the presence of polymer (Gen = 4.5) is virtually identical durin~ the first 10 hours of dialysis to that of the salicylic acid control study.
The following observations are made from the data presented in this example:

35,444-F"A" -82--83- 131fi~56 (1) Full generation PAMAM starburst polymers function as a carrier for salicylic acid.
(2) Full generation PAMAM starburst polymers have sustained release functionality for salicylic acid.
(3) Salicylic acid carrier properties of full generation PAMAM starburst polymers can be controlled by pH.
ExamPle 5: Demonstration of multiple chelation of iron by a sodium propionate terminated sixth generation starburst polyamidoamine.
The sodium propionate terminated sixth generation polyamidoamine (initiated from ammonia) (97.1 mg, 2.45 mol.) was dissolved in 1.5 ml of deionized water. Addition of 0.5 ml of 0.5N HCl reduced the pH to 6.3. Ferric chloride was added (0.5 2~ ml oP 0.1.2M solution? 0.051 mmol) producing a light brown gelatinous precipitate. On heating at 60C for 0.5 hours, the gelatinous precipitate became soluble, resulting in a homogeneous orange solutionO The solution was filtered through Biogel P2 acrylamide gel (10 g9 twice) isolating the orange band (free of halide contamination). Removal of the solvent in vacuo gave the product as an orange film (30 mg). Analysis was consistent with chelation of approximately 20 moles of ferric ions per mole of starburst dendrimer.
3o 35,444-F"A" -83--8~- 13~645~

Table IV
Theoretical Found Na4Fe20H128SB Na5Fe2oH127S~ Na6Fe20H126SB
Na 0.39,0.24 0.25 0.31 0.38 (0.31 0.1%) Fe 3.14,3.113.05 3.05 3.04 (3.12 0.02%) C 47.11 49.87 49.84 49.81 H 7.33 7.31 7.30 7.29 N 14.81 14.49 14.48 14.47 o ~ 25.03 25.02 25.01 Mwt.36632.23 36654.21 36375.18 These results confirm chelation of 20+2 moles of ferric ions per mole of starburst dendrimer.
Example 6: Preparation of a product containing more than one rhodium atom per starburst polymer.

2.5 Gen PAMAM ~ester terminated, initiated from NH3) (0.18 g, 0.087 mmole) and RhCl3 3H20 (0.09 g, 0.3 mmole) were mixed in dimethyl~ormamide (DMF) (15 ml) and heated for 4 hours at 70C. The solution turned crimson and mo~t of the rhodium was taken up. The unreacted rhodium was removed by ~iltration and the solvent removed on the rotary evaporator. The oil formed was chloroform soluble. This was washed with water and dried (MgS04) before removal of solvent to yield a red oil (0.18 g). The NMR spectrum was recorded in CD~13 only minor differences were noted between the chelated and unchelated starburst.

35,444-F"A1' -84--85- 13~64~6 Dilution of some of this CDCl3 solution with ethanol followed by NaBH4 addition resulted in rnodium precipitation. RhCl3-3H20 is insoluble in chloroform and in chloroform starburst solution thus confirming chelation.

Example 7: Preparation of a product containing Pd chelated to a starburst polymer 3.5 Gen PAMAM tester terminated, initiated from NH3) (1.1 g, 0.24 mmole) was dissolved with stirring into acetonitrile (50 ml). Palladium chloride (0.24 g, 1.4 mmole) waq added and the solution was heated at 70-75C (water bath) overnight. The PdCl2 was taken up - 15 into the starbur~t~ After removal of the solvent, the NMR in CDCl3 confirmed that chelation had occurred.
Dilution of the CDC13 solution with ethanol and addition of NaBH4 resulted in precipitation of the palladium. The chelated product (1.23 g) was isolated as a brown oil.

Example 8: Demonstration of multiple chelation of yttrium by a methylene carboxylate terminated second generation starburst polyethyleneimine by trans chelation from yttrium acetate The starburst polyethyleneimine methylene carboxylate terminated material (0.4~ g 52. 5 percent active, remainder sodium bromide, 0.18 mmol active starburst dendrimer), from Example FF, was dissolved in 4.5 ml of deuterium oxide. The resultant pH was 11.5-12. A solution of yttrium acetate was prepared by dissolving yttrium chloride (0.15 g, 0.5 mmol) and sodium acetate (0.41 g, 0.5 mmol) in 1.5 ml of deuterium oxide (2.9 moles of yttrium per mole of dendrimer). Aliquots of 0.5 ml of the yttrium acetate 35,444-F''AI' -85--86- 1 31 ~4 56 solution were added to the dendrimer solution and the 3C NMR spectra recorded at 75.5 MHz~

The 13C NMR spectrum of yttrium acetate shows two resonances, ,'84.7 ppm for the carboxyl carbon and 23.7 ppm for the methyl carbon, compared with 182.1 and 24.1 ppm for sodium acetate, and 177.7 and 20.7 ppm for acetic acid (Sadtler 13C NMR Standard Spectra).-Monitoring the positions of these bands indicateqdegree of chelation with the starburst dendrimer. The most informative signal for the starburst dendrimer which is indicative of chelation is the a-CH2 (of the methylene carboxylate group involved in chelation)~
= 15 which appears at 58.4 ppm in the unchelated dendrimer, and 63.8 ppm in the chelated dendrimer. Upon chelation with yttrium, the spin lattice relaxation times of the time a-CH2 shortens as expected from 0.24 + O.Ols to 0.14 + 0.01s, indicative of chelation.

Following the addition of 0.5 ml of the yttrium acetate solution to the starburst dendrimer, all the yttrium appeared to be chelated by the dendrimer, confirmed by the signals for the acetate being that of sodium acetate. The same observation was noted for the addition of a second 0.5 ml aliquot of the yttrium acetate solution. Upon addition of the third aliquot of yttrium acetate, not all of the yttrium was observed 3 to be taken up as the starburst chelate, the acetate carboxyl resonance was observed to shift to 183.8 ppm indicating that some of the yttrium was associated with the acetate. The integrated area of the chelated -CH2 groupq on the dendrimer increased, indicating that some of the third mole equivalent of yttrium added was 35,444-F"A" -86--87- 1 3 ~

indeed chelated with the dendrimer. These results indicate that the dendrimer can chelate from 2-3 yttrium ions per dendrimer molecule.

Example 9: Demonstration of Multiple Chelation of Yttrium by a methylene carboxylate terminated second generation starburst polyamidoamine by trans chelation from yttrium acetate.
The same experimental methods were used for this study as were used for Example 8. The starburst polyamidoamine methylene-carboxylate terminated material (0.40g 62.5% active, remainder sodium bromide, 0.12 mmol.) was dissolved in 4-5 ml of deuterium oxide.
The resultant pH was 11.5-1~, which was lowered to 9.4 with 6N HCl prior to the experiment. A solution of yttrium acetate was prepared by di~solving yttrium chloride (0.1125g, .37 mmol.) and sodium acetate (0.0915g, 1.1 mmol.) in 1.5 ml of deuterium oxide, thus every 0.5 ml of solution contains one mole equivalent of metal.
The first two mole equivalents of yttrium acetate added were fully chelated by the starburst polyamidoamine. On addition of a third mole equivalent of yttrium, precipitation of the product occurred and as such no NMR data could be obtained. The signals which gave the most information about chelation by the starburst dendrimer were those of the two carbons adjacent to the chelating nitrogen. The chemical shifts of these carbons in the unchelated dendrimer occurred at 59.1 ppm for the a-CH2, and 53.7 ppm for the first methylene carbon of the backbone. Upon chelation these two resonances were observed to shift downfield to 60.8 and 55.1 ppm respectively. The trans chelatiGn shows that two metal ions can be readily 35,444-F"A" -87--~8- 1316~6 chelated per dendrimer molecule, however upon chelation oY some unknown fraction of a third mole equivalent, the product precipitates out of solution.

Example 10: Demonstration of Multiple Chelation of 90Y
by a methylenecarboxylate terminated second generation starburst polyethyleneimine.
Standard solution of yttrium chloride (3x10-2 M, spiked with non-carrier added 90Y) and methylenecarboxylate terminated second generation starburst polyethyleneimine (6x10-2 M) were prepared.
These were reacted together at variou~ metal:starburst ratios in HEPES buf~er. The complex yield was determined by ion exchange chromatography using Sephadex G50 ion exchange beads, eluting with 10%
NaCl:NH40H, 4:1 at pH lO. Noncomplexed metal is removed on the column, complexed metal elutes. Yields were obtained by comparing the radioactivity eluted with that on the column, u~ing a well counter.

35,444-F"A" -88-_~9_ ~3~6~

Table V
Chelation of 2.5 Gen. PEI Acetate wlth 9Y

VO1. Y+3 VQ1. PEI Vol HEPES M: L Theor . 3 Complex M: L Act.
S ~0 370 0 . 1 110 0 . 1 360 0 . ~ 101 0 . 2 350 0 ~ 4 95 0. 4 340 0 . 5 97 0. 5 1 C 30 30 340 0 . 5 102 0 . ~
310 1 . 0 99 1 . 0 120 30 250 2 . 0 100 2 . 0 180 30 180 3 . O g4 2. 8 , 1 ~ 250 ~0 120 ~ . 1 80 3 . 3 300 20 80 7 . 5 4~ 3. 3 300 20 70 5 . 0 40 2 . 0 300 20 70 5 . 0 41 2. 0 2C All volumes in Table V are in microlitres Within the accuracy of the experiments, these results indicate that the 2.5 Gen. starburst PEI acetate can chelate between 2 and 3 metals per polymer giving a soluble complex.
Example 11: Conjugation of 4-isothiocyanatophenyl methylenecarboxylate terminated third generation starburst polyethyleneimine with IgG monoclonal antibody The isothiocyanate, 10 mg (50 ~ moles), from Example DD was dissolved in 500 ~l of 3 mM indium chloride which had been spiked with radioactive indium-111 chloride and the pH was adjusted to 9 with 660 ~l N
f~ 35 NaOH. Aliquots of whole1antibody IgG CC-46 was then mixed with aliquots of the chelated starburstO The 35,444-F"A" -89-go ~31~4~

mixtures were shaken then left for 18 hours. The~
",t mixtures were then anal~ ed by HPLC ~column ~rt Zorbax Biosphere GF-250; eluent 0.25 M ~odium acetate, B pH 6) and a UV detector at 254 nm and a radioactivty detector. Results are shown in Table VI.

Table VI
Starburst-IgG Conjugates 1 2 ~ 4 IgG solution (~l) 20 20 20 20 Chelated Starburst 5 20 50 100 solution (~l) ~ Radioactivity 6 5 5 3 ,- 15 on IgG
% IgC conjugated 2 7 17 22 Example 12: Coniugation of 4-isothiocyanatophenyl methylenecarboxylate terminated third generation starburst polyethyleneimine with Ig6 monoclonal antibody The isothiocyanate from Example DD, 4 mg (20 mole~) was mixed with 200 ~l of 3 mM indium chloride (60 ~ moles). A 20 ~l aliquot of the solution was then spiked with radioactive indium-111 chloride and the pH
adjusted to 9 by the addition of 30 ~l 1 N NaOH and 10 ~1 of 0.1 HCL. The indium chelate was mixed with 150 ~l of CC-49 whole antibody IgG, 10 mg/ml in 50 mM HEPES
bufffer at pH 9.5. After 18 hours at room temperature the antibody was recovered by preparative HPLC (column Dupont Zorbax Biosphere GF 250; eluent 0.25 m sodium acetate, pH 6); and a UV detector at 254 nm and a radioactivity detector. The recovered antibody was concentrated on an Amicon membrane and exchanged into ~r~ r ~

35~444-F"A" -90-~ 316~
6459~ g PBS buffer ~phosphate buf~ered saline pH 7.4 which contains 0.12 M
NaC~, ~.7 mM KCl and 10.0 mM p]losphate). ~le recovered antlbody had speclfic activity of approximately 0.5 ~cl~lOO~g.

Examole 13: In Vivo localization oE 111In labeled star~urst antibody con~ugate.
The usefulness of the labeled starburst antibody con~u-gate prepared in Example 12 was demonstrated by measurlng the up-take of the material by a hurnan tumor xenograft 1n an athymic mouse. Female anthymlc mice were innoculated su~cutaneously with the human colon carcinoma cell line, LS-174T (approxima~ely 4 x 106 cells~animal). Approximately two weeks after innoculation, each animal was ln~ected via the tail vein. The mice were sacri ficed after 17 and 48 hours (five animals at each time point), the tumor and selected tlssues were excised and weighe~, and radio-activity was measured in a gamma counter. After 17 hours 13.5 percent of the lniected dose per gram of tissue had localized at the tumor. After 48 hours 21.6% of the in~ected dose per gram of tissue had localized at the tumor.

~,

Claims (36)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A dense star polymer conjugate which comprises at least one starburst polymer associated with at least one unit of at least one carried pharmaceutical material.

2. The conjugate of Claim 1 wherein the dense star polymer is a dense star polymer dendrimer.

3. The conjugate of Claim 1 or 2 wherein at least one of the carried pharmaceutical materials is a drug, radionuclide, chelator, chelated metal, toxin, antibody, antibody fragment, antigen, signal generator, signal reflector, or signal absorber.

4. The conjugate of Claim 2 wherein there are at least two different carried materials at least one of which is a target director and at least one of which is a bioactive ingredient.

5. The conjugate of Claim 4 wherein the target director is an entity specific for one or more target receptors and the bioactive agent is a radionuclide, drug, or toxin.

6. The conjugate of Claim 4 or 5 wherein the target director is a polyclonal antibody or fragment thereof.

7. The conjugate of Claim 4 or 5 wherein the target director is a monoclonal antibody or fragment thereof.

*Trademark

8. The conjugate of Claim 1 wherein the dendrimer contains discontinuities.

9. A dense star polymer conjugate of Claim 1 of the formula:
(P)x * (M)y (I) wherein each P represents a dendrimer;
x represents an integer of 1 or greater;
each M represents a unit of a carried pharmaceutical material, said carried pharmaceutical material can be the same carried pharmaceutical material or a different carried pharmaceutical material;
y represents an integer of 1 or greater; and * indicates that the carried pharmaceutical material is associated with the dendrimer.

10. The conjugate of Claim 9 wherein M is a drug, pesticide, radionuclide, chelator, chelated metal, toxin, antibody, antibody fragment, antigen, signal generator, signal reflector, or signal absorber.

11. The conjugate of Claim 9 wherein x=1 and y=2 or more.

12. The dense star polymer conjugate of Claim 9 wherein the molar ratio of any ionic M to P is 0.1-1,000:1.

13. The dense star polymer conjugate of Claim 9 wherein the weight ratio of any drug or toxin M to P is 0.1-5:1.

14. A process for preparing (P)x * (M)y (I) wherein each P represents a dendrimer; x represents an integer of 1 or greater; each M represents a unit of a carried pharmaceutical material, said carried pharmaceutical material can be the same carried pharmaceutical material or a different carried pharmaceutical material; y represents an integer of 1 or greater;
and * indicates that the carried pharmaceutical material is associated with the dendrimer, which comprises reacting P with M, at a temperature which facilitates the association of the carried material (H) with dendrimer (P).

15. The process of Claim 14 wherein the temperature is from room temperature to reflux.

16. The process of Claim 14 wherein P is reacted with H in a solvent which is water, methanol, ethanol, chloroform, acetonitrile, toluene, dimethylsulfoxide or dimethylformamide.

17. A conjugate of any one of Claims 1, 2, 4, 5 and 8 to 13 which also has at least one pharmaceutically acceptable diluent or carrier present.

18. The conjugate composition of Claim 17 which also has other active ingredients present.

19. A conjugate of any one of Claims 1, 2, 4, 5, 8 to 13 and 18 for use as a diagnostic agent.

20. A use for the delivery of a pharmaceutical material of at least one dense star polymer conjugate of any of Claims 1, 2, 4, 5, 8 to 13 and 18 containing said material.

21. A use for scavenging therapeutic or diagnostic compounds of a bifunctional dense star polymer conjugate according to Claim 4 or 5 wherein said target director is suitable for localizing the conjugate to a target locus and said bioactive ingredient is a scavenging moiety which can bind a secondarily administered therapeutic or diagnostic compound.

22. A use of Claim 21 wherein the scavenging moiety is a chelant, antigen, or antibody.

23. A process for preparing a dense star polymer conjugate of the formula [(T)e - (C')f]g * (P)x * [(C")h - (M)y]k (III) wherein each C' represents the same or different connecting group;
each C" represents the same or different connecting group;
g, and k each individually represent an integer of 1 or greater;
f and h each individually represent an integer of 0 or greater;
- indicates a covalent bond in instances where a connecting group is present;
each P represents a dendrimer;
x represents an integer of 1 or greater;
T represents a target director;
each M represents a unit example of a carried pharmaceutical material, said carried pharmaceutical material can be the same carried pharmaceutical material or a different carried material;
y represents an integer or 1 or greater; and * indicates that the carried pharmaceutical material is associated with the dendrimer;
which comprises the reaction of P, having reactive moieties, with a pharmaceutical material M and a target director T having, where required, connecting groups C' and C".

24. A process for preparing a dense star polymer conjugate as defined in Claim 1 which comprises the reaction of a starburst polymer P, having reactive moieties, with a connecting group with at least one pharmaceutical material.

25. The process of Claim 24 wherein the connecting group has the formula wherein R is -C(CH3)3. .

26. The process of Claim 24 wherein the P also has a connecting group (handle) attached of the formula where n is 1 or 2, and X is F, Cl, Br, I, SO2Cl, and when n is 1, the NO2 group is in the para position.

27. The process of Claim 26 wherein the connecting group is 4-fluoronitrobenzene.

28. The conjugate of Claim 2 wherein the dense star polymer dendrimer is of the formula (Core) wherein: the core is # of terminal groups per dendritic branch =
G is the number of generations; Nr is the repeating unit multiplicity which is at least 2; Nc is the valency of the core compound; the terminal moiety is determined by the following:
# of terminal moieties per dendrimer =
wherein Nr, G and N are as defined above; and the Repeat Unit has a valency or functionality of Nr + 1 wherein Nr is as defined above.

29. The conjugate of Claim 2 wherein the dense star pulymer dendrimer is of the formula wherein i is 1 to t-1; the core compound is represented by the formula ?(Zc)Nc where ?
represents the core, zc represents the functional groups bonded to ?

and Nc represents the core valency; the repeat unit is represented by the formula XiYi(Zi)Ni wherein "i" is defined as above; the final or terminal units are represented by XtYt(Zt)Nt wherein t represents terminal generation and Xt, Yt, Zt and Nt may be the same as or different from Xi, Yi, Zi and Ni except that there is no succeeding generation connected to the Zt groups and Nt may be less than two; the n function is the product of all the values between its defined limits, such as i - 1 .pi. Nn = (N1)(N2)(N3).. (N1-2)(Ni-1) n = 1 which is the number of repeat units, XiYi(Zi)Ni, comprising the ith generation of one dendritic branch and when i is 1, then .pi.° = 1 n=1

30. A process for preparing A dense star polymer conjugate as defined in Claim 1 which comprises the reaction of a starburst polymer P, having reactive moieties, with an aniline moiety, which may have the NH2 group protected by an N-phthalimide of the formula

31. A process for preparing a dense star polymer conjugate as defined in Claim 1 which comprises the reaction of a starburst polymer P, having reactive moieties including an NH2 group, with a pharmaceutical material, wherein the NH2 group is protected by any protecting group used for amines which is inert under the conditions used for dense star polymer synthesis.

32. A process for preparing a dense star polymer conjugate of Claim 1 wherein the dense star polymer is a dense star polymer dendrimer and the carried material is a lanthanide which comprises chelation of the lanthanide with a dense star polymer polyethyleneimine acetate.

33. A process according to Claim 23 wherein the carried pharmaceutical material is a bioactive agent.

34. A process according to Claim 23 or 24 wherein the connecting group is an aniline moiety.

35. A process according to claim 34 wherein the NH2 group of the aniline moiety is protected.

36. A process according to claim 14 wherein P is reacted with M in a solvent.