Patent Application
20080020043
The invention is directed to the preparation of “dendrimer-like” structures as shown in
These variations will provide for the drug delivery system (DDS) properties.
Drugs, or other biologically active molecules, including but not limited to peptides, proteins, enzymes, small molecules drugs (antibiotics, fungicides, anti-viral, anti-inflammatory, anti-tumor, cardiovascular, etc . . . ), dyes, lipids, nucleosides, etc., may be included in the dendrimer-drug conjugates of the invention. The solubility of a large molecule such as the dendrimer-drug conjugate is achieved through incorporation of hydrophilic spacers, e.g. poly(ethylene glycol) (PEG) via enzyme-degradable or hydrolyzable linkages in such way they form a hydrophilic layer at different levels of the final chemical structure of the dentrimer-drug conjugate of the invention.
In the dendrimer-drug conjugate shown in
The central core (CC) is a reactive core allowing several branch lines to be formed. Preferably CC is selected from the group consisting of an aliphatic, cycloaliphatic or aromatic alcohol, a diol, a triol (e.g. phloroglucinol), a tetrol (e.g. pentaerythritol), a reducing sugar e,g, sorbitol, mannitol, arabitol, glycerol dipentaerythrytol, glycerol oligomers (hexaglycerol), a synthesized polyol, a thiol or polythiol, a polyamine, a halomethylaryl compound represented by the formula
a acid halide (e.g. a aromatic or aliphatic acid halide such as
wherein R′ is an aliphatic radical, n is an integer of from 2 to 6 and X is Br, Cl, I, or another leaving group;
or any other structure built by the combination of one or more of the above molecules
The secondary core (SC) may be identical or different than CC:
Preferably SC is selected from the group consisting of monomers of formula A-R′—B2, wherein R′ is an aromatic (phenyl, naphthyl . . . ), or aliphatic radical, A and B are functional groups capable of forming a covalent bond with either a preceding or subsequent generation of the drug-dendrimer conjugate and preferably selected to provide that only one group (A or B) can react first whereas the second one does not react or remains protected and monomers of formula A-R″—B3 wherein R″ is an aromatic radical and A and B are as defined above.
Example of secondary cores A-R′—B2 used in this invention include:
wherein TBDMS is t-butyl dimethyl silyl and THP is tetrahydropyran.
Examples of secondary cores A-R″—B3 used in this invention include:
wherein R′″ is alkyl.
The secondary core can be bonded, through an enzyme-degradable chemical linkage, to the central core or to hydrophilic segment as shown in
In order to allow the dentrimer-drug conjugates of the invention to exhibit satisfactory water solubility, a hydrophilic chemically defined spacer is incorporated in the dentrimer-drug conjugate providing that said spacer presents low toxicity and is a biocompatible polymer (including linear or non-linear polymers) such as: (poly(ethylene glycol) (PEG), PEG-like spacers, poly(amino acid) (linear poly(lysine), polyvinyl alcohol, polyhydroxyethylmethacrylate, polyacrylamide, polyacrylic acid, polyethyloxazoline, polyvinyl pyrrolidinone, polysaccharides such as agarose, chitosan, alginates, hyaluronic acid, dextrans, etc. and it brings no polydispersity to the final structure which is a critical factor to ensure the synthesis of the preferred drug-dendrimer conjugate of this invention.
For example, preferably linear poly(ethylene glycol) (PEG) is used as the spacer.
The PEG spacer may include different M end-capped groups like amino, ester, carboxylic acid, succinic acid, amide, urethane, thiol, etc . . . (in place of the hydroxyl functionality) to allow further diversity and variability in the molecular construction.
For example, the PEG spacer utilized in the present invention may include different end-capped groups as illustrated below.
Ts is tosyl, M, in this illustrative chemical synthesis scheme, may be D, R, CC, SC, —OH, —SH, —NH2, protecting groups, carboxylic acid, amide, ester, urethane, etc. and n is an integer of from 1 to 7.
Higher molecular weight monodisperse PEG or PEG-like spacers can be obtained by using an iterative process by addition of commercial or modified monodisperse PEG units. PEG monodispersity is controlled by using a chain length (n) of from 1 to 7.
For example, in the following reaction scheme
wherein M and n are as defined above;
P represents protecting groups and m is an integer of from 1 to 7.
The spacer is covalently attached to the structure (“interior”) of the dendrimer structure and to the drug through a degradable linkage moiety, e.g. an enzyme esterase, i.e. a hydrolase.
Spacers are incorporated in the dendrimer structure in such way to form a hydrophilic layer which can be present at different levels in the structure. Thus, in case of their presence at several levels in the structure, they are distributed into successive or different hydrophilic layers or generations.
In the dentrimer structure of
Any substance intended for diagnosis, cure, mitigation, treatment or prevention of disease in humans or animals,
Examples of biologically actives molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, . . . Classes of small molecules drugs that are suitable for use with the invention include, but are not limited to, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, ophthalmic drugs, dermatological drugs and mixtures thereof.
The four key elements presented above, i.e. central core (CC), secondary core (SC), hydrophilic chemically defined spacer (SH) and drug or other biologically active substance (D), will be linked together and will allow for a multiplicity of chemical structures which could be customized depending on the drug delivery system (DDS) characteristics targeted.
Controlled drug delivery from such chemical structures can be achieved by variations of several parameters in the structure's “architecture”. These variations would define the final DDS properties:
Number of drugs present on the structure (periphery).
Dissymmetrical structure: relative drug position, nature of drugs.
Different chemical linkages within same structure.
Core.
Secondary core or cores.
Hydrophilic spacer.
Preferably, the structures of this invention are built in such way each sub-unit or generation i.e. central core, secondary core, hydrophilic spacer and active ingredient are bonded to each other through an enzymatically degradable linkage. Assuming an equal proportion of the active ingredient is brought by each chemical system from structures II to IV, drug release in the body is expected to be respectively slower from structure IV of Figure compared to structures III and II of
Structures containing different drug at different positions (internal or external) can be synthesized. (See
Structures containing sub-units attached by different enzymatically degradable links can be synthesized. Presence of such different chemical links implies different behavior in the presence of enzymes (difference in accessibility, in speed of cleavage . . . ) that inevitably induce differences in drug's release. (See
The invention is further illustrated by the following examples which are illustrative of a specific mode of practicing the invention and are not intended as limiting the scope of the claims.
As previously described, different possibilities would allow defining a specific drug delivery system: the number of actives linked to the structure, their relative position to the central core (internal, external), the nature of the chemical links to the structure.
The dendrimer-drug conjugates of the present invention as shown in
Equal amounts of flurbiprofen bonded to structures II and III of
While different products are expected from the enzymatic activity, the experiment was mainly focused on the detection and quantification of the initial structure (II and III) as well as on the apparition of flurbiprofen.
Structures II and III as well as flurbiprofen released from these two structures are quantitated using high performance liquid chromatography (HPLC). The analytes are eluted from a XTerra® Phenyl column using an eluent gradient composed of water/methanol/formic acid 50 mM. Analytes are detected by UV absorbance at 240 and 280 nm.
The results are shown in
Structures II and III in quantities providing the same amount of flurbiprofen, were incubated with 25 UI of esterase at 37° C. The 25 UI esterase was daily renewed for a total of 4 days. Analysis was performed at times 0, 24 H, 48 H, 72 H and 96 hours after the addition of the enzyme.
The following results were obtained:
After four incubation days under conditions using the same quantities of esterase and flurbiprofen, about 70% of flurbiprofen base was released from structure II while only 10% was released from structure III. This difference in active drug release from the initial structure indicates that the active is cleaved from a simple structure, where all the bonds between the drug and the dendrimer are substantially identical, at a faster rate as compared to a complex structure wherein the bonds vary or the drug is present in different generations of the dendrimer. Furthermore, several peaks have been identified with the structure III that may correspond to intermediate products.
From structure IV, which comprises two Structure III units, covalently bonded together, the rate of dendrimer-drug conjugate having release of the active molecule would be slower. (See the results of this experiment in
The dendrimer-drug conjugate having 3 flurbiprofen molecules bonded to a dendrimer molecule was successfully prepared by the process scheme set forth in
While particular embodiments of the invention have been described it will be understood of course that the invention is not limited thereto since many obvious modifications can be made and it is intended to include within this invention any such modifications as will fall within the scope of the appended claims. For example, as shown in
