WO2007149500A2

WO2007149500A2 - Formulations containing hybrid dendrimers

Info

Publication number: WO2007149500A2
Application number: PCT/US2007/014402
Authority: WO
Inventors: Abhay Singh Chauhan; Sonke Svenson
Original assignee: Dendritic Nanotechnologies, Inc.
Priority date: 2006-06-21
Filing date: 2007-06-20
Publication date: 2007-12-27
Also published as: WO2007149500A9; WO2007149500A3

Abstract

The present invention concerns hybrid dendrimers comprising a mixture, at the same or different ratios, of at least two dendritic polymers or dendronized polymers that have at least one difference between them. The surfaces of the dendritic polymer or dendronized polymer may be further modified by known methods. Also included are formulations of hybrid dendrimers wherein the dendritic polymers have the same drug present at either identical or different loading concentrations but have different release profiles. Such formulations of hybrid dendrimers may have different guest molecules present at either identical or different loading concentrations but have different release profiles. Additionally a method of using a hybrid dendrimer for delivery of a drug or guest moiety in order to provide increased solubility to poorly soluble drugs or guest moieties is provided.

Description

FORMULATIONS CONTAINING HYBRID DENDRIMERS

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to an improved formulation for release of drugs or prodrugs using mixtures of dendrimers or dendronized polymers.

Description of Related Art

Generally, single drug delivery systems cannot achieve a complex release profile because of their inbuilt limitations and require extended formulation studies to ensure reproducibility of such drug release. For example, individual delivery systems can either show burst release or sustained release; however, achieving initial burst followed by sustained release from the same system is a complex task.

Using a combination of two or more delivery systems can cause problems due to interactions between the systems used. For example, liposomes are metastable and can fuse together, combinations of polymers or copolymers can interact and flocculate, complicating the characterization of the respective delivery system. Interactions between combined delivery systems may result in destabilization of one or more of the systems. Thus it becomes difficult to determine the actual release of the drug or prodrug obtained by that system. Hence, there is a need for a rugged, reliable, reproducible and stable delivery system which can be tailored according to the specific therapeutic requirements.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an improved formulation for drug delivery by using dendritic polymers and/or dendronized polymers to achieve a more rugged, reliable, reproducible and stable delivery system which can be tailored according to the specific therapeutic requirements.

The present invention concerns hybrid dendrimers comprising a mixture, at the same or different ratios, of at least two dendritic polymers or dendronized polymers that have at least one difference between them. The surfaces of the dendritic polymer or dendronized polymer may be further modified by known methods. Also included are formulations of hybrid dendrimers wherein the dendritic polymers have the same drug present at either identical or different loading concentrations but have different release profiles. Such formulations of hybrid dendrimers may have different guest molecules present at either identical or different loading concentrations but have different release profiles.

Additionally, a method of using a hybrid dendrimer for delivery of a drug or guest moiety in order to provide increased solubility to poorly soluble drugs or guest moieties is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the loading efficiency of indomethacin of the individual G4-NH2 and G3.5-COONa dendrimers and their resulting hybrid dendrimer (n=2).

Figure 2 shows the loading efficiency of indomethacin of the individual G4-NH2 and G4-PEG dendrimers and their resulting hybrid dendrimer (n=2). Figure 3 shows the loading efficiency of indomethacin of the individual G4-NH2 and G4-PYR dendrimers and their resulting hybrid dendrimer (n=2).

Figure 4 shows the loading efficiency of indomethacin of the individual G4-NH2 and G4-TRIS dendrimers and their resulting hybrid dendrimer (n=2).

Figure 5 shows the loading efficiency of indomethacin of the individual G4-NH2 and G4-AE dendrimers and their resulting hybrid dendrimer (n=2).

Figure 6 shows the loading efficiency of indomethacin of the individual G4-NH2 and G4-AEEA dendrimers and their resulting hybrid dendrimer (n=2).

Figure 7 shows SEC profiles (Intensity in [mVolt] over Time in [min]) of G4-NH2 (I), G4-PYR (2) and the resulting G4-NH2+G4-PYR hybrid dendrimer. Figure 8 shows SEC profiles (Intensity in [mVolt] over Time in [min]) of G4-NH2

(1), G4-PEG (2) and the resulting G4-NH2+G4-PEG hybrid dendrimer.

Figure 9 shows the in vitro release profile of the G4-NH2+G3.5-COONa hybrid dendrimer (n=2).

Figure 10 shows the in vitro release profile of the G4-NH2-K34-TRIS hybrid dendrimer (n=2).

Figure 1 1 shows the in vitro release profile of the G4-NH2+G4-AE hybrid dendrimer (n=2). Figure 12 shows the in vitro release profile of the G4-NH2+G4-PYR hybrid dendrimer (n=2).

Figure 13 shows the in vitro release profile of the G4-NH2+G4-PEG hybrid dendrimer (n=2). Figure 14 shows the in vitro release profile of the G4-NH2+G4-AEEA hybrid dendrimer (n=2).

Figure 15 shows the in vitro release profile of G4-NH2+G4-TRIS hybrid dendrimer, where: Route 1 = indomethacin loading after mixing of the dendrimers (solid circles); and Route 2 = indomethacin loading prior to mixing of the dendrimers (solid squares) (n=2). Figure 16 shows the in vitro release profile of G4-NH2+G4-AE hybrid dendrimer, where: Route 1 = indomethacin loading after mixing of the dendrimers (solid triangles); and Route 2 = indomethacin loading prior to mixing of the dendrimers (solid squares) (n=2).

Figure 17 shows the in vitro release profile of G4-NH2+G4-PYR hybrid dendrimer where: Route 1 = indomethacin loading after mixing of the dendrimers (solid squares); and Route 2 = indomethacin loading prior to mixing of the dendrimers (solid diamonds) (n=2).

Figure 18 shows the loading efficiency of indomethacin of the individual PAMAM G4-NH2 and PEHAM G2.5-COOH dendrimers and their resulting hybrid dendrimers.

Figure 19 shows the in vitro release profile Of(PAMAM G4-NH2)-(PEHAM G2.5- COOH) hybrid dendrimers. Figure 20 shows the effect of mixing ratio on the in vitro release profiles of the

(PAMAM G4-NH2)-(PEHAM G2.5-COOH) hybrid dendrimers.

Figure 21 shows the in vitro release profile Of(PAMAM G4-NH2)-(PEHAM Gl .5- NH) hybrid dendrimers prepared through Route 2, i.e., indomethacin loading prior to mixing of the constituent dendrimers. Figure 22 shows simultaneous release of two drugs, indomethacin and camptothecin, from (PAMAM G4-NH2)-(PEHAM G2.5-COOH) hybrid dendrimers.

Figure 23 shows the in vitro release profile of indomethacin from (PPI G4-NH2)- (PEHAM Gl .5-NH) hybrid dendrimers prepared through Route 2, Le., indomethacin loading prior to mixing of the constituent dendrimers. Figure 24 illustrates a schematic presentation of the hybrid dendrimer formulations of this invention. The constituent dendritic polymers (A) and (B) are composed of dendrimers such as PAMAM, PEHAM, PPI or PEI, or dendronized polymers (C). These dendrimers have at least one difference between them (such as composition, size (generation), surface functionality, or loading) with either the same active at different concentrations or two actives at the same or different concentrations. The interactions between dendritic polymers A, B, and C are either repulsive (1. and 4.) based for example on electrostatic repulsion between dendrimers of same charge as shown in Example I for G4-NH2 and G4-AE, or they are attractive (2. and 3.) based for example on electrostatic attraction between dendrimers of opposite charges as shown in Example 1 for G4-NH2 and G3.5-COONa, or based on hydrogen bonding between dendrimers as shown in Example 1 for G4-NH2 and G4-TRIS.

DETAILED DESCRIPTION OF THE INVENTION Glossary

The following terms as used in this application are to be defined as stated below and for these terms, the singular includes the plural. AE means ethanolamine

AEEA means amidoethylethanolamine BR means branch cell reagent (C) or C means core cm means centimeter(s) Dendritic polymer means all dendritic architectures, including but not limited to PAMAM and PEHAM dendrimers, and dendronized polymers

Dendronized polymers means where the (C) moiety has dendons on its surface, where such cores include, but are not limited to, linear polymers, latex particles, cage molecules such as macrocycles, cyclodextrins, and others, and the dendron can be a portion of a dendritic polymer

DI water means deionized water

DOD means 1,12-diaminododecane

Drug means therapeutic agents, including prodrugs, and diagnostic agents, and combinations of drugs, prodrugs and diagnostic agents EDA means ethylenediamine; Aldrich

G means dendrimer generation, which is indicated by the number of concentric branch cell shells surrounding the core (usually counted sequentially from the core) g means gram(s)

G4-NH2 means EDA core, generation 4, amine surface PAMAM dendrimer G4-SUC means EDA core, generation 4, succinamic surface PAMAM dendrimer G4-PYR means EDA core, generation 4, pyrrol idone surface PAMAM dendrimer G4-PEG means EDA core, generation 4, polyethylene glycol surface PAMAM dendrimer G4-TR1S means EDA core, generation 4, TRIS surface PAMAM dendrimer G4-AE means EDA core, generation 4, ethanolamine surface PAMAM dendrimer G3. S-COONa means EDA core, generation 3.5, sodium carboxylate surface PAMAM dendrimer G4-AEEA means DOD core, generation 4, AEEA surface PAMAM dendrimer Guest molecule or guest means a drug, bioactive agents, metal ions, metals, signal generators, immuno-potentiating agents, agricultural materials, and/or pharmaceutical materials and materials that image and/or trigger a biological response h means hour(s)

HCl means hydrochloric acid

HPLC means high pressure liquid chromatography

Hybrid dendrimers mean dendritic polymers and/or dendronized polymers, where at least 2 such polymers are present or there are 2 similar polymers used that have at least one difference between them such as surface groups or G or BR

IF means interior functionality L means Iiter(s) Loading means: physical encapsulation into the interior of a dendritic polymer; absorption onto the surface of a dendritic polymer or dendronized polymer; or chemical attachment of the drug to the dendritic polymer (surface and/or interior) or dendronized polymer, with or without a linker MeOH means methanol mg means miltigram(s) min means minute(s) mL means milliliter(s)

MWCO means molecular weight cut off nm means nanometers)

-S- N-SIS means nanoscale sterically induced stoichiometry

PAMAM means poly(amidoamine) dendrimers, including linear and branched polymers or dendrimers with primary amine terminal groups or other surface groups, or dendrons

PEHAM means poly(etherhydroxylamine) dendrimer PEHAM G2.5-COOH means pentaerythritol tetraglycidyl ether core, generation 2.5, carboxylate surface PEHAM dendrimer

PEHAM Gl .5-NH means pentaerythritol tetraglycidyl ether core, generation 1.5, piperazine surface PEHAM dendrimer

PEl means poly(ethyleneimine) Percent or % means by weight unless stated otherwise

PPI means poly(propyleneimine) dendrimer

PPI G4-NH2 means 1,4-diaminobutane core, generation 4, amine surface PPI dendrimer

% w/v means Percent weight by volume

RT means room temperature or ambient temperature, about 20-25⁰C SEC means size exclusion chromatography

TRIS means /ri5(hydroxymethyl)aminomethane

UV means ultraviolet spectroscopy μ means micron(s) urn means micrometers) VIS means visible spectroscopy

Chemical structures of Dendritic Polymers

The dendritic polymer structures of the present invention may be any dendritic polymer, including without limitation, PAMAM dendrimers, PEHAM dendrimers, PEI dendrimers, POPAM dendrimers, PPI dendrimers, polyether dendrimers, dendrigrafts, random hyperbranched dendrimers, polylysine dendritic polymers, arborols, cascade polymers, avidimers or other dendritic architectures. There are numerous examples of such dendritic polymers in the literature, such as those described in Dendrimers and other Dendritic Polymers, eds. J.M.J. Frέchet, D. A. Tomalia, pub. John Wiley and Sons, (2001) and other such sources.

These dendritic polymers can be any physical shape, such as for example spheres, rods, tubes, or any other shape possible. The interior structure may have an internal cleavable bond (such as a disulfide) or an internal functionality such as a hydroxide or other group to associate with it. Additionally, the dendritic polymer can be a dendron. This dendron can have any dendritic polymer constituents desired.

General Syntheses Used to Prepare Dendritic Polymers

Most of these dendritic polymers have been taught in the literature. See Dendrimers and other Dendritic Polymers, eds. J.M.J. Frέchet, D. A. Tomalia, pub. John Wiley and Sons, (2001) where most of these structures are discussed, including but not limited to PPI, PAMAM and PEI dendrimers. The PEHAM dendritic polymers have been taught in WO2006/065266 and WO2006/1 15547.

Drug Delivery Systems

Dendritic polymers are fast evolving as dependable drug delivery systems. For example, PAMAM dendrimers are robust and flexible unimolecular entities with multivalent surfaces, providing advantages compared to other existing drug delivery systems. PAMAM dendrimers can be tailored with respect to their core and surface groups. Loading and release of drugs from various PAMAM dendrimers is well documented.

Each dendrimer has a characteristic release profile that is specific to a drug or group of drugs. Two or more dendrimers can be combined to work in tandem in a formulation, thereby achieving a therapeutically desired release profile.

In the present invention the hybrid dendrimers offer a platform to resolve this issue of complex release profiles. The use of different dendrimers in a single formulation can give the desired release profile for a given drug. Dendrimers are stable and will not fuse; and combination of such dendrimers can be characterized as reported for core-shell tectodendrimers. To investigate this potential various combinations of dendrimers were prepared and studied for their loading and release profiles.

The loading and release profiles of hybrid dendrimers indicate that different dendrimers have the ability to work in tandem in a formulation. Numerous possible permutations and combinations of dendrimers will lead to a formulation of a given drug that shows the desired drug delivery profile. The dendrimer platform offers several options:

1. Two or more different dendrimers loaded with the same drug but having different release profiles; 2. Two or more different dendrimers loaded with the same drug but where the drug load is either the same or differs per dendrimer;

3. Two or more identical dendrimers loaded with the same drug at different concentrations; or

S 4. Two or more different dendrimers loaded with different drugs (drug cocktails) where the drug loads are either the same or differ per dendrimer.

The dendrimers that may be used for these formulations are hybrid dendrimers that may be selected from a mixture of dendritic polymers including but not limited to PEHAM 0 dendrimers, PAMAM dendrimers, PPI dendrimers, polylysine, core-shell tectodendrimers, and others, or dendronized polymers (i.e., where the core moiety has dendons on its surface, where such cores include, but are not limited to, linear polymers, latex particles, cage molecules such as macrocycles, cyclodextrins, and others,). The hybrid dendrimers are loaded with the same drug or different drugs as discussed in 1-4 above. A desired drug S release profile can be tailored by the composition of the mixture of dendritic polymers, i.e., the superposition of individual release profiles of each component of the mixture results in the tailored total release profile. Thus, rather than trying to find a new formulation for each drug and each application, a stock of dendritic polymers with known release profiles can be used to prepare a hybrid dendrimer formulation showing the desired drug release profile. 0 This mixture of hybrid dendrimers for the desired formulation can be different dendrimers or dendronized polymers where the dendrimer is: 'a) the same basic dendrimer, such as PAMAM or PEHAM, but of different generations or having different surface groups or different branches, or b) different dendrimers, such as PAMAM and PEHAM used together, or c) mixtures of dendronized polymers, or d) mixtures of dendrimers and dendronized 5 polymers. These hybrid mixtures may be admixtures in a specified ratio to obtain the desired release profile, or may have hydrogen bonding or electrostatic attraction to each other.

The present invention provides compositions comprising of two or more dendritic polymers. With slight variation in core, generation and surface functionality, different 0 dendritic compositions can be produced with characteristic release profiles. Each dendritic polymer in these compositions acts as an individual delivery system or, in case of interactions between the dendritic components via hydrogen bonds or electrostatic attraction, these dendritic polymers can work together. Loading of drug molecules into dendritic polymers can be achieved following two routes:

1. Individual dendrimers are mixed to form a composition followed by loading with the drug; or

S 2. Individual dendritic polymers are first loaded with drug material and then the loaded dendritic polymers are mixed to form a composition.

In one embodiment of the present invention, a library having different dendritic polymers, with or without preloaded drug, is created and instant formulations are prepared 0 by using either route described above. These mixed compositions of hybrid dendrimers are used in the following general applications:

1. Dendritic polymers composed of a mixture of dendrimers of same kind (i.e., mixture of PEHAM dendrimers with different surfaces or different generations or different internal composition such as C, BR and EF, or mixture of PAMAM dendrimers with S different surfaces or different generations) or different kinds {e.g., mixture of PEHAM dendrimer + PAMAM dendrimer, or mixture of PEHAM dendrimer + PPI dendrimer).

2. Dendritic polymers composed of a mixture of dendronized polymers of same kind (i.e., mixture of linear polymers or mixture of dendronized cores) or different kinds (mixture of linear polymers + dendronized latexes, or mixture of dendronized latexes + 0 dendronized cage molecules).

3. Mixtures of dendritic polymers composed of components described under #1 and Wl above.

4. Mixtures composed of two or more dendritic polymers at the same or different mixing ratios. 5 S. The components of the mixture may interact through hydrogen bonding or electrostatic attraction or repulsion or they may be independent of each other like an admixture.

6. Mixture of two or more dendritic polymers loaded with the same drug at identical loading concentration but with different release profiles. 0 7. Mixture of two or more different dendritic polymers loaded with the same drug at different loading concentrations and with different release profiles. 8. Mixture of two or more different dendritic polymers loaded with different drugs (at least two different drugs) at identical loading concentrations but with different release profiles.

9. Mixture of two or more different dendritic polymers loaded with different S drugs (at least two different drugs) at different loading concentrations and with different release profiles.

10. In case of two drugs the drugs are either loaded both into each dendritic polymer of the mixture, or the drugs are separated (i.e., a mixture of dendritic polymers A, B, and C loaded with drugs X and Y forms a mixture of A-X + A-Y + B-X + B-Y + C-X + 0 C-Y or A-X + B-Y + C-X and any combination hereof) with X and Y either having the same concentration in A, B, and C or different concentrations.

11. Drug loading includes physical encapsulation into the interior of the dendritic polymer, adsorption onto the surface of the dendritic polymer or chemical attachment of the drug to the dendritic polymer (surface and/or interior) with or without using a chemical S linker for the attachment. The chemical attachment can be temporary ("prodrug") or permanent.

12. Drug loading can be done either using premixed dendrimers (one drug at identical or different concentrations per dendritic polymer depending on the respective drug- to-dendritic polymer interaction), or dendritic polymers are first loaded with one or more 0 drugs at identical or different concentrations and then mixed at ratios that provide the desired combined release profile.

13. The mixtures of dendritic polymers containing drug as described under #1-12 above can be used also for solubility enhancement of poorly soluble drugs.

14. The m ixtures of dendritic polymers containing drug as described under #1-12 5 above can be used for providing a constant and desired drug-to-drug ratio and drug concentration in the blood (i.e., in cancer treatment using drug cocktails).

15. The mixtures of dendritic polymers containing drug as described under #1-12 above can be used for passive drug delivery based on the size of the dendritic polymers (i.e., Enhanced Permeability and Retention (EPR) effect in leaky tumor vasculature). 0 16. The mixtures of dendritic polymers containing drug as described under #1-12 above can be used for active, targeted drug delivery based on specific ligands attached to the surface of the dendritic polymers that target specific receptors at the desired site of action (i.e., a tumor cell surface.)

17. The mixtures of dendritic polymers containing drug as described under #1-12 above can be used for modifying the pharmacokinetics and phamacodynamics of the drug or S drugs temporary or permanently attached to the dendritic polymer.

Thus this invention provides for a formulation of hybrid dendrimers that have various release profiles, may provide solubility to drugs that would otherwise be insoluble or low solubility or too toxic to administer to an animal or human, can be directed to the desired target in the animal or human, may have desirable EPR characteristics, and be 0 reproducibly made. This dendritic polymer platform of hybrid dendrimers can be applied in multiple drug therapy by increasing formulation stability and preventing undesired problems associated with drug-drug interactions. These formulations of hybrid dendrimers can alter the pharmacokinetic (PK) and dynamic (PD) profiles of a drug.

The formulations of this invention containing hybrid dendrimers are administered to 5 animals (warm-blooded), including humans. The formulation is administered by all usual routes; namely, parenteral, subcutaneous, intramuscular, intravenous, and non-invasive routes selected from oral, mucosal, rectal, vaginal, intrauterine, buccal, sublingual, nasal, ocular, ear, lung, transdermal and topical. The route selected will depend on the disease being treated, the location in the animal body required to administer the formulation, the 0 ease of administration depending on the condition of the patient, whether the drug is for therapy, diagnosis or therapy/diagnosis and the time intervals of the desired drug release. These formulations can be used as pharmacologically-acceptable drug delivery systems selected from controlled drug delivery, sustained drug delivery, targeted drug delivery and intelligent drug delivery. 5 These formulations are administered as sterile or non-sterile formulations selected from solution, suspension, emulsion, elixirs, capsules, cachets, sachets, pills, tablets, granules, powders, creams, solids, ointments, suppositories, lotions, film-forming solution, ointment, creams, gels, solutions, topical aerosols and pastes. These hybrid dendrimers may be formulated into a tablet using binders known to those skilled in the art. Such dosage 0 forms are described in Remington's Pharmaceutical Sciences. 18^th ed. 1990, pub. Mack

Publishing Company, Easton, PA. Suitable tablets include compressed tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, multiple compressed tablets, controlled- release tablets, and the like. Ampoules, ointments, gels, suspensions, emulsions, injections (intramuscular, intravenous, intraperitoneal) may also be used as a suitable formulation. Customary pharmaceutically-acceptable salts, adjuvants, diluents and excipients may be used in these formulations. For agricultural uses these compositions may be formulated S with the usual suitable vehicles and agriculturally acceptable carrier or diluent, such as emulsifiable concentrates, solutions, and suspensions.

These hybrid dendrimers that have attractive interactions can be stabilized by cross- linking of the components at the surface with biodegradable or non-biodegradable bonds, in which drugs are incorporated. 0 The form used will depend on the disease being treated, the location in the animal body required to administer the formulation, the ease of administration depending on the condition of the patient, whether the drug is for therapy, diagnosis or therapy/diagnosis and the time intervals of the desired drug release.

These formulations can be administered alone or in combination with other drug S delivery systems such as liposomal or vesicular systems, nanoparticles, microspheres, microcapsules, cyclodextriπs, calixarene, polymers and supramolecular biovectors.

The guest material that is associated with these hybrid dendrimers may be chosen from a large group of possible candidates. Such materials include, but are not limited to, pharmaceutical materials for in vivo or in vitro or ex vivo use as therapeutic treatment of 0 animals, including humans, or plants or microorganisms, viruses and any living system, which material can be associated with these dendrimers 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, 5 chloramphenicol, cytarabine, daunomycin, doxorubicin, fluorouracil, gentamycin, ibuprofen, kanamycin, meprobamate, methotrexate, novantrone, nystatin, Oncovin, phenobarbital, polymyxin, probucol, procarbabizine, rifampin, streptomycin, spectinomycin, Symmetrel, thioguanine, tobramycin, trimethoprim, and valbanl; toxins, such as diphtheria toxin, gelonin, exotoxin A, abrin, modeccin, ricin, or toxic fragments thereof; metal ions, 0 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 ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ⁸²Rb, ⁸⁹Sr, ⁸⁸Y, ⁹⁰Y, ""¹Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ^{l l5m}In, ¹²⁵1, ¹³¹1, ¹⁴⁰Ba, ¹⁴⁰La, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁴Ir, and ¹⁹⁹Au, preferably ⁸⁸Y, ⁹⁰Y, ""¹Tc, ¹²⁵1, ¹³¹1, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re₅ ⁶⁷Ga, ¹¹¹In, ^115mIn, and ¹⁴⁰La; signal generators, which includes anything that results in a detectable and measurable perturbation of the system due to its presence, such as fluorescing entities, phosphorescence entities and radiation; signal reflectors, such as paramagnetic entities, for example, Fe, Gd, or Mn; chelated metal, such as any of the metals given above, whether or not they are radioactive, when associated with a chelant; signal absorbers, such as contrast agents and electron beam opacifiers, for example, Fe, Gd or Mn; antibodies, including monoclonal antibodies and anti-idiotype antibodies; antibody fragments; hormones; biological response modifiers such as inter leukins, interferons, viruses and viral fragments; diagnostic opacifiers; and fluorescent moieties. Carried pharmaceutical materials include scavenging agents such as chelants, antigens, antibodies or any moieties capable of selectively scavenging therapeutic or diagnostic agents.

When the guest materials are agricultural materials such materials include any materials for in vivo or in vitro treatment or application to plants or non-mammals

(including microorganisms) which can be associated with the hybrid dendrimer without appreciably disturbing the physical integrity of the dendrimer. For example, the carried materials can be 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 ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ⁸²Rb, ⁸⁹Sr, ⁸⁸Y, ⁹⁰Y, ""¹Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ^115mIn, ¹²⁵1, ¹³¹1, ¹⁴⁰Ba, ¹⁴⁰La, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁴Ir, and ¹⁹⁹Au; signal generators, which includes anything that results in a detectable and measurable perturbation of the system due to its presence, such as fluorescing entities, phosphorescence entities and radiation; signal reflectors, such as paramagnetic entities, for example, Fe, Gd, or Mn; signal absorbers, such contrast agents and as electron beam opacifiers, for example, Fe, Gd, or Mn; hormones; biological response modifiers, such as interleukins, interferons, viruses and viral fragments; pesticides, including antimicrobials, algicides, arithelmetics, acaricides, insecticides, attractants, repel lants, herbicides and/or fungicides, such as acephate, acifluorfen, alachlor, atrazine, benomyl, bentazon, captan, carbofuran, chloropicrin, chlorpyrifos, chlorsulfuron cyanazine, cyhexatin, cypermithrin, 2,4-dichlorophenoxyacetic acid, dalapon, dicamba, diclofop methyl, diflubenzuron, dinoseb, endothall, ferbam, fluazifop, glyphosate, haloxyfop, malathion, naptalam; pendimethalin, permethrin, picloram, propachlor, propanil, sethoxydin, temephos, terbufos, trifluralin, triforine, zineb, and the like. Carried agricultural materials include scavenging agents such as chelants, chelated metal (whether or not they are radioactive) or any moieties capable of selectively scavenging therapeutic or diagnostic agents.

The guest materials may also be immuno-potentiating agents. Such materials which are suitable for use in these formulations include any antigen, hapten, organic moiety or organic or inorganic compounds which will raise an immuno-response which can be associated with the hybrid dendrimers without appreciably disturbing the physical integrity of the dendrimers. For example, the carried materials can be synthetic peptides used for production of vaccines against malaria (US Patent 4,735,799), cholera (US Patent 4,751,064) and urinary tract infections (US Patent 4,740,585), bacterial polysaccharides for producing antibacterial vaccines (US Patent 4,695,624) and viral proteins or viral particles for production of antiviral vaccines for the prevention of diseases such as AIDS and hepatitis.

The use of these hybrid dendrimers as carriers for immuno-potentiating agents avoids the disadvantages of ambiguity in capacity and structure associated with conventionally known or synthetic polymer formulations used to give a macromolecular structure to the adjuvant carrier. Use of these hybrid dendrimers as carriers for immuno- potentiating agents, allows for control of the size, shape and surface composition of the conjugate. These options allow optimization of antigen presentation to an organism, thus resulting in antibodies having greater selectivity and higher affinity than the use of conventional adjuvants. It may also be desirable to connect multiple antigenic peptides or groups to the dendrimer, such as attachment of both T- and B-cell epitopes. Such a design would lead to improved vaccines.

It may also be desirable to conjugate pesticides or pollutants capable of eliciting an immune response, such as those containing carbamate, triazine or organophosphate constituents, to said hybrid dendrimers. Antibodies produced to the desired pesticide or pollutant can be purified by standard procedures, immobilized on a suitable support and be used for subsequent detection of the pesticide or pollutant in the environment or in an organism. Furthermore the guest materials suitable for use in these formulation of hybrid dendrimers include any materials other than agricultural or pharmaceutical materials which can be associated with said hybrid dendrimers without appreciably disturbing the physical integrity of the hybrid dendrimer, for example: metal ions, such as the alkali and alkaline- earth metals; signal generators, which includes anything that results in a detectable and measurable perturbation of the system due to its presence, such as fluorescing entities, phosphorescence entities and radiation; signal reflectors, such as paramagnetic entities, for example, Fe, Gd, or Mn; signal absorbers, such as contrast agents and an electron beam opacifiers, for example, Fe, Gd, or Mn; pheromone moieties; fragrance moieties; dye moieties; and the like. Carried materials include scavenging agents such as chelants or any moieties capable of selectively scavenging a variety of agents.

Preferably the guest materials are bioactive agents. As used herein, "bioactive" refers to an active entity such as a molecule, atom, ion and/or other entity which is capable of detecting, identifying, inhibiting, treating, catalyzing, controlling, killing, enhancing or modifying a targeted entity such as a protein, glycoprotein, lipoprotein, lipid, a targeted cell, a targeted organ, a targeted organism [for example, a microorganism, plant or animal (including mammals such as humans)] or other targeted moiety. Also included as bioactive agents are genetic materials (of any kind, whether oligonucleotides, fragments, or synthetic sequences) that have broad applicability in the fields of gene therapy, analysis, modification, activation, anti-sense, silencing, diagnosis of traits and sequences, and the like. These formulations include effecting cell transfection and bioavailability of genetic material comprising a complex of a hybrid dendrimer and genetic material and making this complex available to the cells to be transfected.

These formulations may be used in a variety of in vivo, ex vivo or in vitro diagnostic or therapeutic applications. Some examples are the treatment of diseases such as cancer, autoimmune disease, genetic defects, central nervous system disorders, infectious diseases and cardiac disorders, diagnostic uses such as radioimmunossays, electron microscopy, enzyme linked immunoadsorbent assays, nuclear magnetic resonance spectroscopy, contrast imaging, immunoscintography, and delivering pesticides, such as herbicides, fungicides, repellants, attractants, antimicrobials or other toxins. Non-genetic materials are also included such as interleukins, interferons, tumor necrosis factor, granulocyte colony stimulating factor, and other protein or fragments of any of these, antiviral agents. For the following examples the various equipment and methods were used to run the various described tests for the results reported in the examples described below.

Equipment and Methods

Formulations (general protocol)

PAMAM dendrimer G4 with EDA core and primary amine surface (G4-NH2) was used in most Examples as a template dendrimer and other dendrimers were mixed each time to form a different composition of dendritic polymers, i.e., hybrid dendrimers. All dendrimers were used at 0.2 % w/v concentration and pH was adjusted to ~7.0.

Table 1: PAMAM dendrimers used in hybrid dendrimer formulations

Loading of indomethacin (general protocols)

A. Drug loading of PAMAM hybrid dendrimer formulations

2.0 mL of template dendrimer G4-NH2 was mixed with 2.0 mL of a second dendrimer, briefly sonicated and kept at RT for 1 day for equilibration. All compositions were transparent, except for the hybrid dendrimers between G4-NH2 and G3.5-COONa, which formed a turbid solution. The model drug indomethacin (10.0 mg) was added to all dendrimer formulations, followed by brief sonication and shaking at 37°C for 24 h. Samples were kept for equilibration at RT for 1 day. During this time period the G3.5- COONa containing formulation became transparent while the G4-SUC containing formulation turned turbid; all other formulations remained transparent. Supernatants of each formulation were tested for their indomethacin content using UV spectroscopy at 320 nm wavelength.

B. Drug loading of dendrimers prior to mixing.

For this route, indomethacin was first loaded into the individual hybrid dendrimers, followed by brief sonication and shaking at 37°C for 24h. Samples were kept for equilibration at RT for 1 day. Excess drug was removed by filtration through 0.2μ nylon syringe filters. Supernatants of each dendrimer solution were tested for their indomethacin content using UV spectroscopy at 320 run wavelength. These drug loaded hybrid dendrimers were then mixed with the template dendrimer G4-NH2, briefly sonicated and kept at RT for 1 day for equilibration. The respective indomethacin content of each composition was measured using UV spectroscopy at 320 run wavelength.

Loading of camptothecin (general protocol)

Camptothecin loading into dendrimers was carried out in water. PEHAM G2.5-COOH dendritic polymer was dissolved in aqueous solution (1% w/v) and mixed with excess drug. These suspensions were briefly exposed to ultrasonication, and heated at 60⁰C for 30 min, then stirred overnight at RT. The suspensions were filtered through a 0.2-μm Nylon syringe filter to remove excess drug. The drug content in solution was measured by UV spectroscopy at 370 nm wavelength using a Perkin Elmer™ Lambda 2 UV AQS Spectrophotometer

In vitro release (general protocols)

A. In vitro release protocol of indomethacin

Dendrimer-indomethacin compositions were analyzed for in vitro release by dialysis (Spectra/Por Membrane MWCO-IOOO; Fisher) against 20.0 mL of DI water at 37°C with constant rocking agitation. At scheduled time intervals, the outer compartment was analyzed for indomethacin using UV spectroscopy at 320 nm wavelength.

B. In vitro release protocol of indomethacin and camptothecin mixtures

The PAMAM dendritic polymer was loaded with indomethacin and PEHAM dendritic polymer was loaded with camptothecin. The formulation was evaluated for indomethacin and camptothecin content by UV spectroscopy at 320 and 370 nm respectively. The PAMAM-indomethacin and PEHAM-camptothecin dendrimers were mixed at equal volume ratio and stirred overnight at RT to form PAMAM-PEHAM hybrid dendrimers. The hybrid dendrimers containing indomethacin and camptothecin were simultaneously evaluated by HPLC. The release study was carried out in water. Size Exclusion Chromatography (SEC)

A methanolic solution of dendrimer compositions was evaporated and reconstituted with the mobile phase used in the SEC experiment (1 mg/mL concentration). All the samples were prepared fresh and used immediately for SEC. Dendrimers were analyzed qualitatively by SEC- SEC system (Waters 1515) was operated in an isocratic mode with refractive index detector (Waters 2400) and Waters 717 Plus Auto Sampler. The analysis was performed at RT on two serially aligned TSK gel columns (Supelco), G3000PW and G2500PW, particle size lOμm, 30cm * 7.5 mm. The mobile phase of acetate buffer (0.5M) was pumped at a flow rate of lmL/min. The elution volume of dendrimer was observed to be 11 - 16 mL, according to the generation and surface of dendrimer.

Ultraviolet/Visible Spectrometry (UV-VlS)

UV-VIS spectral data were obtained on a Perkin Elmer Lambda 2 UV/VIS Spectrometer.

The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the present invention.

Example 1 : Loading of indomethacin in hybrid dendrimers

As can be seen from Figures 1-6, PAMAM dendrimers, G4 with (C) of EDA, were generally used and one PAMAM dendrimer, G4 with (C) of DOD and surface of AEEA. The dendrimer formulations were maintained at pH~7 using 0. IN HCl. However, when the dendrimer was a G4-SUC dendrimer, the pH was -5.26 and had incomplete dissolution and had turbidity and inconsistent loading. Formulations containing G4-TRIS, G4-AE, G4- PYR, and G4-PEG dendrimers mixed with G4-NH2 dendrimer to form the hybrid dendrimer showed combined indomethacin loading efficiencies in the range of 400-500 μg/mL, similar to the loading efficiency of G4-NH2 dendrimer alone. However, the loading efficiencies of formulations containing G3.5-COONa and G4-SUC dendrimers are similar to the loading efficiencies of each of those respective dendrimers.

Arithmetical calculation of the total loading efficiency of a hybrid dendrimer formulation F composed of dendrimers A and B could be F = A+B or F < A+B or F> A+B. Hybrid dendrimer formulations in these experiments predominately show an loading efficiency according to F< A+B. This observation indicates attractive interaction between the constituent dendrimers of these formulations. This interaction is reducing the resultant dendrimer surface area available for interaction and loading of drug molecules, leading to lower loading efficiency than expected due to an additive effect. See Figures 1 -6.

An illustration of the various aspects of the properties of these hybrid dendrimers is provided in Figure 24.

Example 2: SEC The G4-NH2+G3.5-COONa and G4-NH2+G4-SUC hybrid dendrimer formulations showed split SEC peaks with slight shifts compared to the single dendrimer solutions. This indicates weak interactions within these hybrid dendrimer formulations. However, only single peaks were observed for G4-NH2 compositions with G4-AE, G4-PYR, G4-AEEA, and G4-TRIS hybrid dendrimers, suggesting strong attractive forces between these hybrid dendrimers. The G4-NH2+G4-PEG hybrid dendrimer formulation revealed two separate peaks corresponding to the individual dendrimers, indicating no interaction or repulsive interactions between these hybrid dendrimers. See Figures 7 and 8.

Thus these SEC results are supporting the loading results. Hybrid dendrimer formulations with single SEC peak (= attractive interaction) showed reduced loading efficiency (F< A+B) compared to the constituent dendrimers.

Example 3: In vitro release profiles

The release profile of the G4-NH2+G3.5-COONa and G4-NH2+G4-SUC hybrid dendrimer formulations showed 100% drug release in 24 h, similar to the release profiles of the individual dendrimers. Drug release in the range of 25-50% over 24 h was observed for hybrid dendrimer formulations between G4-NH2 and each of G4-TRIS, G4-PEG, G4-AE and G4-PYR dendrimers. However for the G4-NH2+G4-AEEA hybrid dendrimer formulation the release rate was 12% drug in 24 h. These formulations showed drug release profiles that are intermediate between the release profiles of the individual dendrimers of the hybrid dendrimer. For example, at 24 h pure G4-NH2 dendrimer releases 2% of indomethacin and G4-TR1S releases 100% of the drug, while the 1 :1 hybrid dendrimer formulation of both releases 37% drug during the same time period. See Figures 9-14. Example 4: Effect of concentration

The effect of concentration on loading efficiency of hybrid dendrimer formulations was studied using the G4-NH2+G4-AEEA hybrid dendrimer. Loading efficiency was tested S at 0.1% (w/v) and 0.2% (w/v) concentrations of each dendrimer. Enhanced indomethacin loading was observed with increasing dendrimer concentration, i.e., the loading efficiency at 0.2% (w/v) was approximately twice as high as the efficiency observed for the 0.1% (w/v) formulation.

0 Example 5: Route of Preparation of hybrid dendrimer formulations

AU formulations described above have been prepared by first mixing the dendritic polymers to form the hybrid dendrimer and then loading the hybrid dendrimer with the drug indomethacin (Route 1). To test this method, the preparation protocol was inverted, i.e., individual dendrimers were first loaded with the drug indomethacin and then mixed to S prepare the desired hybrid dendrimers (Route 2). Hybrid dendrimer formulations comprising G4-NH2 and G4-TRIS, G4-AE and G4-PYR as the second component were prepared and their loading efficiency and release profile studied. AU compositions encapsulated essentially the same amount of indomethacin as the formulations prepared following the first protocol. Similar observations have been made for the in vitro release 0 profiles, which were almost identical to the profiles found for the first protocol, with the exceptions of hybrid dendrimer formulation of G4-NH2-KJ4-TRIS, which showed slightly faster release, and of G4-NH2+G4-AE and G4-NH2+G4-PYR, which showed slightly slower release rates.

Differences between release profiles following Routes 1 or 2 are mainly dominated 5 by the surface functionalities of the constituent dendrimers, i.e., hybrid dendrimers showing strong interactions between the constituent dendrimers show release profiles depending on the route of loading, while hybrid dendrimers showing weak interactions between the constituent dendrimers show essentially the same release profiles independent of the route of loading. See Figures 15-17. 0 Example 6: Loading and release of indomethacin from PAMAM-PEHAM hybrid dendrimer formulations

A. Drug loading into (PAMAM G4-NH2)-(PEHAM G2.5-COOH) hybrid dendrimers Dendritic polymers PAMAM G4-NH2 and PEHAM G2.5-COOH were selected to disclose loading and in vitro release of indomethacin from hybrid dendrimers consisting of different dendrimers. Hybrid dendrimer preparation and indomethacin loading were carried out according to the protocols given above. The loading efficiency of the PAMAM- PEHAM hybrid dendrimers falls in between the loading efficiencies of the two parent dendrimers, and therefore, follows the observations disclosed in Example 1 for PAMAM- PAMAM hybrid dendrimers. See Figure 18.

B. In vitro release from (PAMAM G4-NH2)-(PEHAM G2.5-COOH) hybrid dendrimers

Indomethacin release from (PAMAM G4-NH2)-(PEHAM G2.5-COOH) hybrid dendrimers following Route 1, i.e., after formation of the hybrid dendrimers, showed a profile similar to PEHAM G2.5-COOH dendritic polymer alone. It is assumed that strong interactions between PAMAM and PEHAM dendrimers resulted in the formation of hybrid dendrimers with PAMAM as the core constituent and PEHAM as the shell constituents thus preventing efficient loading of indomethacin into the core PAMAM. See Figure 19. Indomethacin release from (PAMAM G4-NH2)-(PEHAM G2.5-COOH) hybrid dendrimers following Route 2, i.e., drug loading prior to the formation of the hybrid dendrimers, showed modified release profiles, which can be adjusted to the desired release rate by the mixing ratio between PAMAM and PEHAM dendrimers. Indomethacin release is increasingly delayed from the hybrid dendrimer formulations with increasing amount of PAMAM in the hybrid dendrimer formulation (Le., ratios PAMAM:PEHAM equal 2: 1, 1 : 1, and 1 :2). See Figure 20.

Example 7: In vitro release from (PAMAM G4-TRIS)-(PEHAM G 1.5-NH) hybrid dendrimers The hybrid dendrimer formulation was prepared following Route 2. PEHAM Gl .5-

NH showed slow release of indomethacin, while dendritic polymer PAMAM G4-TRIS showed faster release. Indomethacin release from (PAMAM G4-TRIS)-(PEHAM G1.5- NH) hybrid dendrimers showed an intermediate release profile. See Figure 21.

Example 8: Release of indomethacin and camptothecin from PAMAM-PEHAM hybrid dendrimer formulations

Polymeric dendrimer PAMAM G4-NH2 was loaded with indomethacin and polymeric dendrimer PEHAM G2.5-COOH was loaded with camptothecin. Both dendrimers were mixed to for hybrid dendrimers according to Route 2. Release of indomethacin and camptothecin from this formulation was measured simultaneously, giving release profiles that show equal concentrations of both drugs over time (25 h). Such release profile is important for the application of combined drugs (drug cocktails).

Example 9: Release of indomethacin from PPI-PEHAM hybrid dendrimer formulations

Dendritic polymers PPI G4-NH2 and PEHAM Gl.5-NH were first loaded with indomethacin and then mixed to form the hybrid dendrimer formulation as disclosed in

Route 2. The hybrid dendrimer formulation showed slower release of indomethacin than the PPl dendritic polymer formulation, i.e., hybrid dendrimers composed of PPI and PEHAM follow the observations disclosed for PAMAM-PAMAM and PAMAM-PEHAM hybrid dendrimer formulations. See Figure 23.

Although the invention has been described with reference to its preferred embodiments, those of ordinary skill in the art may, upon reading and understanding this disclosure, appreciate changes and modifications which may be made which do not depart from the scope and spirit of the invention as described above or claimed hereafter.

Claims

WHAT IS CLAIMED IS:

I . Hybrid dendrimers comprising a mixture, at the same or different ratios, of at least two dendritic polymers or dendronized polymers that have at least one difference between them.

5 2. The hybrid dendrimer of claim 1 wherein one dendritic polymer is a

PAMAM dendrimer and one is a PEHAM dendrimer.

3. The hybrid dendrimer of claim 1 wherein one dendritic polymer is a PAMAM dendrimer and one is a dendronized polymer.

4. The hybrid dendrimer of claim 1 wherein one dendritic polymer is a PEHAM 0 dendrimer and one is a dendronized polymer.

5. The hybrid dendrimer of claim 3 or 4 wherein the dendronized polymer is latex, linear polymers, cage molecules.

6. The hybrid dendrimer of claim 5 wherein the dendronized polymer is PEL

7. The hybrid dendrimer of claim 1 wherein one dendritic polymer is a PPI S dendrimer and one dendritic polymer is PAMAM dendrimer.

8. The hybrid dendrimer of claim 1 wherein one dendritic polymer is a PPI dendrimer and one dendritic polymer is PEHAM dendrimer.

9. The hybrid dendrimer of claim 1 wherein at least two dendritic polymers are present that differ from each other by one or more physical features selected 0 from surface groups, G, BR, EX, drug, and guest moiety.

10. The hybrid dendrimer of claim 1 where the dendritic polymers or dendronized polymers may interact through hydrogen bonding, electrostatic attraction or repulsion.

I I. A formulation of a hybrid dendrimer of any one of claims 1-10 wherein the 5 dendritic polymers have the same drug present at either identical or different loading concentrations but have different release profiles.

12. A formulation of a hybrid dendrimer of any one of claims 1-10 wherein the dendritic polymers have different guest molecules present at either identical or different loading concentrations but have different release profiles. 0

13. The formulation of claim 11 or 12 wherein the loading of a drug or guest material may be done after the formation of the hybrid dendrimer or in the dendritic polymer prior to formation of the hybrid dendrimer.

14. The formulation of claim 11 or 12 wherein the solubility of the drug or guest material is enhanced.

15. The formulation of claim 1 1 wherein the hybrid dendrimer provides a constant drug-to-drug ratio and drug concentration in the blood.

16. The formulation of claim 15 as a drug cocktail for cancer treatment of an animal.

17. The formulation of claim 11 or 12 for use as a passive drug delivery system based on the size of the hybrid dendrimer for EPR.

18. The formulation of claim 11 or 12 for use as pharmacologically-acceptable drug delivery systems selected from controlled drug delivery, sustained drug delivery, targeted drug delivery and intelligent drug delivery.

19. The formulation of any one of claims 1 1-18 wherein a suitable excipient, carrier or solvent is present.

20. A method of using a hybrid dendrimer of any one of claims 1-19 for delivery of a drug or guest material in order to provide increased solubility to poorly soluble drugs or guest materials.