Patent Application
20060039891
This invention relates to the treatment of cancer in animals, especially humans, using dendritic polymer conjugates having an antineoplastic drug present.
The use of polymers as carriers for drugs, especially those drugs that have low water solubility at physiological pH, are toxic to the normal tissue, or cannot be administered in sufficient dosage, has gained interest in recent years [e.g., H. Ringsdorf, J. Polymer Sci.: Symp. 51, 135-153 (1975)]. A polymer carrier for antineoplastic drugs would provide a useful system for administration of these drugs because of their solubility, toxic and higher dose at delivery characteristics. Several efforts to deliver doxorubicin are illustrative of this effort [e.g., R. Duncan et al., “Preclinical Toxicology of a Novel Polymeric Antitumor Agent: I-copolymer-doxorubicin (PK1)”, Hum Exp. Toxiocol. 17(2), 93-104 (1998); P. A. Vassey et al., “Phase I Clinical and Pharmacokinetic Study of PK1 [N-(2-Hydroxypropyl)methacrylamide Copolymer Doxorubicin]: First Member of a New Class of Chemotherapeutic Agents—Drug-Polymer Conjugates”, Clin. Cancer Res. 5, 83-94(1999); L. W. Seymour et al., “N-(2-Hydroxypropyl)methacrylamide Copolymers Targeted to the Hepatocyte Galactose-receptor;Pharmacokinetics in DBA2 Mice”, Br. J. Cancer 63, 859-866 (1991).
The prospect of using dendritic polymers as caters or carriers for drug delivery has been previously proposed on account of the unique structure and characteristics of these polymer molecules [R. Esfand and D. A. Tomalia, “Poly(amidoamine) (PAMAM) Dendrimers: from Biomimicry to Drug Delivery and Biomedical Applications”, research focus, DDT 6(8), 427-436 (8 Apr. 2001); U.S. Pat. Nos. 5,338,532 and 5,527,524]. More specifically, it has been proposed that the external surface functionality and interior morphological characteristics of dendritic polymer molecules appear to be very promising for developing new methods for controlling drug release and targeted drug delivery systems. However, relatively little work has been done in specific areas of drug delivery. In particular, the use of dendritic polymers as effective caters for specific anti-tumor agents has not heretofore been demonstrated.
Certain platinum containing compounds, particularly carboplatin (cis-diamine(1,1-cylobutanedicarboxylato)platinum (II)) and cisplatin (cis-diamminedichloroplatinum), have been used in the treatment of ovarian cancer, lung cancer, testicular cancer, breast cancer, stomach cancer and lymphoma. However, because of the non-specific toxicity and poor water solubility of these platinum-containing compounds, the use of carboplatin and cisplatin has been relatively limited.
In order to overcome the non-specific toxicity and water solubility problems associated with cisplatin and carboplatin, it has been proposed to use linear polymers as carriers for these drugs. However, the use of linear polymers as caters in drug delivery systems has several disadvantages. A major disadvantage with linear polymer drug carriers is that they are heterogeneous, polydisperse compositions containing various different molecular weight polymer molecules with a limited number of functional groups and/or reactive sites. Because linear polymer compositions are not comprised of molecules having a precisely defined structure, it is more difficult to maintain uniform polymer properties, drug delivery properties, and therapeutic efficacy. As a result it is relatively difficult to obtain governmental regulatory approval of the linear polymer-drug composites. Another disadvantage with the use of linear polymers as drug-carriers is that the location, and hence the availability, of the drug is difficult to control In particular, the drug must either be bound covalently or non-covalently in a random unpredictable manner and the linear polymer structure lacks well-defined cargo space for the drug. The tendency of the drug to become buried in the linear polymer leads to greater unpredictability on account of the non-uniform or heterogeneous properties of the linear polymer molecules, and results in reduced drug efficiency because a significant proportion of the drug molecules are not effectively presented to the cell being treated. In some cases the random coil structure of the linear polymers may even prevent successful drug attachment within the coil and lead to passive entrapment, leading to uncontrolled drug release (e.g., random diffuse system), i.e., lack of uniformity in the timing of the drug release.
Accordingly, it would be highly desirable to provide a precisely defined drug delivery system for cisplatin and carboplatin as well as related antineoplastic agents which exhibit high drug efficiency, high drug carrying capacity, good water solubility, good stability on storage, reduced toxicity, and improved anti-tumor activity in vivo.
U.S. Pat. No. 5,338,532 teaches polymer conjugates comprising dense polymers associated with a carried material [One type of dense star polymers is Starburst® polymers (trademark of The Dow Chemical Company) where the dendrimer is a polyamidoamnine (PAMAM).] A variety of suitable applications for such conjugates are broadly discussed in U.S. Pat. No. 5,332,532, including the use of these conjugates as delivery vehicles for biologically active agents. However, U.S. Pat. No. 5,338,532 does not specifically teach, claim, or even mention the use of polymer conjugates as delivery vehicles for cisplatin, carboplatin, titanocene dichloride and diorganotin dihalides or other antineoplastic agents. U.S. Pat. No. 5,338,532 only exemplifies the use of zero valence metals, and ionic or radioactive metals, specifically exemplifying Fe, Rh, Pd, Y, Fn, Pb, Gd, Mn and Gd.
A recent publication has disclosed the use of dendrimers as carriers for the delivery of cisplatin, i.e., “Dendrimer-platinate: a Novel Approach to Cancer Chemotherapy”, Anti-Cancer Drugs, 10, 767-776 (1999). This publication deals specifically with the formation of a dendrimer-cisplatin conjugate, i.e., a dendrimer-platinate. According to this publication the dendrimer-cisplatin conjugate is formed by a covalent or ionic interaction between the surface of the dendrimer and the platinum of the cisplatin, releasing a chloride ion in the process. This method of association differs from that of the present invention which involves the conjugation of the dendritic polymer with an antineoplastic agent, (e.g., cisplatin, carboplatin, and other metal-containing analogues thereof) through the encapsulation of the antineoplastic agent within the interior of the dendritic polymer. The method of the present invention is advantageous over that of this publication because the encapsulated conjugate of the present invention provides a method of coupling a higher delivery dose of an antineoplastic agent of a broader class than cisplatin together with a lowering of toxicity.
This invention pertains to dendritic polymer conjugates which are useful drug delivery systems for caring cisplatin, carboplatin, oxalipatin, teraplatin, platinium-DACH, ormaplatin, titanocene dichloride, vanadocene dichloride, niobocene dichloride, molybdenocene dichloride, rhenocene dichloride, and diorganotin dihalides or other metal containing antineoplastic agents (hereinafter “antineoplastic dendritic polymer conjugates”); preferably cisplatin and carboplatin and other platin-based analogues (hereafter “platin-based analogue dendritic polymer conjugates”); more preferably cisplatin (hereafter “cisplatin dendritic polymer conjugates”), as antineoplastic agents to malignant tumor as anti-tumor agents. The invention also pertains to methods of treating malignant tumors using these antineoplastic dendritic polymer conjugates, and to a method of preparing an antineoplastic dendritic polymer conjugate useful for carrying platinum (Pt), titanium (Ti), tin (Sn), vanadium (V), niobium (Nb), molybdenum (Mo), or rhenium (Re) containing compounds such as those agents named above (collectively “antineoplastic agents”) to malignant tumors.
The antineoplastic dendritic polymer conjugates comprise a dendritic polymer conjugated to an antineoplastic agent, forming a dendritic polymer antineoplastic conjugate, e.g., especially a platin-based dendritic polymer conjugate. These antineoplastics dendritic polymer conjugates are prepared by obtaining a dendritic polymer having functional groups or chelational groups which are accessible to a antineoplastic agent and capable of interacting with the functional or chelational groups (chelated through a linker to the surface of the dendrimer or preferably in the interior of the dendrimer—“cargo space”), contacting the dendritic polymer with the antineoplastic agent, and thereby allowing for uptake and encapsulation of the antineoplastic agent by the dendritic polymer by means of covalent and/or non-covalent interactions, e.g., physically encapsulated within the interior of the dendrimer, dispersed partially or fully throughout the dendrimer, or linked to the dendrimer with a chelation group, or any combination thereof, whereby the attachment or linkage is by means of covalent bonding, hydrogen bonding, adsorption, adsorption, metallic bonding, dipole-dipole interaction, van der Waals forces, or ionic bonding, or any combination thereof. These antineoplastic dendritic polymer conjugates are administered to an animal having a malignant tumor in an amount which is effective to inhibit growth of the malignant tumor, preferably intravenously (I.V.), although other methods such as oral, parental I.P.), subcutaneous (S.C.), intramuscular, intraarterial or topical administration are also possible.
The antineoplastic dendritic polymer conjugate results in an anti-tumor agent that exhibits unexpected and surprisingly high efficacy, drug carrying capacity, and dosage capabilities. The antineoplastic dendritic polymer conjugate also shows a surprising and unexpected decrease in toxicity, good water solubility, good stability on storage, and improved anti-tumor activity in vivo. Most significantly, these antineoplastic dendritic polymer conjugates were found to be active against B16F10 tumor models, which are known to be resistant to cisplatin at its maximum tolerated dose via I.V. administration (about 1 mg/kg).
The dendritic polymers which may be used to form antineoplastic dendrimer polymer conjugates include generally any of the known dendritic architectures including dendrimers, controlled hyperbranched polymers, dendrigrafts, and random hyperbranched polymers. Dendritic polymers are polymers with densely branched structures having a large number of reactive groups. A dendritic polymer includes several layers or generations of repeating units which all contain one or more branch points. Dendritic polymers, including dendrimers and hyperbranched polymers, are prepared by condensation reactions of monomeric units having at least two reactive groups after attachment. The dendrimers that can be used include those comprised of a plurality of dendrons that emanate from a common core which can be a single atom or a group of atoms. Each dendron generally consists of terminal surface groups, interior branch junctures having branching functionalities greater than or equal to two, and divalent connectors that covalently connect neighboring branching junctures. For a review article of this area see, for example, Donald A Tomalia, et al., Angew. Chem. Int. Engl. 29, 138-175 (1990).
Dendrons and Dendrimers can be Prepared by Convergent or Divergent Synthesis.
Divergent synthesis of dendrons and dendrimers involves a molecular growth process which occurs through a consecutive series of geometrically progressive step-wise additions of branches upon branches in a radially outward molecular direction to produce an ordered arrangement of layered branched cells. Each dendritic macromolecule includes a core branch cell, one or more layers of internal branch cells, and an outer layer of surface branch cells, wherein each of the cells includes a single branch juncture. The cells can be the same or different in chemical structure and branching functionality. The surface branch cells may contain either chemically reactive or passive functional groups. Chemically reactive surface groups can be used for further extension of dendritic growth or for modification of dendritic molecular surfaces. The chemically passive groups may be used to physically modify dendritic interiors or dendritic surfaces, such as to adjust the ratio of hydrophobic to hydrophilic terminals. In this fashion one can improve the solubility of a guest molecule in the interior of the dendritic polymer or the solubilization of the dendritic container in a particular solvent. (See for example for dense star polymers U.S. Pat. Nos. 4,507,466; 4,588,120; 4,568,737; 4,631,337; 4,587,329; and 4,737,550; WO 84/02705; EP 0115771; and EP 0608908; for rod shaped dense star polymers U.S. Pat. No. 4,694,064; EP 02344008; and EP 0556871; for hydrophobic outer shell dense star polymers U.S. Pat. No. 5,560,929; and EP 0680495.)
Convergent synthesis of dendrimers and dendrons involves a growth process, which begins from what will become the surface of the dendron or dendrimer, and progresses radially in a molecular direction toward a focal point or core. (See for example U.S. Pat. No. 5,041,516.) The dendritic polymers may be ideal or non-ideal, i.e., imperfect or defective. Imperfections are normally a consequence of either incomplete chemical reactions, or unavoidable competing side reactions. In practice, real dendritic polymers are generally nonideal, i.e., contain certain amounts of structural imperfections.
The hyperbranched polymers which may be used represent a class of dendritic polymers which contain high levels of nonideal, irregular branching as compared with the more nearly perfect regular structure of dendrons and dendrimers. Specifically, hyperbranched polymers contain a relatively high number of irregular branching areas in which not every repeat unit contains a branch juncture.
The preparation and characterization of dendrimers, dendrons, random hyperbranched polymers, controlled hyperbranched polymers, and dendrigrafts (collectively “dendritic polymers”) is well known. Examples of dendrimers and dendrons, and methods of synthesizing the same are set forth in U.S. Pat. Nos. 4,410,688, 4,507,466; 4,558,120; 4,568,737; 4,587,329; 4,631,337; 4,694,064; 4,713,975; 4,737,550; 4,871,779 and 4,857,599. Examples of hyperbranched polymers and methods of preparing the same are set forth, for example in U.S. Pat. Nos. 5,418,301 and 5,514,764. Examples of dendrigrafts and methods of preparing the same are set forth, for example in an article by D. A. Tomalia and R. Esfand, Chem & Ind., 416-420 (2 Jun. 1997).
The dendritic polymers or macromolecules useful in the practice of this invention are characterized by a relatively high degree of branching, which is defined as the number average fraction of branching groups per molecule, i.e., the ratio of terminal groups plus branch groups to the total number of terminal groups, branched groups and liner groups. For ideal dendrons and dendrimers, the degree of branching is 1; whereas for linear polymers, the degree of branching is 0. Hyperbranched polymers have a degree of branching that is intermediate to that of linear polymers and ideal dendrimers, preferably of at least about 0.5 or higher. The degree of branching is expressed as follows:
where Nx is the number of type x units in the structure. Both terminal (type t) and branched (type b) units contribute to the fully branched structure whilst linear (type 1) units reduce the branching factor; hence
O≦fbr≦1
where fbr=O represents the case of a linear polymer and fbr=1 represents the case of a fully branched macromolecule.
Dendritic polymers also include macromolecules commonly referred to as cascade molecules [e.g., E. Buhleier et al., Synthesis 155-158 (February 1978)], arborols [e.g., U.S. Pat. Nos. 5,376,690 and 5,210,309], arborescent grafted molecules, tectodendrimers [e.g., Srinivas Uppuluri et al., “Tecto(dendrimer) Core-shell Molecules: Macromolecular Tectonics for the Systematic Synthesis of Larger Controlled Structure Molecules” PMSE, Spring Meeting (Mar. 21-25, 1999) 55-56], and the like. Suitable dendritic polymers also include bridged dendritic polymers, ie., dendritic macromolecules linked together either through surface functional groups or through a linking molecule connecting surface functional groups together, and dendritic polymer aggregates held together by physical forces. Also included are spherical-shaped dendritic polymers (e.g., U.S. Pat. Nos. 4,507,466; 4,588,120; 4,568,737; 4,631,337; 4,587,329; and 4,737,550) and rod-shaped dendritic polymers (e.g., U.S. Pat. No. 4,694,064) grown from a polymeric core. Additional dendritic polymers suitable for use with the present invention include all the basic dendritic structures where specific chelating groups or moieties are either in the central core of the dendrimer, and/or located within the interior structure on the dendron arms and/or located on the surface of the dendrimer. All of these above dendrimer terms are to be understood to be included within the term “dendritic polymers”.
The dendritic polymers can be generationally monodisperse or generationally polydisperse. Dendritic polymers in a monodisperse solution are substantially all of the same generation, and hence of uniform size and shape. The dendritic polymers in a polydisperse solution comprise a distribution of different generation polymers. The dendritic polymer molecules which may be used include mixtures of different interior and exterior compositions or functionalities. Examples of suitable dendritic polymers include poly(ether) dendrons, dendrimers and hyperbranched polymers, poly(ester) dendrons, dendrimers and hyperbranched polymers, poly(thioether) dendrons, dendrimers and hyperbranched polymers, poly(amino acid) dendrons dendrimers and hyperbranched polymers, poly(arylalkylene ether) dendritic polymers and poly(propyleneimine) dendrons, dendrimers and hyperbranched polymers. Poly(amidoamine) (PAMAM) dendrimers have been found to be particularly useful for preparing the metal-containing antineoplastic dendritic polymer conjugates of this invention.
Dendritic polymers which are useful include those that have symmetrical branch cells (arms of equal length, e.g., PAMAM dendrimers; for example described in U.S. Pat. No. 5,527,524) and those having unsymmetrical branch cells (arms of unequal length, e.g. lysine-branched dendrimers; for example described in U.S. Pat. No. 4,410,688),) branched dendrimers, cascade molecules [e.g., E. Buhleier et al., Synthesis 155-158 (February 1978)], arborols [e.g., U.S. Pat. Nos. 5,376,690 and 5,210,309], and the like.
*The term “dendritic polymer” also includes so-called “hyper comb-branched” polymers. These comprise non-crosslinked poly-branched polymers prepared by (1) forming a first set of linear polymer branches by initiating the polymerization of a first set of monomers which are either protected against or non-reactive to branching and grafting, during polymerization, each of the branches having a reactive end unit upon completion of polymerization, the reactive end units being incapable of reacting with each other; (2) grafting the branches to a core molecule or core polymer having a plurality of reactive sites capable of reacting, with the reactive end groups on the branches; (3) either deprotecting or activating a plurality of monomeric units on each of the branches to create reactive sites; (4) separately forming a second set of linear polymer branches by repeating step (1) with a second set of monomers; (5) attaching the second set of branches to the first set of branches by reacting the reactive end groups of the second set of branches with the reactive sites on the first set of branches, and then repeating steps (3), (4) and (5) above to add one or more subsequent sets of branches. Such hyper comb-branched polymers are disclosed in European Patent Publication 0473088A2 which are generally referred to as “dendrigraft polymers”. A representative formula for such hyper comb-branched polymer is:
wherein C is a core molecule; each R is the residual moiety of an initiator selected from a group consisting of free radical initiators, cationic initiators, anionic initiators, coordination polymerization initiators and group transfer initiators; A and B are polymerizable monomers or comonomers capable of withstanding the conditions required for branching therefrom or grafting thereto, at least during the polymerization of the {(A)-(B)} linear polymer chain and during its grafting to a prior {(A)-(B)} branch of the {(A)-(B)} core branch; each G is a grafting component and the designation
indicates that G can attach to either an (A) unit or a (B) unit; n is the degree of polymerization of the indicated generation comb-branches; y is the fraction of B units in the indicated generation branch, and has a value of 0.01 to 1; the superscripts 0, 1 and i designate the comb-branch generation level, with i beginning at “2” and continuing for the number of reiterative branch set generations in the polymer; and at least n0 and n′ are ≧2.
For purposes of clarifying terminology, it should be noted that dense star dendrimers are built by reiterative terminal branching, while hyper comb-branched dendrimers are built by reiterative comb-branching. In dense star dendrimers, subsequent generation branches are attached to the terminal moieties of a previous generation, thus limiting the degree of branching to the functionality of the previous generation terminal moiety, which would typically be two or three. In contrast, by branching oligomers upon prior generation oligomer branches in accordance with hyper comb-branched dendrimer, one can dramatically increase the degree of branching from generation to generation, and indeed can vary the degree of branching from generation to generation.
The dendritic polymers which are believed to be most useful are approximately monodispersed. That is, dendritic polymers in a monodispersed solution in which all of the dendritic polymer molecules are substantially of the same generation, and hence of uniform size and shape, are preferred. Monodispersed solutions of dendrimers are particularly preferred.
The dendritic polymers used have internal and/or terminal functional or chelational groups which are accessible to an antineoplastic agent which is capable of associating with the functional or chelational groups, thereby allowing for the uptake of the antineoplastic agent by the dendritic polymer. Dendritic polymers having anionic terminal functional groups are preferred. Examples of anionic terminal function groups include sulfonates, sulfates and carboxylate groups, with carboxylate or carboxylic groups, including the sodium and potassium salts thereof, being particularly preferred. While not wishing to be bound by theory, it is believed that the advantageous results are obtained because the platin form of the Pt permits it to be active, usually in its hydrated form, in the interior of the dendrimer, and at the cancer cell site it is unloaded or released from the dendrimer in its active form, still believed to be the hydrated form, and then the Pt is able to cross-link with the DNA of the cancer cell and thereby inhibit the proliferation of the cancer cells. When the EPR antineoplastic dendritic polymer conjugate is injected upstream of the tumor mass it will enter easily as it size is controlled by the dendrimer and the tumor has a large incoming blood flow and it will not leak to the surrounding area as the vascular size leaving the tumor is more restricted and the dendrimer size too large for those vessels. Thus a high concentration of the drug is kept in the tumor. [See for example U M. Ohndorf et al., Nature 399, 708-712 (17 Jun. 1999).] While not wishing to be bound by theory, it is believed that the advantageous results are obtained because the antineóplastic agents are positively charged and are attracted to the negative charge of the carboxylic groups on the dendrimer and with a higher osmotic pressure on the outside of the dendrimer than in its interior a shunting of the antineoplastic agent occurs to move it into the interior of the dendrimer.
Encapsulation or entrapment is a chemical or physical interaction based on ionic or any other form of association between two compounds (e.g., covalent bonding, hydrogen bonding, adsorption, absorption, metallic bonding, van der Waals forces between a host molecule (dendritic polymer) and a guest molecule (an antineoplastic agent). Encapsulation can be reversible or irreversible. The dendrimer and antineoplastic agent encapsulation of the present invention defines the dendrimer to act as a host, port, or site for the antineoplastic agent, i.e., especially cisplatin or carboplatin. In the area of polymer chemistry because dendrimers have specific structural related properties, encapsulation is a more defined and accurate term. In contrast, with linear polymers where it is not really clear where the drug is bound or associated to the polymer, the term entrapment is more commonly used.
It is also possible to covalently attach an antineoplastic agent to the dendrimer. This covalent attachment may be directly between the interior surface of the dendrimer and the antineoplastic agent or by means of a linker moiety between the surface of the dendrimer and the antineoplastic agent. Some linkers that may be used are described in U.S. Pat. No. 5,527,524; EP 0353450; EP 0570575; and EP 0296522.
Any surface association between the antineoplastic agent and the dendritic polymer forms via a covalent or ionic interaction, thereby allowing for the antineoplastic agent to be associated with the dendritic polymer surface long enough for a second interaction to occur between the interior amine groups of the dendrimer and the antineoplastic agent. Thus, resulting in an encapsulated conjugate by a charged shunt mechanism, resulting in the encapsulation or uptake of the antineoplastic agent by the dendritic polymer. Thus, dendritic polymers having anionic terminal functional groups are preferred. Examples of anionic terminal function groups include sulfonates, sulfates and carboxylate groups, with carboxylate or carboxylic groups and their salts being particularly preferred.
Examples of suitable dendritic polymers which may be used include poly(amidoamine) dendrimers, especially carboxylate terminated poly(amidoamine) dendrimers, and carboxylate terminated poly(propyleneimine) dendrimers, especially where these carboxylic acid groups have formed salts, especially sodium or potassium.
The generation of the dendritic polymer, and hence the size of the dendritic polymer, which may be utilized may vary considerably. For example, generation 3.5 poly(amidoamine) dendrimers (3.5 PAMAM) are acceptable for this use. However, higher and lower generations are also expected to be useful, but especially the range from generation 3.5 to 7.5 for PAMAM dendrimers, having an ethylenediamine (EDA) core.
The antineoplastic agent can be generally any antineoplastic agent, especially a platin-based analogue, which can be reversibly encapsulated within the dendritic polymer or chelated with a linker to the dendritic polymer surface and which exhibits anti-tumor activity when released from the dendritic polymer. An antineoplastic agent is a therapeutic compound used for the treatment of neoplastic diseases, such as ovarian cancer, lung cancer, testicular cancer, breast cancer, stomach cancer and lymphoma. Examples of antineoplastic agent, as used herein, are as defined before. The preferred antineoplastic agent is a platinum containing compound, e.g., a platin-based analogue. More preferred are cisplatin (cis-diamminedichloroplatinum) [see B. Rosenberg et al., Nature 205, 698 (1965), German Patents 2,318,020 and 2,329,485] and carboplatin (cis-diammine(1,1-cyclobutane-dicarboxylato)platinum) [see U.S. Pat. No. 4,140,707]. Other suitable platinum containing compounds include those having a tetravalent platinum atom bonded to the nitrogen of two amine ligands, which may be the same or different, the amine ligands being in cis conformation with respect to each other, the remaining ligands may be capable of interacting with or being displaced by a functional group of the dendritic polymer. An example of such a compound is cis-diamminedicbloroplatinum. A large number of different analogues of cisplatin have been investigated [see for example; J. Respondek and J. Engel, Drugs Of The Future 21(4), 391-408 (1996) and R. B. Weiss and M. C. Christian, Drugs 46, 360-377 (1993)] and many of these different platinum-derivatives are likely to be useful in the present invention and are included within the term “platin-based analogues”.
Antineoplastic dendritic polymer conjugates may be prepared by dissolving a dendritic polymer in a suitable solvent, such as water, contacting the dissolved dendritic polymer with a dissolved antineoplastic agent under conditions sufficient to cause the antineoplastic agent to associate with the dendritic polymer and to form a dendritic polymer-antineoplastic conjugate. A cisplatin to dendrimer (Generation 3.5 PAMAM, EDA core, dendrimer) molar ratio of 35:1 was used in many of the experiments described in the examples. The ratio of cisplatin molecules to dendritic polymer molecules can vary considerably. Dendritic polymer platinates having a cisplatin to dendritic polymer molar ratio of from about 100:1 to about 1:1 have been evaluated and are expected to provide practical advantages. The antineoplastic agents used are encapsulated within the dendrimer which it is believed to be by using a shunt mechanism whereby the anionic groups (e.g., carboxylate groups) of the dendrimer surface are responsible for a weak dendrimer-antineoplastic agent interaction that allows for the antineoplastic agent to be uptaken by the dendrimer (i.e., primary interaction), possibly proceeding through a reaction between the interior nitrogen groups of the dendrimer and the antineoplastic agent (i.e., secondary interaction). This ionic shunt mechanism results in the encapsulation of the antineoplastic agent within the dendritic polymer. In addition to encapsulation it is also believed that some surface association of the antineoplastic agent with the dendritic polymer may occur.
The antineoplastic dendritic polymer conjugate may be administered to animals, especially humans, in a therapeutically effective amount to treat a malignant tumor in the animal. The antineoplastic dendritic polymer may be administered orally or topically, but are preferably administered parentally, such as by subcutaneous (S.C.) injection, intraperitoneal (I.P.) injection, intravenous (I.V.) injection, intraaterial injection or intramuscular injection. An effective amount of a generation 3.5 poly(amidoamine) dendrimer-cisplatin conjugate in which the cisplatin loading is about 25% by weight (i.e., 25% by weight of the conjugate is cisplatin) has been found to be from about 1 milligram per kilogram (1 mg/kg) of body weight to about 15 milligrams per kilogram (15 mg/kg) of body weight for a mouse (DBA2 or C57) for an intraperitoneal injection. Suitable quantities of various antineoplastic dendritic polymer conjugates which are therapeutically effective in the treatment of various malignant tumors in other animals can be determined through routine experimentation and testing.
It is anticipated that the antineoplastic dendritic polymer conjugates will be effective in the treatment of various malignancies in which cisplatin, carboplatin and other antineoplastic agents as anti-tumor agents have been found to be therapeutically affective, including ovarian cancer, lung cancer, testicular cancer, breast cancer, stomach cancer and lymphoma. Also it is anticipated that the antineoplastic dendritic polymer conjugates could be used in combination therapy with other anticancer agents (i.e., synergistic application). In vitro testing and in vivo testing on mice suggest that the antineoplastic dendritic polymer conjugates, especially the platin-based analogues, are also therapeutically effective in the treatment of melanoma and human lymphoblastic leukemia.
Poly(amidoamine) dendrimers (PAMAM, EDA core) (Sigma) were synthesized according to the method of Tomalia et al., Polymer J., 17, 117-132(4) (1985). Dendrimers of generation 3.5 (COONa) and 4 (NH2) were allowed to interact with cisplatin under stirring conditions at room temperature for 4 hours.
The dendrimer-platinum (Pt) was characterized using the OPDA (calorimetric) assay and AAS (total Pt), GPC (Mw and free Pt), IR and NMR.
Pt Release
To study the rate of Pt release and also dendrimer biodegradation the conjugate was incubated in buffers at pH 7.4 and 5.5 and also in the presence of serum and lysosomal enzymes.
Biological Evaluation
In vitro cytotoxicity was assessed against B16F10 melanoma, CCRF (human lymphoblastic leukemia) and Cor-L23 (human lung) cells using the MTT assay. Dendrimer-Pt and free cisplatin were administered I.P. (days 1,2,3 or day 1 only) to DBA2 or C57 mice bearing I.P. inoculated L1210 or B16F10 tumors (respectively). Alternatively drug was administered I.V. to mice bearing S.C. implanted B16F10 when the tumor reached palpable size (50-100 mm2). Animal weight, tumor size and animal survival were monitored (UK guidelines for animal experiments involving neoplasia were followed.)
Materials
Polyamidoamine (PAMAM, EDA core) Starburst® dendrimers (trademark of The Dow Chemical Company) were purchased from Aldrich (UK) Ltd.
The following examples further illustrate the invention but are not considered as a limitation on the scope of the invention.
Method
Poly(amidoamine) dendrimers (cationic and anionic) of increasing generations were incubated with rat erythrocytes obtained from an adult Wistar rat. The interaction of the dendrimer with the erythrocyte was assessed spectrophotometrically by the detection of released hemoglobin, induced by lysis, with a spectrophotometer at 550 nm. Various concentrations of dendrimer, controls (methanol (BDH)), poly-L-lysine (HBr salt—56.5 KD Mw (Sigma)), and dextran (74 KD Mw (Sigma)) (dissolved in physiologically buffered saline) were incubated with the rat erythrocytes (2% w/v solution) for 1 hour at 37° C., and at 10 rpm (shaking water bath). On completion, the erythrocytes were spun in a centrifuge at 1500×g for 10 minutes to pellet the cells and 100 μl of the supernatant was removed and analyzed on the spectrophotometer after blanking against PBS. The results are expressed in
Result
Cationic dendrimers, except generation 1, were lytic, whereas soluble anionic dendrimers (including PAMAM generation 3.5) were not lytic.
Method
B16F10 cells are an adherent murine melanoma cell line. B16F10 cells were seeded at a density of 1×105 cells per ml (1×104 cells per well) in a 96 well flat bottomed microtitre plate (Costar) in RPMI 1640 tissue culture media (Gibco) supplemented with 10% FCS (Gibco). All cellular growth and cytotoxic incubations were carried out in a cell incubator at 37° C. and 5% CO2 atmosphere.
Cell density was assessed using an improved neurenbrow hemocytometer (Sigma). The cells were washed with PBS twice and fresh RPMI media (supplemented with FCS) was added, and the cells were then seeded in a microtitre plate. The cells were left for 24 hours to recover and re-adhere.
All polymers and controls were dissolved in RPMI media (supplemented with FCS) and then sterilized through a 0.2 μm sterile filter (Acrodisk), the first few microliters of the solution being discarded in the case of adherence of the polymer to the filter membrane. Polymer and controls were then added in increasing concentrations to the cells in the microtitre plate. Some cells were left in media only to act as cellular controls. The methanol and poly-L-lysine were negative controls and the dextran was a positive control. The cells were left in the incubator for 72 hours, and checked occasionally for yeast or bacterial contamination. Five hours prior to the incubation time end point, at 67 hours, 20 μl of MTT was added and the cells left for the final 5 hours. Then cellular media was removed, 100 μl of optical grade DMSO (Sigma) was added and the MTT crystals dissolved. The plates were read in a Titerteck plate reader and the results (OD) are expressed in
Result
Cationic dendrimers were cytotoxic (similar to poly-L-lysine) towards the cell line, while anionic dendrimers (including PAMAM generation 3.5, EDA core) were not cytotoxic (similar to dextran).
Method
CCRF-CEM cells are lymphoblastic leukemia and a suspension cell line, i.e. it grows in suspension. CCRF-CEM cells were seeded at a density of 5×104 cells per ml (5×103 cells per well) in a 96 well V-shape microtitre plate (Costar) in RPMI 1640 tissue culture media (Gibco) supplemented with 10% FCS (Gibco). All cellular growth and cytotoxic incubations were carried out in a cell incubator at 37° C. and 5% CO2 atmosphere.
Cell density was assessed using an improved neurenbrow hemocytometer (Sigma). The cells were centrifuged at 1000×g and resuspended in fresh media (supplemented with FCS) before the cell density was assessed. The cells were then seeded in a microtitre plate. The cells were left for 24 hours to recover and re-adhere.
All polymers and controls were dissolved in RPMI media (supplemented with FCS) and then sterilized through a 0.2 μm sterile filter (Acrodisk), the first few microliters of the solution being discarded in the case of adherence of the polymer to the filter membrane. Polymer and controls were then added in increasing concentrations to the cells in the microtitre plate. Some cells were left in media only to act as cellular controls. The methanol and poly-L-lysine were negative controls and the dextran was a positive control. The cells were left in the incubator for 72 hours, and checked occasionally for yeast or bacterial contamination. Five hours prior to the incubation time end point, at 67 hours, 20 μl MTT was added, and the cells left for the final 5 hours. Then cellular media was removed, 100 μl of optical grade DMSO (Sigma) was added and the MTT crystals dissolved. The plates were read in a Titerteck plate reader and the results (OD) are expressed in
Result
Cationic dendrimers were cytotoxic (similar to poly-L-lysine) towards the cell line, while anionic dendrimers (including PAMAM generation 3.5) were not cytotoxic (similar to dextran).
Method
HepG2 is a hepatocellular carcinoma and is an adherent cell line, i.e. it grows in a monolayer. HepG2 cells were seeded at a density of 1×105 cells per ml (1×104 cells per well) in a 96 well flat bottomed microtitre plate (Costar) in MEM tissue culture media (Gibco) supplemented with 10% FCS (Gibco). All cellular growth and cytotoxic incubations were carried out in a cell incubator at 37° C. and 5% CO2 atmosphere.
Cell density was assessed using an improved neurenbrow hemocytometer (Sigma). The cells were washed with PBS twice and fresh RPMI media (supplemented with FCS) added, the cells were then seeded in a microtitre plate. The cells were left for 24 hours to recover and re-adhere.
All polymers and controls were dissolved in RPMI media (supplemented with FCS) and then sterilized through a 0.2 μm sterile filter (Acrodisk), the first few microliters of the solution being discarded in the case of adherence of the polymer to the filter membrane. Polymer and controls were then added in increasing concentrations to the cells in the microtitre plate. Some cells were left in media only to act as cellular controls. The methanol and poly-L-lysine were negative controls and the dextran was a positive control. The cells were left in the incubator for 72 hours, and checked occasionally for yeast or bacterial contamination. Five hours prior to the incubation time end point, at 67 hours, 20 μl of MTT was added and the cells left for the final 5 hours. The cellular media was removed and 100 μl of optical grade DMSO (Sigma) was added and the MTT crystals dissolved. The plates were read in a Titerteck plate reader and the results (OD) are expressed in
Result
Cationic dendrimers were cytotoxic (similar to poly-L-lysine, MW 56 KDa) towards the cell line, while anionic dendrimers (including PAMAM gen. 3.5) were not cytotoxic (similar to dextran, MW 78 KDa).
Method
0.8207 Gram (6.40×105 mol) of poly(amidoamine) Starburst® dendrimer (generation 3.5, EDA core with carboxylate surface groups) was dissolved in 20 ml of 18Ω water (Barnstead-Nanopure). 0.6601 Gram (2.22×10−3 mol) of cisplatin was dissolved in 300 ml of 18Ω water (Barnstead-Nanopure). Once the cisplatin was fully dissolved in the water, the dendrimer was added dropwise to the stirred solution of cisplatin, under nitrogen, over a period of 45 minutes (a molar ratio of cisplatin to dendrimer of approximately 35: 1.). The solution was isolated from light and stirred under nitrogen for 24 hours at room temperature. The unreacted or excess cisplatin was removed using Centricon Plus-80 MWCO 5000 (Millipore, Bioseparations). Upon recovery of the retentate, the isolated solution was lyophilized for 48 hours (The Labconco FreeZone 4.5 Lit.) to give the hygroscopic dendrimer-platinate as a fine white powder.
Results
The weight percent was determined at 19.25 wt % Pt.
Method
0.6964 Grams (5.39×10−5 mol) of poly(amidoamine) Starburst® dendrimer (generation 3.5, EDA core) was dissolved in 20 ml of 18Ω water (Barnstead-Nanopure). 0.666 g (1.79×10−3 mol) of carboplatin was dissolved in 300 ml 18Ω water (Barnstead-Nanopure). This mixture was gently heated (35° C.) until the carboplatin was completely in solution Once the carboplatin was fully dissolved in the water, the dendrimer was added dropwise, under nitrogen, and stirred over a period of 45 minutes (a molar ratio of carboplatin to dendrimer of 33:1). The solution was isolated from light and stirred under nitrogen at room temperature for 24 hours. The unreacted or excess carboplatin was removed using Centricon Plus-80 MWCO 5000 (Millipore, Bioseparations). Upon recovery of the retentate, the isolated solution was lyophilized for 48 hours (The Labconco FreeZone 4.5 Lit.) to give the hygroscopic dendrimer-platinate as a fine white powder.
Results
The weight percent was determined at 20.47 wt % Pt.
Method
Poly(amidoamine) Starburst® dendrimers, EDA core, of generation 3.5, 4.5, and 5.5 with sodium carboxylic surface groups were each dissolved in 20 ml of 18Ω water (Barnstead-Nanopure) and then were reacted with cisplatin. A molar ratio of cisplatin to dendrimer of 33:1, 74:1, 127:1, respectively was used. The cisplatin was dissolved in 300 ml of 18Ω water (arnstead-Nanopure) and gently heated (35° C.) until the cisplatin was completely in solution. Once the cisplatin was fully dissolved in the water, the dendrimer was added dropwise, under nitrogen, and stirred over a period of 45 minutes. The solution was isolated from light and stirred under nitrogen at room temperature for a reaction time of 24 hours. The unreacted or excess cisplatin was removed using Centricon Plus-80 MWCO 5000 (Millipore, Bioseparations). Upon recovery of the of retentate, the isolated solution was lyophilized for 48 hours (The Labconco FreeZone 4.5 Lit.) to give the hygroscopic dendrimer-platinate encapsulate as a fine white powder.
Results
The weight percent was determined at 19.25, 16.82, 16.81 wt % Pt for the 3.5, 4.5, and 5.5 poly(amidoamine) generation dendrimers, respectively. The data appears to suggest that the increased generations slightly lowers the rate of platin loading in the dendrimer-platin conjugates probably because of the compact (physical crowding) surface groups. However, even with the use of a poly(amidoamine) dendrimer of generation 5.5 with sodium carboxylate surface groups a loading of 16.81 wt % Pt is obtained. Considering the molecular weight of the PAMAM dendrimers (generation 3.5, MW 12931; 4.5, MW 26252; and 5.5, MW 52913), the weight percent in terms of molar ratio indicates an increased uptake of platin from lower to higher generations.
Method
Reactions were carried out where a poly(amidoamine) Starburst® dendrimer of generation 3.5 with sodium carboxylate surface groups were dissolved in 20 ml of 18Ω water (Barnstead-Nanopure) and reacted with cisplatin at a molar ration of 32:1. The cisplatin was dissolved in 300 ml of 18Ω water (Barnstead-Nanopure) and gently heated (35° C.) until the cisplatin was completely in solution. Once the cisplatin was fully dissolved in the water, the dendrimer was added dropwise, under nitrogen, and stirred over a period of 45 minutes. The solution was isolated from light and stirred under nitrogen at room temperature for reaction times of 4 hours, 24 hours, and 48 hours. The unreacted or excess cisplatin was removed using Centricon Plus-80 MWCO 5000 (Millipore, Bioseparations). Upon recovery of the retentate, the isolated solution was lyophilized for 48 hours (The Labconco FreeZone 4.5 Lit.) to give the hygroscopic dendrimer-platinate encapsulate as a fine white powder.
Results
The weight percent was determined at 5.81, 19.25, 20.26 wt % Pt for the reaction times of 4, 24 and 48 hours, respectively. The data appears to suggest that the longer reaction times (i.e., 24 hours versus 4 hours) favors a higher platin loading in the dendrimer-platin conjugates. However, when the reaction time was increased to 48 hours, platin loading remained essentially unchanged. This indicates that the optimum loading is reached using a 24 hour reaction time.
Method
Poly(amidoamine) Starburst® dendrimers of generation 3.5 with varying surface groups were dissolved in 20 ml of 18Ω water (Bamstead-Nanopure) and were reacted with cisplatin. A molar ratio of cisplatin to dendrimer of 32:1, 16:1, 32:1, respectively was used and surface groups consisted of amine groups, acetamide groups, and extended carboxylic groups, respectively. The extended carboxylic groups were prepared by reacting the generation 3 amine groups with succinic anhydride in DMSO. This involved the formation of an amide goup on the surface amine (e.g., from methylmethacrylate) with a concurrent ring opening of the succinic anhydride to produce extended carboxylic acid groups (e.g., carboxylated surface). The cisplatin was dissolved in 300 ml of 18Ω water (Barnstead-Nanopure) and gently heated (35° C.) until the cisplatin was completely in solution. Once the cisplatin was fully dissolved in the water, the dendrimer was added dropwise, under nitrogen, and stirred over a period of 45 minutes. The solution was isolated from light and stirred under nitrogen at room temperature for a reaction time of 24 hours. The unreacted or excess cisplatin was removed using Centricon Plus-80 MWCO 5000 (Millipore, Bioseparations). Upon recovery of the of retentate, the isolated solution was lyophilized for 48 hours (The Labconco FreeZone 4.5 Lit.) to give the hygroscopic dendrimer-platinate encapsulate as a fine white powder.
Results
These conjugates resulted in very low platin loading, 6.25, 0.98, and , 0.01 wt % Pt, respectively. These data suggest that the first interior amine groups, as well as the carboxylate groups on the surface of the dendrimer, may play a significant role in the uptake of the platinate.
Method
The reaction of cisplatin with dendrimers appears to still contain unbound cisplatin, even after several hours reaction time. Therefore, as a part of the synthetic procedure in the preparation of dendrimer-platinates, the unbound cisplatin must be removed. Operating on the general premise that the cisplatin was covalently attached to the dendrimer (however later known not to be the case in several preparations) a number of commercially available ultrafiltration devices were tested as a means to remove the unbound cisplatin. Specifically tested were: (1) an Amicon stirred cell with a 3000 MWCO or a 10000 MWCO membrane where fluid is forced through the membrane under nitrogen pressure, (2) Amicon Centriprep devices with 3000 MWCO filters where fluid is forced through the membrane by centrifugation, and (3) Amicon Centricon plus devices with 5000 MWCO filters where fluid is forced through the membrane by centrifugation. Theoretically, the retentate can be repeatedly washed in these devices thus continuously diluting out any remaining cisplatin. The dendrimer-cisplatin conjugates of this example were synthesized in a similar manner as before, and all the reaction reported used the same lot of dendrimer, the same ration of cisplatin to dendrimer, and the same reaction times.
Results
The retention of cisplatin by the dendrimer decreased with increased purification times and increased wash volumes. The results are summarized in the table below. The results indicate that the weight percent of platinum found in the final product depends on the filtration technique, as well as the volume of solvent used to wash the retentate in the purification technique. The practical significance of these date is as follows: (1) the data strongly suggest that there is a significant loss of platin from the conjugate during the purification process, thereby indicating that the platin is not irreversibly or covalently bound to the dendrimer; and (2) the loss of platin during filtration may roughly approximate the rate of release. This result is consistent with a reversible encapsulation of the cisplatin rather than a surface attachment.
Influence on the purification technique on Pt loading of the dendrimer-platinate
SC = Stirred Cell,
CTP = Centiprep
It also appears from these data that a fast wash of the conjugate does remove the unbound surface Pt and perhaps also some bound surface Pt; however, a long wash will cause the encapsulated Pt to be removed from the interior of the dendrimer.
Method
Known amounts of cisplatin and dendrimer were placed in two buffered solutions 25 (PBS at pH 7.4 and Citrate-Phosphate at pH 5.5) to simulate different biological compartments (the plasma/extracellular and the lysosomal compartments, respectively). The solution was sealed in a dialysis bag with a MWCO of 10 KD. Then the bag was placed in a container filled with the respective buffered solution. The solutions were then placed in heated water bath at 37° C. At regular intervals, samples from the buffer solutions were removed and analyzed in triplicate (over a period of 74 hours). At the end of the experiment, a sample was taken from within the bag. All the samples were analyzed using atomic absorption spectroscopy as described previously.
Result
The amount of platinum released at pH 5.5 was slightly greater than that released at pH 7.4. However, as shown in
Method
Cells were seeded at a density of 1×105 cells per ml (1×104 cells per well) in a 96 well flat bottomed microtitre plate (Costar) in RPMI 1640 tissue culture media (Gibco) supplemented with 10% FCS (Gibco). All cellular growth and cytotoxic incubations were carried out in a cell incubator at 37° C. and 5% CO2 atmosphere.
Cell density was assessed using an improved neurenbrow hemocytometer (Sigma). The cells were washed with PBS twice and fresh RPMI media (supplemented with FCS) added, the cells were then seeded in a microtitre plate. The cells were left for 24 hours to recover and re-adhere. If cells were in a suspension they were spun at 1000×g and resuspended in fresh media.
The dendrimer-platinate and cisplatin were dissolved in RPMI media (supplemented with FCS) and then sterilized through a 0.2 μm sterile filter (Acrodisk), the first few microlitres of the solution being discarded in the case of adherence of the polymer to the filter membrane. Then dendrimer and cisplatin were added in increasing concentrations to the cells in the microtitre plate. Some cells were left in media only to act as cellular controls. The cells were left in the incubator for 72 hours, and checked occasionally for yeast or bacterial contamination. Five hours prior to the incubation time end point, at 67 hours, 20 μl of MTT was added and the cells left for the final 5 hours. Then cellular media was removed and 100 μl of optical grade DMSO (Sigma) was added and the MTT crystals dissolved. The plates were read in a Titerteck plate reader and the results (OD) are expressed in
Result
The dendrimer-platinate was less cytotoxic than the cisplatin alone by several orders of magnitude.
Method
Li210 or B16F10 cells were injected at a cell density into a mouse (DBA2 or C57 respectively, 25 g) at a cell density of 1×105 (0.9% saline solution) into the intraperitoneal (I.P. 100 μl) cavity. Twenty-four hours later, the dendrimer-platinate and cisplatin (on one day or on three consecutive days) were injected at a concentration according to the weight of the mouse (e.g. 1 mg/kg-15 mg/kg). The mouse body weight and general toxicity was also monitored according to UK guidelines in the use of animals used in neoplasia studies. At the end point the gross morphology of the organs was noted.
Result
This pharmacology demonstrated the maximum tolerated does of the dendrimer-platinate (25-50 mg/kg). As shown in
Method
B16F10 cells were injected at a cell density of 1×105 (0.9% saline solution) into the left or right flank of the C57 mouse S.C. The mouse was then left until the tumor was visible at a palpable size of between 50-100 mm2. Then the platin-based analogue dendrimer and cisplatin were injected I.V. into the tail vein at the respective doses. The animal was monitored and the tumor size measured using calipers and recorded on a daily basis. When the animal tumor size was between 300-400 mm2, the animal was culled. The tumor excised and weighed and gross morphology of the organs noted.
Result
The platin-based analogue dendrimer was active against the S.C. tumor and demonstrated a significant difference in the final tumor weight and survival time, as shown in
Method
C57 mice were injected S.C. with B16F10 cells at a cell density of 1×105 cells per mouse. The tumor was allowed to reach a palpable size before injecting the platin-based analogue dendrimer I.V. At specific time points (0-24 hours) the animal was culled and key organs (liver, kidney, and blood) including the tumor were isolated and weighed. The organs were solubilized in concentrated nitric acid (10M) and hydrogen peroxide added to decolorize the solution during boiling. The solutions were made up to a fixed volume (25 ml) and then analyzed using AAS after addition of lanthanum (La) (excess) to free up bound platinum (Pt).
Result
Compared to cisplatin alone, the platin-based analogue dendrimer was found to accumulate preferentially in the tumor by at least 3×, relatively quickly after the injection. The results are shown in
Conclusion
Antineoplastic dendritic polymer conjugates have greater water solubility than cisplatin, were 3-5 times less toxic, and have greater anti-tumor activity in vivo.
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.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US01/48568 | 10/29/2001 | WO | 4/21/2004 |
