[go: up one dir, main page]

EP1501522A2 - Vorblockierung mit nicht-ha gags erhöht die wirksamkeit von ha-konjugierten krebsmitteln - Google Patents

Vorblockierung mit nicht-ha gags erhöht die wirksamkeit von ha-konjugierten krebsmitteln

Info

Publication number
EP1501522A2
EP1501522A2 EP03750067A EP03750067A EP1501522A2 EP 1501522 A2 EP1501522 A2 EP 1501522A2 EP 03750067 A EP03750067 A EP 03750067A EP 03750067 A EP03750067 A EP 03750067A EP 1501522 A2 EP1501522 A2 EP 1501522A2
Authority
EP
European Patent Office
Prior art keywords
cancer
derivative
agent
dox
hyaluronic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03750067A
Other languages
English (en)
French (fr)
Other versions
EP1501522A4 (de
Inventor
Glenn D. Prestwich
Lurong Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Georgetown University
University of Utah Research Foundation Inc
Original Assignee
University of Utah Research Foundation Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2002/014402 external-priority patent/WO2002090390A1/en
Application filed by University of Utah Research Foundation Inc filed Critical University of Utah Research Foundation Inc
Publication of EP1501522A2 publication Critical patent/EP1501522A2/de
Publication of EP1501522A4 publication Critical patent/EP1501522A4/de
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • a major challenge in cancer therapy is to selectively deliver small molecule anti- cancer agents to tumor cells.
  • One ofthe most promising methods involves the combination or covalent attachment ofthe cytotoxin with a macromolecular carrier ⁇
  • Many kinds of drug carriers including soluble synthetic and natural polymers , liposomes , microspheres 4 , and nanospheres 5 ' 6 have been employed to increase drug concentration in target cells.
  • soluble synthetic and natural polymers including soluble synthetic and natural polymers , liposomes , microspheres 4 , and nanospheres 5 ' 6 have been employed to increase drug concentration in target cells.
  • a sustained therapeutic concentration can be maintained at tolerable doses.
  • Water-soluble polymer-anti-cancer drug conjugates seem to offer great potential because they can traverse compartmental barriers in the body 7 and therefore gain access to a greater number of cell-types.
  • a variety of water-soluble polymers such as human serum albumin (HSA) , dextran , lectins , poly(ethylene glycol) (PEG) I0 , poly(styrene-co-maleic anhydride) (SMA) n , poly(N-hydroxylpropylmethacrylamide) (HPMA) n , and poly(divinylether-co-maleic anhydride) (DINEMA) 13 have been used to prepare polymeric anti-cancer prodrugs for cancer treatment.
  • HSA human serum albumin
  • PEG poly(ethylene glycol)
  • SMA poly(styrene-co-maleic anhydride)
  • HPMA poly(N-hydroxylpropylmethacrylamide)
  • DINEMA poly(divinylether-co-maleic anhydride) 13
  • poly (styrene-co-maleic acid)-neocarzinostain conjugate (SMA ⁇ CS) was approved for the treatment of liver cancer in Japan ⁇ ' 14 .
  • the linking of doxorubicin to HPMA gives a new prodrug with improved in vitro tumor retention, a higher therapeutic ratio, and avoidance of multi-drug resistance 12 .
  • This system has passed the Phase I clinical trial and is currently in Phase II trials against ovarian cancer 15 .
  • the conjugate of HPMA copolymer-camptothecin was also pre-clinically evaluated and is now in Phase I .
  • Anti-cancer polymer-drug conjugates can be divided into two targeting modalities: passive and active.
  • the biological activity ofthe passive targeting drug delivery systems is based on the anatomical characteristics of tumor tissue, and allows polymeric prodrugs to more easily permeate tumor tissues and accumulate over time. This is one ofthe chief reasons for the success of polymeric drugs, it is often referred to as the enhanced permeability and retention (EPR) effect. Maeda proved that macromolecules can accumulate more efficiently in solid tumors than free drugs n .
  • Active targeting drug delivery systems can be achieved using specific interactions between receptors on the cell surface and the introduction of targeting moieties conjugated to the polymer backbone. In this way, active therapeutic agents conjugated to polymers can be selectively transported to tumor tissues.
  • the active approach therefore takes advantage ofthe EPR effect, but further increases therapeutic index through receptor-mediated uptake by target cancer cells.
  • N-acylated galactosamine 18 and monoclonal antibodiy fragments 19 were valuable targeting moieties for HPMA-DOX conjugates, selectively increasing the cytotoxicity ofthe polymer-drug conjugates to tumor cells.
  • Hyaluronic acid also known as hyaluronan, Figure 1
  • HA Hyaluronic acid
  • Figure 1 a linear polysaccharide of alternating D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (Glc ⁇ Ac) units, is present in the extracellular matrix, the synovial fluid of joints, and the scaffolding that comprises cartilage 20 .
  • It is an immunoneutral building block for preparing biocompatible and biodegradable biomaterials 21"25 , and has been employed as both a vehicle and an angiostatic agent in cancer therapy 26 ⁇ 28 .
  • Mitomycin C and epirubicin were coupled to HA by carbodiimide chemistry and the HA-mitomycin adduct was selectively toxic to a lung carcinoma xenograft 29 .
  • Taxol® bioconjugate 30 ' 31 has been described, which showed good selectivity in cell culture studies. It is evident that directly correlates uptake with cytotoxicity using a fluorescently-labeled HA-Taxol® derivative, and it was demonstrated that toxicity is due to hydrolytic release ofthe parent drug.
  • HA serves a variety of functions within the extracellular matrix, including direct receptor-mediated effects on cell behavior. These effects occur via intracellular signaling pathways in which HA binds to, and is internalized by, cell surface receptors.
  • HA binding proteins Several cell membrane-localized receptors (HA binding proteins) have been identified including: CD44, RHAMM, INd4, and the liver endothelial cell clearance receptor 32"35 .
  • HA-protein interactions play crucial roles in cell adhesion, growth and migration 36"38 , and HA acts as a signaling molecule in cell motility, inflammation, wound healing, and cancer metastasis .
  • the structure and regulation of HA receptors ° is a growing area of structural and cellular biology that is critical to understanding how HA-protein interactions enhance metastasis.
  • HA 41 Most malignant solid tumors contain elevated levels of HA 41 , and these high levels of HA production provide a matrix that facilitates invasion 42 .
  • high HA levels correlate with poor differentiation and decreased survival rate in some human carcinomas.
  • HA is an important signal for activating kinase pathways 43 ' 44 and regulating angiogenesis in tumors 45 .
  • HA intemalization is mediated via matrix receptors, including CD44, which is a transmembrane receptor that can communicate cell-matrix interactions into cells and can alter the matrix in response to intracellular signals.
  • CD44 matrix receptor that is a transmembrane receptor that can communicate cell-matrix interactions into cells and can alter the matrix in response to intracellular signals.
  • the pathological enrichment of HA in tumor tissues suggests that manipulation ofthe interactions between HA and its receptors could lead to dramatic inhibition of growth or metastasis of several types of tumor.
  • Antibodies to CD44, soluble forms of CD44 or RHAMM, HAse, and oligomers of HA have all been used effectively to inhibit tumor growth or metastasis in animal models.
  • intemalization of ⁇ H-labeled HA revealed that intracellular degradation of HA occurs within a low pH environment, such as that of lysosome.
  • Targeting of anti-cancer agents to tumor cells and tumor metastases can be accomplished by receptor-mediated uptake of bioconjugates of anti-cancer agents conjugated to HA 29"31 , followed by the release of free drugs through the degradation of HA in cell compartments. Isoforms of HA receptors, CD44 and RHAMM are over-expressed in transformed human breast epithelial cells 47 , human ovarian tumor cells 48 , and other cancers 49 5 0
  • Targeting of drug delivery aims to increase the concentration of drugs in specific tissues such as tumor sites, and to reduce the drug distribution in other tissues, such as the normal organs. Effective targeting can enhance the therapeutic effect and minimize the toxicity of delivered therapeutics.
  • Several approaches have been utilized for this purpose.
  • the antibodies against erbB2, vesicular endothelium growth factor receptor or transferrin have been used to conjugate molecules, such as anti-tumor agents, to target the molecules to cells and tissue expressing the cognate receptors.
  • anti-tumor agents have also been conjugated with biopolymers or lipid to provide a longer circulation time, controlled release and high retention in specific target tissues, such as tumor sites, because the permeability of vessels is higher in tumor than in normal tissues.
  • HA Hyaluronic acid
  • the following in one aspect, relates to compounds comprising an anti-cancer agent, a carrier molecule, and hyaluronic acid or a derivative thereof, wherein the anti-cancer agent, the carrier molecule, and the hyaluronic acid or a derivative thereof are attached to one another via a covalent bond.
  • the following relates to compounds comprising hyaluronic acid and methods of delivery of these compounds related to a blocking step with non-HA GAGs, such as chondroitin sulfate.
  • non-HA GAGs such as chondroitin sulfate.
  • the following also relates to methods of making and using these compounds.
  • Figure 1 shows a tetrasaccharide fragment of HA with the repeating disaccharide units.
  • Figure 2 shows the possible attachments ofthe anti-cancer agent, the carrier molecule, and the hyaluronic acid or derivative thereof to one another.
  • Figure 3 shows a synthesis of HA-DOX conjugates.
  • Figure 4 shows a structure of HPMA-HA-DOX conjugates.
  • Figure 5 shows data for an In vitro cytotoxicity of HPMA-HA-DOX conjugates against HBL-100 human breast cancer cells. Cell viability of HBL-100 cells as function of DOX equivalent concentration. The cytotoxicity of polymer conjugates (targeted and non- targeted) were determined using MTT assay.
  • Figure 6 shows a binding of targeted HPMA-HA-DOX conjugate on human ovarian cancer SK-ON-3 cells surface, (a) transmission image; (b) fluorescence (50 ⁇ g/ml HA equivalent of HPMA-HA-DOX at 0°C for 2hr).
  • Figure 7 shows a time course of intemalization of targeted HPMA-HA-DOX conjugates (50 ⁇ g/ml HA equivalent) on human ovarian cancer SK-ON-3 cells in comparison with non-targeted HPMA-DOX conjugate.
  • Figure 8 shows in vitro cytotoxicity of DOX, non-targeted HPMA-DOX conjugate, targeted HPMA-HA-DOX with 17% and 36% HA loading against human prostate cancer cell-line DU-145.
  • Figure 9 shows that HA-Taxol effectively reduce the growth of tumors in mice model.
  • the 4T1 mouse breast cancer cells (10 6 /site) were subcutaneously injected into either BABL/c mice and allowed to grow for 2 days for the tumor to be established. Then, the mice bearing with tumor were randomly divided into three groups (5 mice/group) and i.p. injected with 0.4 ml of : 1) saline alone as vehicle control; 2) 4 mg/kg of Taxol; or 3)HA-Taxol containing Taxol equal to 4 mg/kg, respectively.
  • the injection was carried out every other day for two to four weeks.
  • the tumor sizes were measured twice a week. At the end of experiment, the mice were sacrificed and the tumor were harvested, photographed and weighted.
  • Figure 10 shows results with other tumor models.
  • the human TSU bladder cancer cells were subcutaneously injected into flank of nude mice and the treatment procedures were similar to the experiment carried out with 4T1 tumor model.
  • Results from the TSU tumor model were similar to that obtained from 4T1 tumor model showing that mice treated with HA-Taxol had slower tumor growth than those treated with vehicle or Taxol alone.
  • Figure 11 shows that chondroitin sulfate reduces the organ up-take of HA and enhances the tumor up-take of HA.
  • Figure 3 A shows the difference in the up-take of HA between the tumors and major organs (liver, lung, heart, brain, spleen, kidney and muscle), the mice bearing tumors were intravenously injected with 0.2 ml of biosynthesized 3 H-HA (4 x 10 5 cpm ⁇ g HA/ml) and sacrificed 24 hours later. The tumors and organs were collected, weighted, and homogenized with ultrasound to make tissue homogenates. The protein concentration was normalized to 0.1 mg/ml.
  • FIG. 3B shows two groups (5 mice/group) and intravenously injected with 0.3 ml of saline (as control) or chondroitin sulfate (100 mg/ml) to block the binding sites of HA in the major organs. Two hours later, 0.2 ml of biosynthesized 3 H-HA (4 x 10 5 cpm ⁇ g HA/ml) was intravenously injected into mice.
  • FIG. 3 C shows that the H-HA in tumor to major organs in mice pretreatment with chondroitin sulfate was higher than that of untreated mice.
  • Figure 3D shows the results of monitoring the liver for HA up-take for two days the amount of 3 H-HA in the liver of mice treated with chondroitin sulfate was much lower than that in mice treated with vehicle alone.
  • Figure 3E shows the ratio of tumor to liver uptake for treatment of animals with or without a pretreatment of chondroitin sulfate.
  • Figure 12 shows that the pre-treatment with chondroitin sulfate enhances the therapeutic effect of HA-Taxol.
  • Mice bearing 4T1 tumors received i.p injections of 0.4 ml of either saline (as control) or chondroitin sulfate (100 mg/ml) followed by HA-Taxol (8 mg/ml) two hours later. This procedure was carried out every other day for 20 days, and mice received a total often injections. The mice were recorded for their survival days and the survival rate was calculated.
  • Figure 13 shows HA conjugated uptake of TSU and 4T1 cells.
  • Figure 14 shows the interaction of HA with tumor cells CD44, including expression of DC44 in 4 TI cells, binding to H-HA, and degradation of H-HA. Also shown is that the CD44 mediated degradation of H-HA could be inhibited by excess cold HA, anti-CD44, neutralization antibody (KM201) and lysosomal inhibitor chloroquinone.
  • Figure 15 shows the in vivo distribution of HA. Both tumor and lymph nodes contain the highest amount of HA as compared to other organs.
  • Figure 16 shows 4T1 primary tumor and lymph node metastases. Both the popliteal lymph node and inguinal lymph node metastases are shown.
  • Figure 17 shows the results from pathohistological analysis, showing that while the lymph nodes from the control group had spontaneous metastases, there was no tumor cells detected in the HA-Taxol treated group.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use ofthe antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • Free anti-cancer agents typically enter cells via passive, or non-energy-requiring, mechanisms. This can lead to loss of drug efficacy as a result ofthe action ofthe evolution ofthe multidrug resistance gene (MDR) due to the P-glycoprotein product, which pumps free drugs out ofthe cell.
  • MDR multidrug resistance gene
  • Polymeric drugs enter cells by pinocytosis or endocytosis rather than membrane fusion, and polymeric drags are less susceptible to inducing MDR.
  • Polymeric drugs also exhibit enhanced permeability and retention (EPR), e.g., the leaky vasculature of tumors allows macromolecular drugs to "concentrate" in the tumor tissues.
  • EPR permeability and retention
  • the EPR effect improves targeting to malignant cells over normal cells; however, the macromolecular drugs have reduced overall cytotoxicity to all cells relative to the free drug.
  • polymeric (macromolecular) drugs have reduced systemic side effects relative to the free drug.
  • the cytotoxicity to cancer cells can be enhanced, without increasing toxicity to normal cells, by using a targeting agent, e.g., an antibody to a tumor antigen. These compounds possess these attributes, increasing the delivery of anticancer agents.
  • the disclosed compositions enhances both the targeting to a specific cell as well as the uptake by the targeted cancer cells relative to other targeting strategies for small molecule or macromolecular anticancer drugs.
  • the anti-cancer agent, the carrier molecule, and the hyaluronic acid or derivative thereof can be attached to one another by a covalent bond.
  • a non-limiting set of exemplary linkages are depicted in Figure 2.
  • X is the tethered moiety ofthe anti-cancer agent
  • Y is the tethered moiety ofthe carrier molecule
  • Z is the tethered moiety of hyaluronic acid or the derivative thereof.
  • a “tethered moiety" can be any portion of a starting molecule that becomes a portion of a molecule produced in a reaction with the starting molecule.
  • hyaluronic acid could be depicted as Z-COOH. If Z-COOH was reacted with another molecule, such as A, and the product formed from this reaction was Z-A, then Z would be considered a tethered moiety .
  • Z' would also be considered a tethered moiety .
  • Z-COOH reacts with a dihydrazide to produce a derivative of hyaluronic acid
  • Z remains the same and is part of the derivatized hyaluronic acid.
  • Z is the tethered moiety ofthe original hyaluronic acid.
  • the anti-cancer agent, the carrier molecule, and the hyaluronic acid or derivative thereof can be directly attached to one another.
  • the anti-cancer agent and/or hyaluronic acid or derivative thereof are directly attached to the carrier molecule via a covalent bond ( Figures 2(a) and (b), respectively).
  • the anti-cancer agent is directly attached to the carrier molecule via a covalent bond
  • hyaluronic acid or derivative thereof is directly attached to the anti-cancer agent residue.
  • hyaluronic acid or a derivative thereof is directly attached to the carrier molecule via a covalent bond
  • the anti-cancer agent is directly attached to the hyaluronic acid or derivative thereof.
  • the anti-cancer agent, carrier molecule, and the hyaluronic acid or a derivative thereof can be indirectly attached to one another by a linker.
  • a linker L denotes the residue ofthe linker
  • linkers include, but are not limited to, succinates, disulfide-containing compounds, and diol-containing compounds.
  • the linkers may also include short peptides with specific targeting sequences for lysosomes and for lysosomal degradation, such as Gly-Phe-Leu-Gly.
  • Other examples include, for prostate cancer, linkages targeted to prostate cells and to a prostate-specific antigen (PSA), which has sequence-specific proteolytic capabilities.
  • PSA hydrolyzes His-Ser- Ser-Lys-Leu-Gln and glutaryl-4-hydroxyprolyl-Ala-Ser-cyclohexaglycyl-Gln-Ser-Leu.
  • the linkers are typically cleavable so that the anti-cancer agent can be released, for example, under reducing conditions, oxidizing conditions, or by hydrolysis of an ester, amide, hydrazide, or similar linkage forms the covalent bond between the linker and the anti-cancer agent.
  • the type of linker may augment the selective cytotoxicity (and thus improve the therapeutic index) aspect by permitting selective release ofthe anticancer agent inside the cells targeted by the targeting moiety (carrier molecule or HA).
  • an anti-cancer agent to hyaluronic acid or a derivative thereof that is indirectly attached to the carrier molecule via a linker. Additionally, it is possible to attach hyaluronic acid or a derivative thereof to an anti-cancer agent that is indirectly attached to the carrier molecule via a linker.
  • the anti-cancer agent and hyaluronic acid or a derivative thereof can be attached to one another via a linker molecule.
  • Figures 2(i) and (j) depicted in Figures 2(i) and (j).
  • the anti-cancer agent and the hyaluronic acid or derivative thereof, respectively are directly attached to the carrier molecule.
  • the anti-cancer agent, the carrier molecule, and the hyaluronic acid or derivatives thereof used to produce the compounds are discussed below.
  • Disclosed herein are methods wherein the HA-anticancer agent-carrier molecule are administered after the or concurrently or before the addition of a non-HA GAG or derivative, which can act as a blocking agent for non-specific HA interactions.
  • GAGs glycosaminoglycans
  • non-HA preblocking agents can be CS-A (chondroitin 4-sulfate) and CS- C (chondroitin 6-suflate), heparin, heparin sulfate, dextran sulfate, keratan, or keratan sulfate.
  • CS-A chondroitin 4-sulfate
  • CS- C chondroitin 6-suflate
  • heparin heparin sulfate
  • dextran sulfate keratan
  • keratan sulfate keratan sulfate
  • Preadministration ofthe non-HA GAG, such as CS by oral or iv dosing to achieve an adequate serum level (approximately 5 to 500 ug/ml) can protect non-targeted organs, especially the liver.
  • the non-HA agents can be added prior to the therapeutic composition, concurrently with the therapeutic composition, or after the therapeutic composition. It is understood that their effectiveness can vary depending on the amount of time that the blocking step can occur. For example, if the blocking step occurs before the addition ofthe HA therapeutic composition, then more effective blocking can occur, however, as the therapeutic reagent is taken up over time, some benefit of blocking can be achieved even if the blocking agent is added after the administration ofthe therapeutic composition. 2. Anti-cancer Agents
  • any anti-cancer agent can be directly or indirectly attached to the carrier molecule and the hyaluronic acid or derivatives to be aided in transport across the cellular membranes.
  • the anti-cancer agent is any small molecule that targets intracellular function, such as protein kinase inhibitors including but not limited to Gleevac.
  • radionuclides including, but not limited to, 1-131, Y-90.
  • In-111, Tc-99m can be used.
  • Gd+3 compounds can be used.
  • meso e- chlorin and cis-platin derivatives can be used as the anti-cancer agent.
  • anticancer agents that can be used with the disclosed compositions can be found in, for example, United States Patent No. 5,037,883, which is herein incorporated by reference as well as any publications and patents, or patent applications, cited therein which contain anti-cancer agents.
  • Other anti-cancer agents such as, cytotoxic agent, a chemotherapeutic agent, a cytokine, antitubulin agents, and a radioactive isotope, can also be used in the disclosed compounds.
  • Anticancer agents such as, vincristine, vinblastine, vinorelbine, and vindesine, calicheamicin, QFA, BCNU, streptozoicin, and 5-fluorouracil, neomycin, podophyllotoxin(s), TNF-alpha, .alpha v beta 3 colchicine, taxol, , a combretastatin antagonists, calcium ionophores, calcium-flux inducing agents, and any derivative or prodrug thereof can also be used herein.
  • United States patent Nos. 6,348,209, 6,346,349, and 6,342,221 are also disclosed for agents related to anti-cancer compounds.
  • the anti-cancer agent comprises 5-fluorouracil, 9-aminocamptothecin, or amine-modified geldanomycin.
  • the anti-cancer agent is doxorabicin.
  • the anticancer agent can be Taxol®.
  • anti cancer agents such as the anti-growth factor receptor antibodies (e.g., Herceptin), are understood to not typically have a need for transport across a cell membrane, and therefore, would typically be used in combination with the disclosed compounds and compositions.
  • Carrier Molecules Any carrier molecule can be used. Typically carrier molecules will be polymer molecules. Typically the carrier molecule is a large macromolecule of at least 5,000 daltons. The carrier molecule can range from 2,000 daltons to 25,000 daltons, or from 25,000 daltons to 100,000 daltons, or from 100,000 daltons to 1,000,000 daltons. It is preferred that the carrier molecule be in the range of 10,000 to 25,000 daltons. The carrier molecule typically aids in the transport of anti-cancer agent across the cell membrane.
  • the anti-cancer agent when directly or indirectly attached to the carrier molecule it typically crosses a cell membrane better than the anti-cancer agent alone.
  • carriers and macromolecular carriers known in the art that will function as the carrier molecule. Examples of carrier molecules are also described in, for example, United States Patent Nos: 5,415,864 for "Colonic-targeted oral drug-dosage forms based on crosslinked hydrogels containing azobonds and exhibiting PH-dependent swelling;” 5,258,453 for "Drug delivery system for the simultaneous delivery of drags activatable by enzymes and light;” 5,037,883 for "Synthetic polymeric drags;” 4,074,039 for "Hydrophilic N,N-diethyl acrylamide copolymers;” 4,062,831 for "Copolymers based on N-substituted acrylamides, N-substituted methacrylamides and N,N-disubstituted acrylamides and the
  • the carrier molecule comprises a polymer produced by the polymerization of an ethylenically unsaturated monomer.
  • monomers include, but are not limited to, acrylates and methacrylates.
  • the carrier molecule is a polymer produced from the polymerization of N-(2- hydroxypropoyl)methacrylamide, which is referred to herein as HPMA.
  • Hyaluronic Acid and Derivatives Thereof There are many uses and derivatives of Hyaluronic acid (HA), which is a macromolecule. HA and derivatives of HA are conjugated to molecules, such as anticancer agents. HA, derivatives of HA, their uses and synthesis are disclosed in, for example, see United States Patent ⁇ os: 6,096,727 for "Method for treating wounds using modified hyaluronic acid crosslinked with biscarbodiimide," 6,013,679 for "Water-insoluble derivatives of hyaluronic acid and their methods of preparation and use," 5,874,417 for "Functionalized derivatives of hyaluronic acid," 5,652,347 “Method for making functionalized derivatives of hyaluronic acid,” 5,616,568 “Functionalized derivatives of hyaluronic acid” 5,502,081 "Water-insoluble derivatives of hyaluronic acid and their methods of preparation and use, " as well as United States Provision
  • the hyaluronic acid is modified with a dihydrazide compound such as adipic dihydrazide.
  • Hyaluronic acid is a polysaccharide of at least 4 disaccharide repeat units of HA, e.g., at least 1,000 daltons. HA and derivatives thereof can range from 1,000 daltons to
  • HA and its derivatives be at least 1,000 daltons.
  • the lower limit ofthe molecular weight is, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000
  • the upper limit is 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000, where any lower limit can be combined with any upper limit.
  • Hyaluronic acid typically aids in the transport ofthe anti-cancer agent across the cell membranes through an active mode of transport.
  • Hyaluronic acid typically aids in the transport ofthe anti-cancer agent across the cell membranes through an active mode of transport.
  • HA is a linear polysaccharide with alternating repeats of D-glucuronic acid and N- acetyl-D-glucosamine.
  • the conjugation of small anti-tumor drug with HA will avoid the quick clearness by kidney and confer a long circulating time to new derivative.
  • CD44 one of hyaluronan (HA) surface receptor, is highly expressed in variety of tumors which will facilitate the taking up of HA-drugs.
  • HA hyaluronan
  • the disclosed compounds can be characterized in that they allow for the uptake of anti-cancer agents by cells using typically different mechanisms than used by the anticancer agent alone. This efficiency can be measured in a number of ways. There are many ways to determine whether the efficiency and/or specificity ofthe uptake is increased by hyaluronic acid and/or the carrier molecule. For example, one can block the HA mediated transport and look at the change in saturation ofthe cells. One can do this by performing the assays with saturating HA present, using HA specific antibodies which block the HA function, using cells without HA receptors, and using cells that over express HA receptors like cancer cells. Typical increases of efficiency and/or specificity can be greater than or equal to at least 2 fold, 5 fold, 10 fold, 25 fold, 50 fold, 100 fold, 500 fold, 1000, fold 5000 fold or 10,000 fold.
  • the compounds have greater specificity for uptake and retention in the targeted cancer cells. This increased specificity is consistent with the specific hyaluronic acid receptors which import hyaluronic acid into cells.
  • Typically disclosed compounds have a 5 to 100 fold greater specificity than either the anti-cancer-carrier molecule or anti-cancer- hyaluronic acid systems.
  • This specificity can be assayed in a number of ways. For example, the intrinsic fluorescence ofthe anti-cancer agent doxorabicin may be observed directly by fluorescence microscopy in anti-cancer agent-carrier molecule systems and the disclosed compounds.
  • the compounds can be prepared using techniques known in the art. As described, there are three components used to produce the compounds: the anti-cancer agent, the carrier molecule, and hyaluronic acid or a derivative thereof. Any ofthe components previously described can be reacted with one another in any possible combination to produce the compounds. Also contemplated is the use of two or more anti-cancer agents, carrier molecules, or hyaluronic acid or its derivatives thereof when producing the compounds. In addition, it is sometimes preferred to couple (i.e., react) two ofthe three components together to produce a new reaction product or intermediate, then chemically connect the intermediate with the third component. For example, the anti-cancer agent can react with the carrier molecule to produce an anti-cancer/carrier molecule.
  • the anti-cancer agent can react with hyaluronic acid or a derivative thereof to produce an anti- cancer/hyaluronic acid molecule, and hyaluronic acid or a derivative thereof can react with the carrier molecule to produce a hyaluronic acid/ carrier molecule.
  • hyaluronic acid or a derivative thereof can react with the carrier molecule to produce a hyaluronic acid/ carrier molecule.
  • Each of these intermediates can be reacted with an individual component (e.g., the reaction of anti- cancer/hyaluronic acid molecule with carrier molecule) or, alternatively, each ofthe intermediates can react with one another to produce the compound (e.g., reaction of anti- cancer/hyaluronic acid molecule with the anti-cancer/carrier molecule).
  • the compound can be produced by (1) reacting the anti-cancer agent with the carrier molecule to produce a carrier/anti-cancer molecule and (2) reacting the carrier/anti-cancer molecule with hyaluronic acid or the derivative thereof.
  • the carrier molecule HPMA is reacted with doxorabicin (DOX) to produce HPMA-DOX, then HPMA-DOX is reacted with hyaluronic acid modified with adipic dihydrazide to produce HPMA-DOX- HA.
  • DOX doxorabicin
  • HPMA-DOX hyaluronic acid modified with adipic dihydrazide
  • the reaction requires compatible reactive functionalities and generally includes a linker connecting the two tetherable moieties.
  • the compound in another embodiment, can be produced by (1) reacting the anticancer agent with hyaluronic acid or the derivative thereof to produce an anti- cancer/hyaluronic acid molecule; (2) reacting the anti-cancer agent with the carrier molecule to produce a carrier/anti-cancer molecule; and (3) reacting the anti- cancer/hyaluronic acid molecule with the carrier molecule/anti-cancer molecule.
  • hyaluronic acid is reacted with doxorabicin to produce HA-DOX, then HA-DOX is subsequently reacted with HPMA-DOX to produce HA-DOX-HPMA.
  • the anti-cancer agent, carrier molecule, and hyaluronic acid can be attached to one another directly or indirectly via a linker.
  • the attachment of each component to one another can vary depending upon the types of components selected and the order in which the components are permitted to react with one another.
  • two or more compounds can be produced simultaneously when the anti-cancer agent, the carrier molecule, and the hyaluronic acid or a derivative thereof are reacted with one another.
  • the molecular weight ofthe carrier molecule and/or the hyaluronic acid or its derivatives will vary for each compound in the composition.
  • the attachment ofthe anti-cancer agent, carrier molecule, and hyaluronic acid or its derivatives to one another may vary from one compound to another in the composition.
  • the anti-cancer agent may be modified once it is attached to the carrier molecule or hyaluronic acid or its derivative thereof Also contemplated is the formation of compositions composed of one or more compounds and free anti-cancer agent. For example, an excess of anti-cancer agent is used relative to the carrier molecule and/or the hyaluronic acid to produce these compositions.
  • An exemplary compound for the disclosed methods is an anti-tumor compound.
  • Taxol 2-OH has been linked via a succinate ester to adipic dihydrazide (ADH)-modified HA. Once this HA-Taxol conjugate is internalized by tumor cells, the active form of Taxol could be hydrolytically released via the cleavage ofthe labile 2' ester linkage.
  • ADH adipic dihydrazide
  • the disclosed compounds can be used for targeted delivery of anti-cancer agents to cells.
  • the disclosed methods also increase the efficiency and specificity of delivery of hyaluronic acid (HA) containing compounds and compositions. These compounds and compositions can be used thus, to treat a variety of disorders that require the delivery of anti-cancer or similar agents. It is understood that any ofthe compounds disclosed can be used in this way.
  • HA hyaluronic acid
  • the compounds will be administered in pharmaceutically acceptable forms and in doses wherein delivery occurs. Typically the compounds would be administered to patients in need of delivery ofthe anti-cancer agent or a similar compound. It is understood that the goal is delivery ofthe compound and that through delivery affect the cells ofthe patient in need ofthe anti-cancer agent or similar agent.
  • the conjugated anti-cancer agents can be given to a subject.
  • Any subject in need of receiving an anti-cancer agent can be given the disclosed conjgated anticancer agents.
  • the subject can, for example, be a mammal, such as a mouse, rat, rabbit hamster, dog, cat, pig, cow, sheep, goat, horse, or primate, such as monkey, gorilla, orangutan, chimpanzee, or human.
  • the conjugated anti-cancer agents can be used for inhibiting cancer cell proliferation.
  • Inhibiting cancer cell proliferation means reducing or preventing cancer cell growth.
  • Inhibitors can be determined by using a cancer cell assay. For example, either a cancer cell line can be cultured on 96-well plates in the presence or absence ofthe conjugated anti-cancer agent or anti-cancer agent alone or anti-cancer agent prepared differently then the disclosed compositions (for example, just anticancer agent and carrier) for any set period of time. The cells can then be assayed.
  • the conjugated anti-cancer compounds are those that will inhibit 10%> or 15%> or 20%> or 25%> or 30% or 35% or 40% or 45% or 50% or 55% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% ofthe cells growth relative to any ofthe controls as determined by the assay.
  • compositions which inhibit metastatic tumor formation in this type of assay disclosed herein as well as compositions that reduce metastatic tumor formation by at least 10% or 15% or 20% or 25% or 30% or 35% or 40% or 45% or 50% or 55% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% of a control compound.
  • conjugated anti-cancer agents can be administered to cells and/or cancer cells which have HA receptors.
  • HA-Taxol Disclosed herein is the in vivo effect of HA-Taxol.
  • Two highly tumorigenic cancer cell lines 4T1 mouse breast cancer and TSU human bladder cancer, were examined for their abilities to bind, internalize and degrade HA via their functional CD44. These cells were used to form tumor xenograft models in mice, then treated with HA-Taxol via i.v. injection.
  • the biosynthetically labeled 3 H- HA was i.v. injected into mice bearing tumors and 24 hours later, the radioactivity of 3 H- HA in the homogenates from tumors and different organs were determined with ⁇ -counter.
  • CS chondroitin sulfate
  • Disclosed are methods comprising administering a blocking agent prior to the addition of an HA conjugated molecule, such as an HA conjugated anti-cancer agent.
  • the blocking agent can be administered to the organism concurrently, at least 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150minutes, 180 minutes, 240 minutes, 300 minutes, or 360 minutes, 420 minutes, 10 hours, or 15 hours prior to the addition ofthe HA conjugated molecule.
  • the blocking agent is added such that receptors capable of binding the blocking agent can bind the blocking agent.
  • preadministration of CS by oral or iv dosing to achieve an adequate serum level can protect non-targeted organs, for example, the liver, kidney, or the lymph nodes, unless these are tumor containing tissues.
  • the blocking agent can be added concurrently or even after the addition ofthe HA agent. This is arises because the administration ofthe HA does not occur immediately, and therefore, some benefit of adding a blocking agent can occur thus, even if the blocking agent has not been added prior to the addition of the HA agent. It is understood, however, that the longer the blocking agent is added after the addition ofthe HA agent, the less effective the blocking agent will be, however, even significant delay can have some effect as competition for the non-specific sites can occur, releasing bound or inactivated HA-agent. It certain embodiments the blocking agent is added within 1 hour, 2 hours, 4, hours, 8 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, or 7 days of adding the HA agent.
  • compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers.
  • a non-limiting list of different types of cancers is as follows: lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumours, myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers in general.
  • a representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas ofthe mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, or pancre
  • Compounds disclosed herein may also be used for the treatment of precancer conditions such as cervical and anal dysplasias, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.
  • precancer conditions such as cervical and anal dysplasias, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.
  • Metastasis the leading death cause of breast cancer, mainly starts from lymph path.
  • the surgical therapy can remove the primary tumor and some metastatic lymph nodes, however, in most cases, the lymphatic metastases are so spread out and so difficult to find that they can not be all removed.
  • the residual lymphatic metastases of breast cancer are treated mainly by irradiation or/and chemotherapy.
  • these two approaches have the disadvantages: 1) they target systemically or a board range of local tissues, not specific in the tumor sites; 2) they cause the adverse side effects, such as systemically impairing the body immunity and the regeneration of blood cells which results in life-threatening infections, and locally destroying the normal tissue structure that results in the permanent damages (such as scaring, arm/hand edema).
  • the current therapy still can not control progression of breast cancer metastasis and the survival rate is not dramatically improved as compared to that of ten years ago.
  • HA Due to its large molecular weight, HA mainly enters the lymph path, and is taken up by endothelial cells which express high level of CD44, the native high affinity receptor for
  • Lymphatic fluid contains a large amount of HA, which appears to serve as a chemo- attract force for lymphocytes, since they have such a high level of CD44 as their "homing receptor" to guide their way back to the lymph node. Cancer cells also utilize the CD44 to make their way to the lymph node. HA plays a critical role in attracting cells to the lymph node. Since the lymph path naturally collects HA, this lymph path specific draining property of HA can be used as a carrier to deliver anti-tumor drug specifically to the lymph path, where the metastatic cancer cells have settled. 2.
  • the dosage ranges for the administration ofthe compounds are those large enough to produce the desired effect in which delivery occurs.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent ofthe disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary from about 1 mg/kg to 30 mg/kg in one or more dose administrations daily, for one or several days.
  • the dose, schedule of doses and route of administration may be varied, whether oral, nasal, vaginal, rectal, extraocular, intramuscular, intracutaneous, subcutaneous, or intravenous, to avoid adverse reaction yet still achieve delivery. 3.
  • Pharmaceutically Acceptable Carriers are provided, whether oral, nasal, vaginal, rectal, extraocular, intramuscular, intracutaneous, subcutaneous, or intravenous, to avoid adverse reaction yet still achieve delivery. 3.
  • Pharmaceutically Acceptable Carriers may be varied, whether oral, nasal, vaginal, rectal, extraocular, intramuscular, intracutaneous, subcutaneous, or intravenous, to avoid adverse reaction yet still achieve delivery.
  • Any ofthe compounds can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • compositions are known to those skilled in the art. These most typically would be standard carriers for administration of compositions to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • the compositionscould also be administered intramuscularly or subcutaneously.
  • Other compounds will be administered according to standard procedures used by those skilled in the art.
  • Molecules intended for pharmaceutical delivery may be formulated in a pharmaceutical composition.
  • Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which may also contain buffers, diluents and other suitable additives.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions as described herein can also be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, gly colic acid, lactic acid, pyravic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, ace
  • CD44 can be activated to function as an hyaluronic acid receptor in normal murine T-cells. Eur. J. Immunol. 22, 2719-2723.
  • Hyaluronan receptors are expressed on human malignant mesothelioma cells but not on normal mesothelial cells. Cancer Res. 54,
  • Tanabe KK Saya H.: The CD44 adhesion molecule and metastasis. Crit Rev Oncog. 1994; 5 (2-3): 201-12.
  • HA-DOX hyaluronic acid
  • DOX dihydroxypropoyl methacrylamide
  • HPMA-HA-DOX N-(2-hydroxypropoyl)methacrylamide
  • HPMA-HA-DOX conjugate was visualized by confocal fluorescence microscopy in comparison to non-targeted HPMA-HA-DOX system, providing compelling evidence for the uptake ofthe targeted conjugates through receptor-mediated pathway.
  • Taxol (HA-Taxol) has a selective toxicity toward several human cancer cell lines.
  • the human TSU bladder cancer cells and mouse 4T1 breast cancer cells expressed HA receptor (CD44) and were capable to bind, internalize and degrade the HA.
  • CD44 HA receptor
  • the inhibitory effect of HA-Taxol was greater than Taxol alone.
  • the results of i.v. injection of 3 H-HA indicated that several normal tissues, especially liver and kidney also had a high capability for taking up HA conjugates.
  • chondroitin sulfate that can bind to some HA binding proteins was injected into mice 2 hours prior to the administration of 3 H-HA. This approach did reduce the HA taken up by liver and increased the amount of HA accumulated in tumors. When mice bearing tumors were treated with CS and then HA-Taxol, their survival time was longer than those treated with vehicle or HA- Taxol alone.
  • mice receiving i.v. injection of 3 H-HA showed that tumors and lymph nodes had the highest concentration.
  • the 3 H-HA up-taken in the blood-enriched peri-tumors was at least 3 times higher than that in the central portion of tumors.
  • 3 H-HA was prepared as previously described with some modification of Dr. Underhill' s method . Briefly, the rat fibrosarcoma cells were cultured in 10 of 100 mm dishes with 10%> fetal calf serum-90%> DMEM to 80%> confluence and then charged to media 2% fetal calf serum-98%> DMEM supplemented with 2 mCi of 3 H-acetate for 2 days. The conditional media was digested with proteinase and dialyzed extensively against distilled water. The biosynthetic 3 H-HA in dialyzed media was precipitated by cetylpryridinium chloride, washed with alcohol and redissolved in saline.
  • the radioactivity ofthe preparation was 5.4 X 10 4 cpm/ ⁇ g HA and the 3 H-HA was 144 ⁇ g/ml.
  • the preparation of 3 H-HA was sterilized with 0.2 ⁇ M filter and stored in -20°C for use. b) Preparation of HA-Taxol
  • Prestwich (Luo Y, Ziebell MR, Prestwich GD: A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells. Biomacromolecules. 2000; 1(2):208-18 ). Briefly, the fermentation-derived HA (Clear Solutions Biotechnology, Inc. Stony Brook, NY) was partially digested by hyaluronidase to size about 12,000 Dalton and dialyzed with a tubing (Mw cutoff 3,500 Da) to get rid of very small Mw of HA. A 5 -fold adipic dihydrazide (ADH) was added to 50 mg of HA to make ADH-HA.
  • ADH adipic dihydrazide
  • Taxol-NHS ester was synthesized with two steps.
  • Taxol®-2'- hemisuccinate was made as following procedure: 38 mg of succinic anhydride was added to 270 mg of Taxol followed by addition of 36 ⁇ l of pyridine. The mixture was stirred at room temperature for 3 days and purified on silica gel (wash with hexane; elute with ethyl acetate). Then, 1.51 g Taxol® -hemisuccinate and 0.83 g of SDPP (N-hydroxysuccinimido diphenyl phosphate) in 30 mL acetonitrile was added with 0.67 ml of triethylamine. The reaction was stirred for 6 h at room temperature and then concentrated in vacuo. The residue was dissolved in 5 ml ethyl acetate and purified on silica gel.
  • Taxol- ⁇ HS ester 345 mg
  • HA- ADH HA- ADH
  • 250 ml water 250 ml water
  • the reaction mixture was stirred at room temperature for 12 days.
  • the purity of HA-Taxol was monitored by GPC analysis. Taxol loading was determined by UN absorbance
  • the loading and transferring of equal amounts of protein were confirmed by staining ofthe membrane with a solution of Ponceau S (Sigma, St. Louis MO).
  • the membranes were blocked with 5%> fat free milk in phosphate buffer saline (PBS, pH 7.4) for 30 min and then incubated overnight with 0.2 ⁇ g/ml of BU52 monoclonal antibody against standard CD44 After washing, the membrane was incubated with peroxidase labeled anti-mouse IgG for one hour, followed by a chemo-luminicent substrate and exposed to ECL Hyperfilm MP (Amersham, Piscataway, NJ).
  • 4T1 and TSU tumor cells were cultured in 24 well plate to 80%> confluence, washed with PBS and lysed with 1 ml of DOC buffer (0.1 % Na deoxycholate, 0.5 M NaCl, 0.02 M Tris-HCl, pH 8.0). The equal amount of lysate (200 ⁇ l) were mixed with or without 100 ⁇ g of HA, and then added 20 ⁇ l (3 ⁇ g) of 3 H-HA. After shaken at room temperature for 30 minutes, 300 ⁇ l of saturated (NH ) SO 4 was added to the reaction tubes, followed by 25 ⁇ l of nonfat milk. The tubes were spun at 12000 rpm for 5 min.
  • DOC buffer 0.1 % Na deoxycholate, 0.5 M NaCl, 0.02 M Tris-HCl, pH 8.0.
  • the equal amount of lysate (200 ⁇ l) were mixed with or without 100 ⁇ g of HA, and then added 20 ⁇ l (3 ⁇ g) of 3 H-HA. After
  • the pellets in the tubes were washed twice with 50%> of (NH 4 ) 2 SO 4 , dissolved in 0.2 ml H O, transferred to scintillation tubes, mixed with 1.2 ml of scintillation solution and counted for the radioactivity with ⁇ - counter.
  • the assay was carried out as described (CBU) with some modification.
  • the cells were treated as described (CBU) with some modification.
  • 80% confluent in 24 well plate were changed to 1 ml fresh media containing 3 H-HA (4 X 10 5 cpm/7.5ug HA/ml) and incubated at 37°C for 48 hours.
  • the media contained 200 ⁇ g/ml of KM201 (a neutralization antibody against CD44) or 0.1 mM of chloroquine (an inhibitor of lysosomal enzymes).
  • the media were collected, which contained some ofthe released and degraded 3 H-HA.
  • the cells were frozen and thawed three times and then spin at 12,000 rpm for 30 min to obtain the degraded 3 H-HA inside cells.
  • the media and the supernatant from cells were centrifuged with Centricon 30 (Amicon, Danvers, MA).
  • the un-degraded high MW 3 H-HA was retained in the upper chamber.
  • the 500 ⁇ l of degraded low MW 3 H-HA passing through the filter membrane was mixed with 6 ml of scintillation solution and counted for the radioactivity.
  • mice 4T1 cells
  • nude mice for TSU cells
  • the tumor bearing mice were randomly divided into three groups and then i.p. injected with 0.4 ml of : 1) saline alone as vehicle control; 2) 4 mg/kg of Taxol; or 3)HA- Taxol containing Taxol equal to 4 mg/kg, respectively.
  • the injection was carried out every other day for two weeks.
  • the tumor sizes were measured twice a week.
  • the mice were sacrificed and the tumor were harvested, photographed and weighted.
  • mice received i.p injections of 0.4 ml of either PBS (as control) or chondroitin sulfate (100 mg/ml) followed by HA-Taxol (8 mg/ml) two hours later. This procedure was carried out every other day for 20 days, and mice received a total often injections. The mice were recorded for their survival during a 33 day experimental period s and the survival rate was calculated.
  • mice tumor bearing with tumor were injected 0.2 ml of 3 H-HA.
  • the tumors and organs were collected, weighed, homogenized with ultrasound to make tissue lysate at a protein concentration of 0.1 mg/ml.
  • 300 ⁇ l of tissue lysate were mixed with 2.5 ml of scintillation solution and counted for the radioactivity.
  • CD44 mediates the binding and degradation of HA by tumor cells.
  • the expression level of CD44 was examined.
  • CD44 is the HA receptor on the cell surface, and is considered the basic target of HA carried drags.
  • a significantly high amount of CD 44 was expressed by both TSU and 4T1 tumor cells (Fig 9A).
  • the cells were lysed in DOC buffer and 100 ⁇ g of lysate proteins was incubated with 20 ⁇ l of 3 H-HA with or without HA.
  • the results showed that the lysate proteins, including detectable CD44, bound to 3 H- HA, which could be competitively reduced by "cold" HA.
  • HA carried drag could be taken up by CD44 receptor by addition of high MW 3 H-HA to the media of culture cells, and allowed the cells to bind, up- take, and degrade the high MW 3 H-HA to low MW derivatives.
  • This mixture was then separated by filter membrane with MW cut of 30,000 Dalton.
  • This process could be blocked by cold 3 H-HA and by CD44 neutralization antibody, KM201, suggesting that the process is mediated by CD44-HA interaction.
  • the functional lysosomes are required for this process, since the blocking of lysosomal enzymes with chloroquine also reduce the degraded 3 H-HA.
  • HA-Taxol effectively reduce the growth of tumors in mice model: Chondroitin sulfate reduces the organ up-take of HA and enhances the tumor up-take of HA:
  • mice pretreated with chondroitin sulfate can increase the accumulation of HA in tumors, which can be utilized to enhance the therapeutic effect of HA-Taxol.
  • Example 2 Doxyrubicin HA-HPMA
  • Fermentation-derived HA (sodium salt, r 1.5 MDa) was provided by Clear
  • Testicular hyaluronidase (HAse), Dulbecco's phosphate-buffered saline (DPBS) and cell culture media were purchased from Sigma (St. Louis, MO).
  • Doxorabicin (DOX) was a kind of gift from Dr. A. Suarato, Pharmacia-Upjoin, Milano, Italy. Fluorescence images were recorded on a Bio-Rad (Hercules, CA) MRC 1024 laser scanning confocal imaging system based on a Zeiss (Oberkochen, Germany) Axioplan microscope and a krypton/argon laser.
  • HBL-100 a human breast cancer cell-line
  • D-MEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • SK-ON-3 a human ovarian cancer cell-line was cultured in D-MEM/F12 + 10% FBS
  • HCT-116 a colon tumor cell- line, was maintained in culture in ⁇ -MEM (Minimal Essential Medium, Eagle) + 10% FBS.
  • HA was characterized by gel permeation chromatography (GPC) was on the following system: Waters 515 HPLC pump,Waters 410 differential refractometer, and Waters 486 tunable absorbance detector.
  • GPC gel permeation chromatography
  • Waters Ultrahydrogel 250 and 2000 columns (7.8 mm ID x 30 cm) (Milford, MA) were used for GPC analysis, the eluent was 150 mM pH 6.5 phosphate buffer/MeOH 80:20 (v/v), and the flow rate was 0.5 mL/min.
  • the system was calibrated with HA standards supplied by Dr. O. Wik (Pharmacia).
  • HPMA copolymer conjugates were characterized by GPC on a Pharmacia FPLC with Superose analytical column, pH 7.4 PBS buffer was used as eluent with a flow rate of 0.4 ml/min.
  • Cell viability in cell culture was determined by thiazoyl blue (MTT) dye uptake protocols measured at 540 nm, which was recorded on a BIO-RAD M-450 microplate reader (Hercules, CA).
  • Laser scanning confocal microscopy was carried out on a Keller type Bio-Rad MRC 1024 with LASERSHARP acquisition software. Fluorescence images were taken using FITC settings with the 488 nm excitation line and a 522 nm 32 bandpass filter was used to collect the images.
  • LMW HA low molecular weight (LMW) HA and HA hydrazide derivative (HA- ADH).
  • LMW HA was obtained by the degradation of high molecular weight HA (1.5 MDa) in pH 6.5 phosphate-buffered saline (PBS) buffer (4 mg/mL) with HAse (10 U/mg HA) as previously described, and purified by dialysis against H 2 O 30 .
  • Hydrazide-derivatized HA (HA- ADH) was prepared 30 ' 51 using a modified purification method that gives preparations free of small molecules 30 .
  • LMW HA 50 mg was dissolved in water to give a concentration of 4 mg/mL, and then a fivefold excess of ADH was added into the solution.
  • the pH ofthe reaction mixture was adjusted to 4.75 by addition of 0.1 ⁇ HC1.
  • 1 equiv of EDCI was added in solid form.
  • the pH ofthe reaction mixture was maintained at 4.75 by addition of 0.1 N HC1.
  • the reaction was quenched by addition of 0.1 N NaOH to adjust the pH of reaction mixture to 7.0 for different reaction time.
  • the reaction mixture was then transferred to prefreated dialysis tubing (Mw cutoff 3,500) and dialyzed exhaustively against 100 mM NaCl, then 25% EtOH/H2 ⁇ , and finally H2O.
  • HA- ADH The purity of HA- ADH was monitored by GPC.
  • the purified polymer solution was then filtered through 0.2 ⁇ m cellulose acetate membrane, flash frozen, and lyophilized.
  • the loading of ADH on the polymer backbone was determined by ⁇ H NMR in D2O 51 . 37 mg of HA- ADH was obtained with 9 mol% > and 18 mol% > loading based on available carboxylates modified respectively, with the reaction time to be 12 min and 20 min.
  • DOX-NHS active ester form
  • N-hydroxysuccinimido diphenyl phosphate (SDPP) was prepared from 10 mmol of diphenylphosphoryl chloride, 10 mmol of N-hydroxysuccinimide, and 10 mmol triethylamine in 6 mL of CH2CI2 as previously described 30 ' 53 .
  • Crude SDPP was titrated with ether, dissolved in ethyl acetate, washed (2 x 10 mL H2O), dried (MgSO4), and concentrated in vacuo to give SDPP with mp 89-90°C (85%).
  • DOX- hemisuccinate and 18.5 mg (1.5 equiv) of SDPP in 2 ml DMF was added with 60 ⁇ L
  • HPMA copolymer-bound DOX HPMA-DOX or P(GFLG)-DOX; P is the
  • HPMA copolymer backbone was synthesized as previously described S4 ' 55 .
  • a lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was used as the oligopeptide side chain.
  • the conjugate was synthesized using a two step procedure 56 .
  • the polymer precursor HPMA-(GFLG)-O ⁇ p was prepared by radical precipitation copolymerization of HPMA and N-methacryloylglycylphenylalanylleucylglycine p- nitrophenyl ester 55 .
  • DOX was bound to the polymer precursor by aminolysis 57 .
  • 200 mg HPMA-(GFLG)-O ⁇ p and 21.9 mg doxorabicin (DOX) hydrochloride were dissolved in 1.0 ml DMSO, and 50 ⁇ l of Et3N was added. The mixture was stirred at room temperature for 1 hr, and precipitated in acetone/ether (3/1) mixture solvent. The red polymer solid was collected and washed with acetone, ether, dried under vacuum to give 210 mg product.
  • the HPMA-(GFLG)-DOX-ONp conjugate contained 1.1 mol% of DOX.
  • HPMA-HA-DOX conjugates were prepared by the conjugation of HA- ADH (9 mol% and 18 mol% hydrazide modification) to the above HPMA-(GFLG)-DOX-ONp with ONp residue.
  • HA-(GFLG)-DOX-ONp copolymer-drag conjugate prepared previously was dissolved in 2.0 ml DMSO, and 90 mg HA- ADH of 18 mol%> hydrazide modification was dissolved in 1.0 ml water and 2.0 ml DMSO. The two solutions were mixed together and stirred it overnight at room temperature. Aminoethanol (100 ⁇ l) was added to destroy unreacted ONp active ester.
  • SKOV-3 and HCT-116 cells was determined using a 96-well plate format in quadruplicate with increasing doses range from 0.001-10 mg/mL of DOX equivalent. Each well contained approximately 20,000 cells in 200 ⁇ L cell culture media. Thus, a 2- ⁇ L aliquot of the stock solution was added to each well, and cells were continuously incubated at 37 °C, 5% CO for 3 days with the test substance, and cell viability was determined using MTT dye uptake at 540 nm. Response was graded as percent live cells compared to untreated controls 58 . Dose-response curves were constructed, an d the concentration necessary to inhibit the growth ofthe cells by 50% relative to the non- treated control cells (IC 50 dose) was determined.
  • HPMA-HA-DOX conjugates by cancer cells by confocal fluorescence microscopy.
  • SKON-3 cells were incubated in a cell culture flask, harvested by trypsinization, and transferred into a 8-well cell culture slide. 20,000 cells were seeded in each well ofthe slide and cultured for 48 hr. The cultured medium was replaced with medium containing HPMA-HA-DOX conjugates, the concentration was adjusted to 50 ⁇ g/ml of HA equivalent. Meanwhile, HPMA-DOX conjugate with equal amount of DOX drag to HPMA-HA-DOX was used as a control. Cells were cultured with the conjugates for various time intervals. Unbound conjugate was removed by washing the cell layer 3 times with DPBS.
  • DOX conjugate at 0°C for 2hr (a condition under which no internationalization occurs), followed by the DPBS washing and paraformaldehyde fixing described above.
  • the cell surface binding conjugate was determined by the fluorescence images.
  • Fluorescence microscopy Cells were examined by using an inverted microscope (Nikon) and a Bio-Rad (Hercules, CA) MRC 1024 laser scanning confocal microscope. Cell images were collected by using a x 60 oil immersion objective, no postacquisition enhancement of images was performed. DOX fluorescene image acquisition was accumulated via the BHS block of filters (excitation 488 nm and emission through a 522 nm 32 bandpass filter). A coverslip was mounted on a microscope slide containing fixed cells with ProLong Antifade Kit (Molecular Probes, Eugene, OR) as the mounting medium. Fluorescence images were scaled to 256 gray levels.
  • LMW HA was generated in this study for three reasons: (i) proton NMR allowed rapid quantification ofthe modification, (ii) LMW HA and its derivatives give injectable, non- viscous solution at concentrations up to 10 mg/mL, and (iii) LMW HA has a longer plasma half-life and is readily cleared by renal ultrafiltration.
  • the LMW HA was prepared by partial degradation of high molecular weight HA (1.5 MDa) with testicular HAse 60 in pH 6.5 PBS buffer at 37 °C.
  • HA-ADH with different ADH loadings were prepared by carbodiimide coupling chemistry 30 ' 31 , in which the extent of ADH modification was controlled through use of specific molar ratios of hydrazide, carboxylate equivalents, and carbodiimide.
  • the purity and molecular size distribution ofthe HA-ADH were measured by GPC, and the substitution degree of ADH was determined by the ratio of methylene hydrogens to acetyl methyl protons as measured by iH NMR 51 .
  • HA-ADH with ADH loadings of 9 mol% and 18 mol% were obtained and used in preparing the HA-DOX and HPMA-HA-DOX conjugates.
  • HA-DOX conjugates was synthesized by the conjugation of HA-ADH to the activated DOX-NHS ester to give a non-cleavable hydrazide linkage between the DOX drag and the HA polymer carrier.
  • the HA-DOX conjugates were purified by gel filtration on a Sephadex G-25 column using PBS buffer as the eluent, following by dialysis against H 2 O.
  • the DOX composition ofthe HA-DOX conjugates used in the in vitro cytotoxicity test were 2.3 wt% and 3.5 wt% which were made from 9 mol% and 18 mol% ADH loading of HA-ADH, respectively.
  • This cell targeted delivery system was designed with HA on the side chain ofthe
  • HPMA copolymer serving as a targeting moiety to cancer cell surface, and DOX linked to the polymer carrier through an lysosomal enzyme degradable peptide linkage 12 .
  • HPMA- HA-DOX conjugates were synthesized by the conjugation of HA-DOX with HPMA-DOX copolymer with active O ⁇ p residue.
  • HA-ADH with 9 mol%> and 18 mol% hydrazide modification were used in the conjugation.
  • the conjugates were purified by gel filtration on a Sephadex LH-20 column.
  • HA loading was determined by mass balance.
  • DOX conjugates were assessed for their dose-dependent growth inhibitory effect on human breast cancer HBL-100 cells, human ovarian cancer SKON-3 cells and human colon cancer HCT-116 cells which have been reported to overexpress HA receptors on the tumor cell surface.
  • Cells were exposed to various DOX concentration (DOX equivalent for polymer- drug conjugates) to determine the concentration necessary to inhibit the tumor cell growth by 50% relative to non-treated control cells (IC 50 dose).
  • IC 50 dose Typical curves describing the dependence of cell viability on the concentration of DOX equivalent covalently bound to the polymer conjugates, were presented in Figure 5.
  • the IC 50 doses for the free DOX drag and the conjugates were listed in Table 1.
  • the IC 50 doses against HBL-100 cells were 0.52 ⁇ M and 1.67 ⁇ M for the targeted HPMA-HA-DOX conjugates with 36 wt% and 17 wt% HA loading, respectively, in comparison ofthe 18.7 ⁇ M for the non-targeted HPMA-DOX conjugate and 0.15 ⁇ M for free DOX drag.
  • the cytotoxicity of targeted HPMA-HA-DOX conjugates had a magnitude increase over the non-targeted HPMA-DOX conjugate.
  • the cytotoxicity ofthe conjugates were even slightly higher than the non-targeted HPMA-DOX conjugate.
  • the IC 50 doses against SKON-3 cells were 157 ⁇ M and 141 ⁇ M for HA-DOX conjugates, comparing to 58.2 ⁇ M for non-targeted HPMA-DOX conjugate, and 9.2 ⁇ M for targeted HPMA-HA- DOX conjugate (36 wt%).
  • Two possible factors would contribute to the loss of cytotoxicity: the conjugation decreases the activity of DOX drag; the non-cleavable hydrazide linkage between DOX and HA polymer carrier.
  • fluorescein-HA was employed to study HA uptake in a variety of systems, e.g., cells expressing CD44 variants 40,41 ' 61"6 9 uptake by tumor cells for correlation with metastatic potential 50 ' 65 , intemalization by chondrocytes 46 , and as a measure of liver endothelial cell function 66 .
  • RH AMM-mediated uptake and trafficking of HA by transformed fibroblasts was observed with Texas Red-HA, and BODIPY-labeled HA was employed to distinguish HA uptake in cancer vs. untransformed cell-lines 30 ' 31 .
  • SKOV-3 Cells chilled to 0°C was incubated with HPMA-HA-DOX for 2hr. After fixing and washing, a well-developed cluster of cells was chosen for the fluorescence microscope analysis. Cells were sectioned optically using confocal microscopy, fluorescence images were taken via the BHS block of filters of excitation 488 nm and emission 522 nm, along with the transmission images.
  • Figure 6 provided a particularly dramatic illustration ofthe initial binding ofthe HPMA-HA-DOX conjugate on the SKON- 3 cells surface where the overexpressed HA binding receptor-CD44 located.
  • the anchoring ofthe targeted HPMA-HA-DOX on the cell surface prior to the cellular uptake through the specific binding between HA and HA binding proteins provides the opportunity ofthe enhanced intemalization ofthe polymer conjugates by receptor-mediated endocytosis.
  • the intemalization of polymer conjugates directly determined the cytotoxicity of conjugate system.
  • the cellular uptake ofthe targeted HPMA-HA-DOX conjugates were also followed by the confocal fluorescence microscopy.
  • SKON-3 cells were incubated with the HPMA-HA-DOX conjugates (36 wt% and 17 wt% HA loading) of 50 ⁇ g/ml HA equivalent for various intervals, before the fluorescence images were taken.
  • the non-targeted HPMA-DOX of equal amount of DOX equivalent was used as a control.
  • Confocal fluorescence images of HPMA-HA-DOX uptake by SKOV-3 cells were presented in Figure 7. Initially the 2 hr images, HPMA-HA-DOX polymer conjugates could be seen mainly on the cell membrane; over the course of 8 hr, it was gradually taken up into the cells. 24 hr and 32 hr later, cells showed the polymer conjugates in most subcellular compartments.
  • the data reported herein indicate that the cytotoxicity of HPMA-HA- DOX polymer conjugates requires cellular uptake ofthe bioconjugate followed by the release ofthe active free DOX drag by the lysosomal enzyme cleavage ofthe GFLG tetra- peptide spacer.
  • Targeting of a variety of anti-cancer agents to tumor cells and tumor metastases could be achieved by receptor-mediated uptake of an HA containing-anti-cancer agent conjugate, followed by the intracellular release ofthe active drag and subsequent cell death.
  • the ability to "seek and destroy" micrometastases is one ofthe most compelling and attractive potential outcomes for the disclosed HA containing-anti-tumor bioconjugates.
  • FIG. 8 depicts the in vitro cytotoxicity results ofthe HPMA-HA-DOX bioconjugates.
  • the cytotoxicity of targeted HPMA-HA-DOX bioconjugates were dramatically higher than non-targeted HPMA-DOX conjugate (Table 2), and 8- to 12-fold higher than the free DOX drag against this prostate cancer cell-line.
  • CD44 was examined to determine if it was expressed by 4T1 cells and if it could interact with HA.
  • CD44 was detected by Western blotting.
  • the binding activity of CD44 was determined by mixing 30 ⁇ g of 4T1 lysate with 3 H-HA (as test) or 3 H-HA plus 50 folds excess of cold HA (as specificity control).
  • the functional CD44 mediated 3 H-HA degradation was determined by incubating 4T1 cells with 3 H-HA for 72 hours and then separating degraded small MW 3 H-HA from intact HA high MW 3 H-HA by MW cut Centricon spin (Culty et al., J Cell Biol. 1992; 116(4): 1055-62.).
  • CD44 was expressed in 4T1 cells (Fig. 14 A) and it could bind to 3 H-HA (Fig. 14B) and degrade 3 H-HA (Fig. 14C). The binding was specific as it could be inhibited by excess of cold HA (Fig. 14B). The CD44 mediated degradation of 3 H-HA could be inhibited by excess clod HA, anti-CD44 neutralization antibody (KM201, Akima et al.; J Drag Target 1996; 4(1): 1-8) and lysosomal inhibitor chloroquine (Fig. 14C), showing that CD44 did mediate the uptake and degradation of HA.
  • the H-HA was i.v. injected into mice bearing with 200 mm size of breast cancer xenografts on their mammary fat pat. After 24 hours, the mice were sacrificed, the organs were homogenized and 20 mg of homogenates from different tissues was measured for 3 H-HA by ⁇ -counter. The results (Fig 15) showed that both tumor and lymph node contained the highest amount of HA as compared to other organs.
  • the 4T1 breast cancer cells were injected into foot pat of syngenic BABL/c mice. Three days after inoculation, the mice were randomly divided into three different treatment groups: 1) saline alone as vehicle control; 2) 4 mg/kg of Taxol; or 3) HA-Taxol containing Taxol equal to 4 mg/kg. About 0.2 ml of above agents were administrated by subcutaneous (s.c.) injection at the middle of leg three times a week, from where HA-drug could be drained/ absorbed into popliteal and inguinal lymph path, in which the spontaneous metastases were expected to take place.
  • s.c. subcutaneous
  • the s.c. injection was chosen because 3 H-HA injected s.c. was mainly drained/absorbed from the injection sites into lymphatic pathway as evidenced by the increased local H-HA and reduced plasma H-HA when the lymphatic structure was surgically destroyed.
  • the specific distribution of HA to the lymph nodes was also observed with 14 C-labelled HA and fluorescent HA by other groups (Akima et al.; J Drag Target 1996; 4(1): 1-8).
  • the subcutaneous administration of HA specially targeting the lymph path shows the effectiveness of using HA as carrier to destroy lymphatic metastases.
  • mice After three weeks of s.c. injection of HA-Taxol, the mice were sacrificed and the primary tumors, the popliteal and inguinal lymph nodes (as represented in Fig. 16) were collected. The lymph nodes were carefully dissected out from surrounding fat, measured for the weights and processed for pathology.
  • HA-Taxol could prevent the tumor cells from their settle- down and growth in both the near and the far distant lymph nodes.
  • the conjugation process was carried out according to Akima's method (Akima et al; J Drag Target 1996; 4(1): 1-8) with some modification.2 mg mitomycin C powder (Sigma) was added to 4 mg of hyaluronan (Lifecore, MW 1.2 xlO 6 kDa) in 35% DMF (dimethyformamide, pH 5.0). After mixing well, 4 mg of water-soluble l-ethyl-3-(-dimethylaminopropyl) carbodiimide (ED AC) was added and then reacted overnight at room temperature. Then, the unconjugated mitomycin C was separated from HA-mitomycin C by dialysis ofthe reaction mixture against distilled water.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dermatology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
EP03750067A 2002-05-06 2003-05-06 Vorblockierung mit nicht-ha gags erhöht die wirksamkeit von ha-konjugierten krebsmitteln Withdrawn EP1501522A4 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US37852902P 2002-05-06 2002-05-06
WOPCT/US02/14402 2002-05-06
US378529P 2002-05-06
PCT/US2002/014402 WO2002090390A1 (en) 2001-05-04 2002-05-06 Hyaluronic acid containing bioconjugates : targeted delivery of anti-cancer drugs to cancer cells
PCT/US2003/014087 WO2003094929A2 (en) 2002-05-06 2003-05-06 Preblocking with non-ha gags increases effectiveness of ha conjugated anticancer agents

Publications (2)

Publication Number Publication Date
EP1501522A2 true EP1501522A2 (de) 2005-02-02
EP1501522A4 EP1501522A4 (de) 2007-12-05

Family

ID=29422599

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03750067A Withdrawn EP1501522A4 (de) 2002-05-06 2003-05-06 Vorblockierung mit nicht-ha gags erhöht die wirksamkeit von ha-konjugierten krebsmitteln

Country Status (3)

Country Link
EP (1) EP1501522A4 (de)
AU (1) AU2003267872A1 (de)
WO (1) WO2003094929A2 (de)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1401875A4 (de) * 2001-05-04 2005-01-26 Univ Utah Res Found Hyaluronsäure enthaltende biokonjugate: gezielte abgabe von antikrebsmitteln an krebszellen
DE102009035586A1 (de) 2009-07-31 2011-02-03 Fresenius Kabi Deutschland Gmbh Intravesikale Instillationslösungen zur Behandlung von Blasenkrebs
WO2013119061A1 (ko) 2012-02-07 2013-08-15 포항공과대학교 산학협력단 경피 전달용 히알루론산-단백질 컨쥬게이트의 제조 방법 및 이에 따라 제조된 경피 전달용 히알루론산-단백질 컨쥬게이트
BR112015031950A2 (pt) * 2013-06-20 2017-07-25 Morphotek Inc métodos para tratamento de câncer de ovário
CN105688221B (zh) * 2016-01-15 2019-01-18 武汉理工大学 Ha/rgd双受体介导多靶点给药系统的制备方法
CN112315940B (zh) * 2019-07-18 2022-12-06 暨南大学 一种促肿瘤凝血和酶/atp双重响应性释药的纳米粒子及其制备方法与应用
US20210145989A1 (en) * 2019-09-30 2021-05-20 Navidea Biopharmaceuticals, Inc. Compositions and related methods for blocking off-target localization of mannosylated dextrans and other cd206 ligands
CN114652852B (zh) * 2020-12-22 2024-12-27 浙江大学 诱导肿瘤细胞发生自发钙化的分子及其应用
WO2023150695A1 (en) 2022-02-04 2023-08-10 Navidea Biopharmaceuticals, Inc. Altering net charge on mannosylated dextrans to maximize target tissue uptake and off target competitive blocking

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5356883A (en) * 1989-08-01 1994-10-18 Research Foundation Of State University Of N.Y. Water-insoluble derivatives of hyaluronic acid and their methods of preparation and use
US5616568A (en) * 1993-11-30 1997-04-01 The Research Foundation Of State University Of New York Functionalized derivatives of hyaluronic acid

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LUO Y. ET AL.: "A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells" BIOMACROMOLECULES, vol. 1, no. 2, June 2000 (2000-06), pages 208-218, XP002308016 *
LUO Y. ET AL.: "Targeted delivery of doxorubicin by HPMA copolymer-hyaluronan bioconjugates" PHARMACEUTICAL RESEARCH, vol. 19, no. 4, April 2002 (2002-04), pages 396-402, XP008028981 *
SAMUELSSON C. AND GUSTAFSON S.: "Studies on the interaction between hyaluronan and a rat colon cancer cell line" GLYCONCONJUGATE JOURNAL, vol. 15, no. 2, February 1998 (1998-02), pages 169-175, XP001119513 *
See also references of WO03094929A2 *

Also Published As

Publication number Publication date
WO2003094929A3 (en) 2004-03-11
AU2003267872A1 (en) 2003-11-11
WO2003094929A2 (en) 2003-11-20
EP1501522A4 (de) 2007-12-05

Similar Documents

Publication Publication Date Title
US20090104143A1 (en) Hyaluronic acid containing bioconjugates: targeted delivery of anti-cancer drugs to cancer cells
Luo et al. Synthesis and selective cytotoxicity of a hyaluronic acid− antitumor bioconjugate
Shi et al. Synergistic active targeting of dually integrin αvβ3/CD44-targeted nanoparticles to B16F10 tumors located at different sites of mouse bodies
Nakamura et al. Two step mechanisms of tumor selective delivery of N-(2-hydroxypropyl) methacrylamide copolymer conjugated with pirarubicin via an acid-cleavable linkage
Choi et al. Hyaluronic acid-based nanocarriers for intracellular targeting: interfacial interactions with proteins in cancer
Luo et al. A hyaluronic acid− taxol antitumor bioconjugate targeted to cancer cells
Martin et al. Glycosidase activated prodrugs for targeted cancer therapy
US5622718A (en) Alginate-bioactive agent conjugates
Journo-Gershfeld et al. Hyaluronan oligomers-HPMA copolymer conjugates for targeting paclitaxel to CD44-overexpressing ovarian carcinoma
JP5537763B2 (ja) 放出システムとしてのポリアセタール薬物抱合体
Li et al. pH/reduction dual-responsive hyaluronic acid-podophyllotoxin prodrug micelles for tumor targeted delivery
AU2009302387B2 (en) HPMA - docetaxel or gemcitabine conjugates and uses therefore
CN102686243B (zh) 用于抗癌剂递送的聚合物系统
Xu et al. Targeted polymer-drug conjugates: current progress and future perspective
Misra et al. Utilization of glycosaminoglycans/proteoglycans as carriers for targeted therapy delivery
Park et al. Self-assembled nanocomplex of PEGylated protamine and heparin–suramin conjugate for accumulation at the tumor site
EP1501522A2 (de) Vorblockierung mit nicht-ha gags erhöht die wirksamkeit von ha-konjugierten krebsmitteln
Roy et al. Recent progress in the development of polysaccharide conjugates of docetaxel and paclitaxel
Brown The development of hyaluronan as a drug transporter and excipient for chemotherapeutic drugs
Tang et al. Synthesis, characterization, and in vitro and in vivo evaluation of a novel pectin–adriamycin conjugate
US20050169883A1 (en) Preblocking with non-ha gags increases effectiveness of ha conjugated anticancer agents
Caliceti et al. Polysaccharide-based anticancer prodrugs
US8778892B2 (en) Compounds and methods for treatment of solid tumors
Shi Synergistic Active Targeting to B16F10 Tumors by αvβ3/CD44-Targeted Nanoparticles Loaded with Docetaxel
Popa et al. Polymer-Drug Conjugates

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20041116

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: GEORGETOWN UNIVERSITY

Owner name: UNIVERSITY OF UTAH RESEARCH FOUNDATION

A4 Supplementary search report drawn up and despatched

Effective date: 20071107

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 35/00 20060101ALI20071031BHEP

Ipc: A61K 31/74 20060101AFI20031127BHEP

17Q First examination report despatched

Effective date: 20081219

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20101201