WO2009059984A2 - Hydrophobisation réversible pour une administration de médicament de premier passage - Google Patents

Hydrophobisation réversible pour une administration de médicament de premier passage Download PDF

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WO2009059984A2
WO2009059984A2 PCT/EP2008/064971 EP2008064971W WO2009059984A2 WO 2009059984 A2 WO2009059984 A2 WO 2009059984A2 EP 2008064971 W EP2008064971 W EP 2008064971W WO 2009059984 A2 WO2009059984 A2 WO 2009059984A2
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drug
cells
antitumor drug
tumor
prodrug
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PCT/EP2008/064971
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WO2009059984A3 (fr
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Vladimir G. Budker
Sean D. Monahan
Vladimir Subbotin
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F. Hoffmann-La Roche Ag
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Publication of WO2009059984A3 publication Critical patent/WO2009059984A3/fr

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    • 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
    • A61K47/54—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 compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • A61K47/54—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 compound
    • 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 variety of methods and routes of administration have been developed to deliver pharmaceuticals to their site of action.
  • One of the general problems associated with drug delivery is balancing the ability to cross cell membranes with solubility in water. If a drug is too hydrophilic, it will be unable to cross the hydrophobic environment of the lipid cell membrane. If a drug is too lipophilic, it will aggregate or have limited solubility in an aqueous environment. A lipophilic drug could also be confined to the cell membrane if it does reach the cell. Most of the drug formulations are therefore amphiphilic, containing both hydrophilic and hydrophobic characteristics, or are formulated with the use of excipient(s) to aid in the delivery of the drug.
  • liver cancers Several types of diseases, notably several types of cancers, have been treated with a regional treatment regiment. For example, the regional treatment of liver cancers has been explored.
  • the liver is the predominant site for metastatic disease progression from a variety of tumor origins, including colorectal carcinoma, melanoma, and neuroblastoma, and is the primary site for hepatocellular carcinoma (HCC) and cholangiocarcinoma.
  • HCC hepatocellular carcinoma
  • cholangiocarcinoma cholangiocarcinoma.
  • Traditional systemic chemotherapy has demonstrated poor antitumor benefit and only marginal increases in survival.
  • resection and transplantation remain the only curative options for patients with progressive liver disease. However due to disease recurrence, or vascular invasion and the presence of multifocal disease, these options might not be medically available.
  • liver neoplasms grow, tumors reaching a diameter of 5-7 mm are predominantly perfused by a neovascularized hepatic arterial route.
  • Normal liver parenchyma is supplied mainly from the portal vein (75%).
  • HAI direct hepatic artery infusion
  • Another regional therapy has been developed consisting of transcatheter hepatic artery chemotherapy (TAC) via the femoral artery (bolus injection).
  • TAC transcatheter hepatic artery chemotherapy
  • TAC transcatheter hepatic artery chemotherapy
  • TAC embolization
  • TACE embolization
  • Ovarian cancer is the second most common pelvic tumor and the leading cause of death from a gynecologic malignancy. Because of the lack of symptoms in the early stages, two thirds of patients present with advanced late-stage disease. Despite advances in surgical oncology, chemotherapy, and molecular biology, overall 5-year survival rates are still poor (approximately 30%).
  • Intraperitoneal chemotherapy was introduced for peritoneal disseminated disease in an effort to direct high levels of chemo therapeutics to the peritoneal exposed tumor surface area.
  • This treatment regime has been additionally modified as intraperitoneal perfusion chemotherapy (IPPC).
  • IPPC removes unabsorbed drug from the peritoneal cavity in order to decrease systemic toxicity and allow for higher dose administration of the chemotherapeutics.
  • IPC Intraperitoneal chemotherapy
  • chemotherapeutics to mediate cytotoxic activity is dependent on sufficient intracellular drug accumulation in the target cell.
  • Intracellular drug levels are a function of the amount of drug are drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux).
  • Drug uptake is determined by membrane transport, occurring through poorly defined mechanisms of passive diffusion and/or energy-dependent active transport. It has been proposed that approximately one-half of all drug uptake takes place by passive diffusion and the other half occurs by facilitated transport. It has been thought that lipid membranes represent a barrier for hydrophilic drug movement, but are not a barrier for hydrophobic drugs.
  • Hydrophobization or lipidization has generated interest for both drug and peptide/protein delivery.
  • Drug hydrophobization utilizing relatively stabile modifications such as esters and amides was shown to increase drug interactions with cellular membranes and has correlated with improved cellular uptake and lowered IC50 values.
  • concerns remain involving both compound aggregation and embolization, and the sequestering of the drug in the cell membrane.
  • the present invention comprises a rapidly reversible hydrophobized antitumor drug for use in a method for treating a tumor comprising mixing said rapidly reversible hydrophobized antitumor drug with a pharmaceutically acceptable carrier solution to form a delivery solution, wherein (a) the rapidly reversible hydrophobized antitumor drug comprises a hydrophobic group covalently linked to an antitumor drug via a rapidly reversible linkage, and (b) the rapidly reversible linkage is stable in a suitable solvent or in a lyophilized powder, and, (c) the rapidly reversible linkage is unstable in said pharmaceutically acceptable carrier solution and in said delivery solution.
  • antitumor drug formulations for use in a method for delivering drugs into cells via a first-pass effect comprising: reversibly attaching one or more hydrophobic moieties to the drug via a very labile linkage to form a rapidly reversible hydrophobized antitumor drug (prodrug).
  • the prodrug is synthesized in or dissolved in a suitable solvent.
  • the resultant prodrug is stable in said suitable solvent, but is unstable in a suitable pharmaceutically acceptable carrier solution.
  • a delivery solution is formed, wherein the rapidly reversible linkage is unstable.
  • the transient hydrophobic conversion of a drug into a prodrug for delivery to cells via first-pass delivery Rapidly reversible hydrophobization (RRH) increases cellular uptake of the prodrug in a first pass setting via increased membrane permeability of the prodrug.
  • the rapidly reversible hydrophobized antitumor drug i.e. the prodrug
  • Rapid reversibility or lability of the hydrophobic linkage of the drug provides for limited duration of this enhanced membrane permeability. Cleavage of the hydrophobic moiety after the association of the prodrug with the cell allows interaction of the unmodified drug with cellular components.
  • Cleavage of the hydrophobic moiety on the prodrug outside the cell decreases the ability of the drug to enter cells and thus decreases undesired effects of the drug, such as toxicity, in non-target, i.e., non-f ⁇ rst-pass, cells.
  • the half-life of the hydrophobic attachment is comparable with the time necessary for first-pass delivery following single-bolus injection or the time necessary for drug diffusion after topical application.
  • the prodrug is capable of this enhanced membrane association and permeability of a target cell for only a limited period of time.
  • the said suitable solvent consist of an organic solvent.
  • the labile linkage consists of a hydro lytically labile bond.
  • the carrier solution consists of an aqueous solution.
  • the linkage attaching the hydrophobic group to the drug consists if a linkage that is cleaved by a component of said carrier solution.
  • the linkage is stable in a compatible organic solvent but hydro lytically unstable in an aqueous environment.
  • the linkage attaching the hydrophobic group to the drug is more stable (longer half-life) in a basic environment but less stable as the pH is lowered. Because of the instability of the hydrophobic modification, prodrug that enters a cell rapidly reverts to the original drug molecule which is then free to interact with target molecules. Prodrug that does not interact with cell membranes during first- pass rapidly reverts to the less membrane permeable drug through loss of the hydrophobic moiety. Reversion limits delivery of the drug into non-targeted cells and tissues thus limiting systemic toxicity.
  • the described drug modifications and processes can be used to enhance cellular accumulation of a chemotherapeutic drug in tumor tissue and thereby decreasing the amount of the delivered dose that non-targeted cells are exposed to, thereby decreasing systemic toxicity.
  • the chemotherapeutic, or anti-neoplastic is transiently converted into a lipophilic or hydrophobic prodrug by attaching one or more hydrophobic moieties to the drug by labile bonds. Conversion of the drug to a prodrug promotes greater interaction with a cellular membrane. Rapid hydrolysis of the chemical linkage under physiological conditions restores the drug to the more membrane impermeable state associated with the parent drug. Transient lipophilic conversion facilitates enhanced drug uptake by tumor tissue and subsequent antitumor efficacy during first-pass delivery, while preserving low systemic toxicity by reversion to the parent drug prior to systemic exposure.
  • the hydrophobic modifications utilized in the prodrug formation are very labile, allowing for facile regeneration of the active drug within the cell. Because first-pass delivery serves to deliver more of the prodrug / drug to regional target cells, such as tumor cells, lowering of the overall dosing of the drug may be possible.
  • the rapidly labile prodrugs which are more cell permeable than the drug, rapidly revert to the less membrane permeable drug, thereby exposing non-target cells to the drug form rather than the more membrane permeable prodrug. The result is a transient increase the therapeutic index of conventional chemotherapeutics while maintaining low systemic toxicity.
  • the lipophilic character of the prodrug will depend on the number and hydrophobicity of groups attached. Sufficient hydrophobicity is added to the drug to increase delivery of the resultant prodrug to cells. Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding. Typically, such chemical groups are not water soluble, and tend not to hydrogen bond. Hydrocarbons are hydrophobic groups. If the hydrophobic group comprises an alkyl chain, the length of an alkyl chain group will affect the hydrophobicity of the group.
  • preferred hydrophobic groups compatible with the described invention may be selected from the group comprising: an alkyl chain of 4 to 30 carbon atoms, which may contain sites of unsaturation; an alkyl group containing an alkyl chain and alkyl rings (aromatic and/or non aromatic); and steroids.
  • the linkages can also be designed such that they posses different lability rates in order to influence prodrug stability in vitro and in vivo.
  • Limited stability of the drug modification allows for a local high concentration of modified drug that is able to enter cells in a first-pass region. A too rapid half-life results in ineffective target cell uptake. Conversely, a half-life of the prodrug that is too long leads to increased delivery of drug to non-target cells and tissues, potentially leading to systemic toxicity.
  • the lability of the described linkages is potentially controllable through the choice of the pharmaceutically acceptable carrier solution. For example, the pH of the carrier solution can be adjusted with the use of an appropriate buffer in order to control the half- life of the prodrug.
  • attachment of additional groups can not only increase the hydrophobicity of the drug, but also effectively increase the time required for complete hydrolysis.
  • Controlling the incubation time of the drug between initial mixing with the carrier solution and initial contact with cells can also be used to influence the amount of time the lipophilic prodrug is present with cells.
  • the rate of hydrolysis of the prodrug may be retarded upon interaction with the cellular membranes.
  • the kinetic lability required for optimal delivery can be controlled through temperature or composition of the pharmaceutically acceptable carrier solution, the volume of the injection, the concentration of the injected prodrug, and the total amount of prodrug delivered.
  • An amine-containing drug has a nitrogen atom in the molecule that is amenable to modification.
  • the amine can be a primary, secondary, or tertiary amine, or another nitrogen derivative such as an aniline.
  • Other reactive groups on the drug may also be utilized for rapidly reversible attachment of a hydrophobic group. The requirement is that the hydrophobation be rapidly reversible and that reversal, cleavage of the hydrophobic group or groups from the drug, yields an active drug.
  • the labile linkage of the prodrug is selected from the list consisting of silazane and maleamic acid.
  • Amine containing drugs can be modified with silazanes.
  • DMODSiCl chlorodimethyloctadecylsilane
  • the function of this group is to transiently attach hydrophobic groups to the drug molecule.
  • the invention is meant to include other silazane derivatives.
  • silazanes can be employed to impart transient hydrophobicity (for example, including but not limited to: trimethylsilyl and tert-butyl-dimethylsilyl groups).
  • silazane or silylamine
  • Silazanes are known to hydrolyze rapidly in the presence of water to yield the original amine and a silanol or disilyl ether. Silazanes have generally been utilized in the field of ceramics or in organic synthesis as reagents for the silylation of other functional groups, most notably, the hydroxyl group. Because of its high lability, this modification has not found utility in biological applications. However, more stable heterosilanes have been employed as prodrugs.
  • Examples include: a trimethylsilyl ether of testosterone; silabolin, a per-trimethylsilylated derivative of dopamine; carbosilane drugs; and silicon used as part of a delivery system. These examples employ a stable bond (carbon-silicon) or a slowly hydrolyzed bond (silicon-oxygen), not a rapidly hydrolyzed bond as found in the silazane.
  • Silyl ethers have long been utilized as removable protecting groups in organic synthesis. The bond is hydro lytically labile under acidic conditions to yield an alcohol and a silanol or disilyl ether.
  • Silyzanes control the hydrolysis rate of silyl ethers, for example the sterics of the silicon atom (i.e. the bulk of groups attached to silicon), and the pH of the solution. Silyzanes (with the exception of the known stable variants) hydrolyze much more readily than the corresponding oxygen variants (the silyl ethers).
  • DMODSiCl can be reacted with methylamine to form dimethyloctadecylsilyl-methylamine.
  • This silazane can then be added to cisplatin or Pt(DMSO) 2 - 1,1 -eye Io butanedicarboxylate to yield a labile cisplatin derivative.
  • Silylation of a heterocyclic nitrogen atom is also possible.
  • Amine containing drugs can also be modified with maleic anhydrides possessing hydrophobic groups.
  • maleic anhydrides possessing hydrophobic groups.
  • Maleic anhydrides have been previously utilized for reversible amine modification.
  • the resulting maleamic acids are known to be stable under basic conditions, but hydrolyze rapidly under acidic conditions.
  • 2-propionic-3-methylmaleic anhydride (a carboxylic acid derivative) has been tested with glycinylalanine.
  • the purpose of the maleic anhydride is to transiently attach a hydrophobic groups to a drug molecule.
  • maleic anhydrides can be employed to impart transient hydrophobicity.
  • labile bonds are known to those skilled in the art, that could be utilized to attach a hydrophobic group or moiety to a drug molecule.
  • the invention is also meant to encompass the use of hydrophobic drug modifications with these other types of hydro lytically labile bonds, when the derived prodrugs are then delivered via the delivery methods described in the present invention.
  • additional labile bonds that may be used to attach the hydrophobic moiety to the drug include, but are not limited to: imines, ortho esters, acetals, aminals, silyl esters, and phosphosilyl esters.
  • a reversible or labile bond is a covalent bond other than a covalent bond to a hydrogen atom that is capable of being selectively broken or cleaved under conditions that will not break or cleave other covalent bonds in the same molecule. More specifically, a reversible or labile bond is a covalent bond that is less stable (thermo dynamically) or more rapidly broken (kinetically) under appropriate conditions than other non- labile covalent bonds in the same molecule. Cleavage of the labile bond results in the formation of two molecules.
  • cleavage or lability of a bond is generally discussed in terms of half- life (ty 2 ) of bond cleavage, or the time required for half of the bonds to cleave.
  • Orthogonal bonds are bonds that cleave under conditions that cleave one and not the other. Two bonds are considered orthogonal if their half- lives of cleavage in a defined environment are 10-fold or more different from one another.
  • reversible or labile bonds encompass bonds that can be selectively cleaved more rapidly than other bonds a molecule.
  • the invention encompasses hydrophobically modified drug formulations in which the half-life of the modification is less than or equal to 5 min in the delivery or carrier solution.
  • the rapidly reversible linkage has a half-life less than 2 minutes. In another preferred embodiment, the rapidly reversible linkage has a half-life less than 1 minute. In another preferred embodiment, the rapidly reversible linkage has a half-life less than 30 seconds. In another preferred embodiment, the rapidly reversible linkage has a half-life less than 20 seconds. Lability is preferably selected to correspond to the time necessary to deliver the modified drug in a first pass setting.
  • Electron withdrawing groups are atoms or parts of molecules that withdraw electron density from another atom, bond, or part of the molecule wherein there is a decrease in electron density to the bond of interest (donor).
  • Electron donating groups are atoms or parts of molecules that donate electrons to another atom, bond, or part of the molecule wherein there is an increased electron density to the bond of interest (acceptor). The electron withdrawing/donating groups need to be in close enough proximity to effect influence, which is typically within about 3 bonds of the bond being broken.
  • Another strategy for increasing the rate of bond cleavage is to incorporate functional groups into the same molecule as the labile bond.
  • the proximity of functional groups to one another within a molecule can be such that intramolecular reaction is favored relative to an intermolecular reaction.
  • the proximity of functional groups to one another within the molecule can in effect result in locally higher concentrations of the functional groups.
  • intramolecular reactions are much more rapid than intermolecular reactions. Reactive groups separated by 5 and 6 atoms can form particularly labile bonds due to the formation of 5 and 6- member ring transition states.
  • Examples include having carboxylic acid derivatives (acids, esters, amides) and alcohols, thiols, carboxylic acids or amines in the same molecule reacting together to make esters, carboxylic and carbonate esters, phosphate esters, thiol esters, acid anhydrides or amides. Steric interactions can also change the cleavage rate for a bond.
  • Appropriate conditions are determined by the type of labile bond and are well known in organic chemistry.
  • a labile bond can be sensitive to pH, oxidative or reductive conditions or agents, temperature, salt concentration, the presence of an enzyme, or the presence of an added agent.
  • increased or decreased pH may be the appropriate conditions for a pH-labile bond.
  • the pH of the carrier solution can be adjusted in order to effect the half- life of the prodrug formulation.
  • oxidative conditions may be the appropriated conditions for an oxidatively labile bond.
  • reductive conditions may be the appropriate conditions for a reductively labile bond.
  • the rate at which a labile group will undergo transformation can be controlled by altering the chemical constituents of the molecule containing the labile group. For example, addition of particular chemical moieties (e.g., electron acceptors or donors) near the labile group can affect the particular conditions (e.g. , pH) under which chemical transformation will occur.
  • chemical moieties e.g., electron acceptors or donors
  • a labile linkage is a chemical compound that contains a labile bond and provides a link or spacer between two other groups.
  • the groups that are linked may be chosen from compounds such as biologically active compounds, membrane active compounds, compounds that inhibit membrane activity, functional reactive groups, monomers, and cell targeting signals.
  • the spacer group may contain chemical moieties chosen from a group that includes alkanes, alkenes, esters, ethers, glycerol, amide, saccharides, polysaccharides, and heteroatoms such as oxygen, sulfur, or nitrogen.
  • the spacer may be electronically neutral, may bear a positive or negative charge, or may bear both positive and negative charges with an overall charge of neutral, positive or negative.
  • a method for treating a tumor in a mammal comprising a) covalently linking a hydrophobic group to an antitumor drug via a rapidly reversible linkage thereby forming a rapidly reversible hydrophobized antitumor drug wherein the rapidly reversible hydrophobized antitumor drug is synthesized in or dissolved in a suitable solvent in which the rapidly reversible linkage is stable; b)mixing the rapidly reversible hydrophobized antitumor drug in the suitable solvent with a pharmaceutically acceptable carrier solution to form a delivery solution, wherein the rapidly reversible linkage is unstable in the delivery solution; and, c) administering said delivery solution to the mammal.
  • perfusion refers to the deliberate introduction of fluid into a tissue.
  • the fluid can be introduced into a vessel, tissue lumen, body cavity, such as the peritoneal cavity or in vitro cell container.
  • the perfused tissue is isolated such that the introduced fluid does not reach non-target tissues.
  • the isolated tissue can be flushed both before and after the perfusion to remove bodily fluid or introduced fluid from the tissue or region.
  • Perfusion has been used to deliver anti-cancer agents into the blood vessels and tissues of an organ (liver or lung) or region of the body (usually an arm or a leg) using circulating bypass machines. Such a procedure is performed to treat cancer that has spread but is limited to an organ or region of the body.
  • the prodrug (dissolved in drug dissolving solvent) is mixed with an aqueous carrier solution in a mixing chamber and delivered to a the tissue to be perfused.
  • An outflow line permits the prodrug delivery solution to perfuse through the cavity and exit through the outflow line. Because the prodrug and drug (resulting from loss of the hydrophobic group(s)) are removed from the tissue, it is possible to utilize prodrugs with a longer half-lives than in cases where the material is not removed following delivery. When the prodrug - drug is not removed, it is preferred to have a prodrug with a shorter half-life in order to protect downstream cells from the highly cell permeable prodrug. In the case of isolated perfusion, the prodrug is removed from the area of interest, thereby protecting cells outside the target region.
  • Rapidly reversible prodrugs may by synthesized in organic or other appropriate solvents.
  • the described prodrugs are stable in the solvents but unstable in a carrier or delivery solution, such as an aqueous solution (for hydro lytically labile bonds).
  • the reaction to form the modified drug can be conducted in a variety of solvents, however, a pharmaceutically acceptable injectable solvent is preferred.
  • a solvent in which the modified drug can be purified from other components of the modification reaction for example, hydrolyzed hydrophobic group, drying agents, and bases
  • drugs can be modified according to the invention.
  • the drug is modified through an amine group on the drug.
  • these drugs may be selected from the list comprising: chemotherapeutics, anti-neoplastic, doxorubicine (adriamycin), cisplatin (cis-diamminedichloroplatinum(II)), melphalan, and the tubulin polymerization agent paclitaxel.
  • Additional functional groups that can be modified include alcohols, thiols, phosphates, and carboxylates.
  • an active derivative of the parent drug, which contains a functional group suitable for modification may also be used.
  • modified drugs include: cisplatin derivatives containing a heterocyclic nitrogen, anthracycline derivatives of doxorubicin, and amino or furanosyl substituted 5- fluorouracil.
  • labile prodrugs comprising: co-injecting the prodrug in an organic or other suitable solvent (a drug carrier solvent) together with a aqueous pharmaceutically acceptable carrier solution, though a mixing chamber.
  • the prodrug described herein comprise hydrophobic groups attached by very hydro lytically reactive linkages that requires synthesis and storage in organic solvents.
  • toxicity concerns prohibit the direct delivery of drugs to cells in undiluted organic solvents. Therefore, mixing the organic solvents with a pharmaceutically acceptable aqueous carrier solution just prior to delivery by co-injection though a mixing chamber is performed.
  • a method for delivering a hydrophobic drug or prodrug to a cell comprising: providing a prodrug that is stable in its dissolving solvent, and injecting the prodrug in its dissolving solvent into a suitable mixing chamber designed to mix the dissolving solvent with a aqueous carrier solution to form a combined delivery solution just prior to delivery of a combined delivery solution to the cell.
  • the aqueous carrier solution is a solution in which the prodrug is not stable.
  • a suitable mixing chamber rapidly mixes the prodrug dissolving solvent with the aqueous carrier solution without producing laminar flow of the dissolving solvent and aqueous solvents.
  • the critical components of a suitable mixing chamber include: means by which to accurately deliver predetermined volumes of drug carrier solvent and aqueous carrier solution, means to rapidly and intimately mix the drug carrier solvent and aqueous carrier solution, and a means of delivering the combined liquid (delivery solution) to cells.
  • Some commercial mixing chambers can result in laminar flows, without effective mixing of the drug carrier solvent with the carrier solution. If the drug carrier solvent is an organic solvent, incomplete mixing results in exposure of some cells to higher concentration of organic solvents that can lead to membrane damage. If the mixing is too slow, then the prodrug may be cleaved prior to contact with the cells.
  • Any mixing chamber that provides adequate and rapid mixing of the drug carrier solvent with the aqueous carrier solution is suitable for use with the present invention.
  • An example of a suitable mixing chamber is the colliding flow mixing microchamber shown in FIG. 3.
  • the aqueous carrier solution and the drug carrier solvent are injected into a mixing chamber (C) though conduits (A) and (B) respectively.
  • the direction of flow (b) of the drug carrier solvent into chamber (C) is in the opposite direction of the flow of the aqueous carrier solution into chamber (C), facilitating mixing of the two liquids.
  • the combined delivery solution is then delivered to cells through vessel conduit (D) and instillation port (E).
  • the volume of drug carrier solvent is generally much less than the volume of carrier solution.
  • Conduits (A), (B), and (D) may be rigid or flexible and may be made of any material than is suitable to convey the respective solutions and drugs.
  • the length of conduit (D) may be varied in length to alter the amount of time the prodrug is in the aqueous carrier solution prior to delivery to the cells, thus modulating the half- life of the prodrug in the presence of the cells.
  • Suitable instillation ports (E) may be selected from the list comprising syringe needles and catheters.
  • delivery solutions comprise about 1/10 th volume prodrug in solvent mixed with about 1 volume aqueous carrier solution (such as, but not limited to, Ringer's or isotonic glucose (ITG)).
  • aqueous carrier solution such as, but not limited to, Ringer's or isotonic glucose (ITG)
  • the total volume of prodrug-containing solvent to be delivered should be less than that which would cause toxicity from the solvent.
  • the volume of carrier solution should be chosen to provide adequate total volume for the target area and provide adequate dilution of the prodrug-containing solvent. For larger animals, target areas, or cell containers, increased total volume is appropriate.
  • the membrane permeability and lability of the prodrug can be measured by monitoring the uptake of the prodrug by liposomes.
  • the eleunt from a suitable mixing chamber can be delivered to a solution containing liposomes whose composition approximates the plasma membrane of the target cells.
  • the liposomes are then purified and the level of drug in the liposomes is measured.
  • the liposomes can contain DNA to facilitate determination of drug uptake. In this manner, the acceptable volumes of solvent and carrier solution, as well as effectiveness of the mixing chamber can be analyzed.
  • the described processes and prodrugs are readily compatible with known techniques such as regional hepatic artery infusion (HAI) therapy, intraperitoneal chemotherapy (IPC), intraperitoneal perfusion chemotherapy (IPPC), transcatheter hepatic artery chemotherapy (TAC), transcatheter hepatic artery chemotherapy with embolization (TACE), and isolated organ or tissue perfusion.
  • HAI regional hepatic artery infusion
  • IPC intraperitoneal chemotherapy
  • IPPC intraperitoneal perfusion chemotherapy
  • TAC transcatheter hepatic artery chemotherapy with embolization
  • isolated organ or tissue perfusion In isolated perfusion applications, potential systemic toxicity of the drug is further reduced because unabsorbed hydro lyzed drug is flushed from the isolated tissue (such as a peritoneal cavity) prior to restoration of normal fluid flow through the tissue.
  • the describe processes and prodrugs are also compatible with topical delivery of the drug.
  • the process may be described as a single bolus delivery of the prodrug, the process is not limited to a single administration. The process may be repeated to provide for increased levels of drug delivery.
  • the term single bolus delivery is meant to be descriptive of first-pass delivery of the drug following an injection/application of a predetermined quantity of the prodrug.
  • the antitumor drug is used for treating a solid tumor, in another preferred embodiment, the antitumor drug is used for treating a vascularized tumor.
  • the antitumor drug is used for treating a tumor selected from the group consisting of: single cells, microinf ⁇ ltrates, microtumors, larger tumors, tumor suspended in a peritoneal cavity, tumor attached to an organ or tissue, and tumor invading an organ or tissue.
  • the described prodrugs and methods is be used to generate an antitumor response against a variety of tumors, both primary and secondary, including, but not limited to, carcinoma, hepatoma, hepatocellular carcinoma, colon carcinoma, melanoma, ovarian carcinoma, peritoneal cancer, disseminated peritoneal ovarian cancer, and neuroblastoma.
  • the utility of single bolus delivery is dependent on the ability of the drug agent to be preferentially exposed to the neoplastic tissue and penetrate the tumor cell membrane during first-pass delivery.
  • Modification of anticancer drugs through labile attachment of hydrophilic moieties transforms relatively membrane impermeable drugs into lipophilic prodrugs that facilitate increased intracellular drug concentrations and enhanced anticancer responses.
  • cancer cells and microtumors are invariably present together with the detectable and operable metastases. Their presence and continuous defoliation from primary and secondary malignancies represent one of the main impediments to the successful treatment of cancers such as peritoneal disseminated ovarian cancer.
  • the disclosed prodrug formulations target all exposed cells, single cells, microinfiltrates, microtumors, and surface cells of larger peritoneal tumors, tumor cells suspended in peritoneal cavity or attached to or invading an organ or tissue.
  • the described prodrugs also exhibit increased penetration of the drug into tumors compared to conventional drugs. We have observed drug penetration up to 500 ⁇ m (about 25 cell layers) within seconds.
  • the described formulations provide for improved delivery of anticancer drugs to cancer cells in a variety of states. The described invention could therefore be utilized following cytoreductive surgery in efforts to slow or minimize reappearance of tumors.
  • hepatocytes are targeted with a single bolus injection to the portal vein (occluded blood flow). Following a single bolus injection into the hepatic artery of normal mouse liver, targeting was evident in the hepatic artery endothelial and smooth-muscle cells, and in a few neighboring hepatocytes and sinusoidal cells. All biliary and gall bladder arteries, as well as bladder epithelium also were targeted. Bile duct cells together with some hepatocytes are targeted following a single bolus injection to the bile duct.
  • Urinary tract cells are targeted (ureter transitional epithelium nuclei, renal pelvis transitional epithelium nuclei, including beginning renal pelvis, that is the source for transitional cell carcinoma, and a majority of collecting tubules, and other epithelial compartments) following a single bolus injection to the ureter.
  • a single bolus injection into the carotid artery of a normal mouse resulted in the targeting of brain endothelial cells and both neurons and glial cells.
  • Topical administration of prodrug results in delivery to the cells to which the prodrug is directly applied. For example, topical application to the cornea or to a skin or into the lumen of the intestine results in drug delivery to the cornea epithelium, or epidermis, or enterocytes respectively.
  • the loco- regional delivery of RRH-therapeutics is uniquely suited to the treatment of primary and metastatic liver neoplasms.
  • the described formulations and process may be combined with co-delivery of compounds known to modulate drug efflux pump efficiency. This co-delivery serves to increase drug retention in the cell.
  • the present invention is also applicable to the modification and delivery to cells of mixtures of drugs, also known as drug libraries.
  • Most drugs contain nitrogen or oxygen atoms within the molecule that aid in the solubility of the drug in aqueous solutions. These atoms can be hydrophobized according to the procedures outlined in this specification.
  • the drug library can be taken up in an appropriate organic solvent such as DMF or DMSO, and be subjected to hydrophobic modification such as outlined for a single compound.
  • the derived prodrug library can then be applied to cells as outlined for a single prodrug.
  • the present invention is also applicable to a method for the hydrophobic modification of a drug or mixture of drugs via the attachment of a labile hydrophobic group to the drug wherein the hydrophobic group is labile in response to a reaction of an agent.
  • the hydrophobic group can contain a disulfide bond which, upon entry to the cell, will be cleavable by the cellular agent glutathione.
  • Hydrophobic groups compatible with the described invention contain a disulfide bond at or within 4 carbon atoms of the point of attachment of the group to the drug molecule that is susceptible to reduction by glutathione.
  • the disulfide system also possesses a hydrophobic group on one side of the disulfide bond that may be selected from the group comprising: an alkyl chain of 4 to 30 carbon atoms, and can contain sites of unsaturation, an alkyl group containing an alkyl chain and alkyl rings (aromatic and/or non aromatic), and steroids.
  • the linkages can also be designed such that they posses different lability rates in order to influence prodrug stability in vitro and in vivo.
  • kits suitable for treating a tumor comprises a rapidly reversible hydrophobized antitumor drug in a suitable solvent, a pharmaceutically acceptable carrier solution.
  • the kit additionally comprises a mixing chamber.
  • the term drug in the present invention is also meant to include the pharmaceutically acceptable salt of the drug.
  • Pharmaceutically acceptable salt means both acid and base addition salts.
  • a pharmaceutically acceptable acid addition salt is a salt that retains the biological effectiveness and properties of the free base, is not biologically or otherwise undesirable, and is formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, pyruvic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, trifluoro acetic acid, and the like.
  • a pharmaceutically acceptable base addition salt is a salts that retains the biological effectiveness and properties of the free acid, is not biologically or otherwise undesirable, and is prepared from the addition of an inorganic organic base to the free acid.
  • Salts derived from inorganic bases include, but are not limited to, sodium, potassium, calcium, lithium, ammonium, magnesium, zinc, and aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary secondary, and tertiary amines, such as methylamine, triethylamine, and the like.
  • FIG. 1 Structures for PI (I), RRH-PI (BDMODS-PI (II), C12PMMA-PI (III)) prodrugs, stable PI derivative (C12CON-PI (IV)), and C12PMMA-melphalan (V).
  • FIG. 2 Illustrations of the chemical structures of: Cicplatin (CP), BDMODS-CP, Melphalan, BDMODS-MeIp halan, the maleamic acid derivative CDMC12-Melphalan, Doxarubicin, DMODS-Dox, and the maleamic acid derivative CDMC12-Dox.
  • CP Cicplatin
  • BDMODS-CP Melphalan
  • BDMODS-MeIp halan the maleamic acid derivative CDMC12-Melphalan
  • Doxarubicin DMODS-Dox
  • CDMC12-Dox the maleamic acid derivative CDMC12-Dox.
  • FIG. 3 Passive mixing chamber with colliding flows.
  • the carrier solution aqueous carrier solution and RRH-prodrug in solvent (drug carrier solvent) are delivered to the chamber by independent syringe pumps, with passive mixing from colliding flows.
  • A Diagram
  • B Photo.
  • FIG. 4 Delivery of PI (I), BDMODS-PI (II), and C12CON-PI (IV) to various cell cultures.
  • Drug / RRH-Prodrug (20 ⁇ l of 7.48 mM solution in DMSO) was mixed with ITG (200 ⁇ l), and added to the cells. After 30 sec, the drug solution was removed (aspirated) and 2 ml growth media was again added to the cells. The cells were immediately imaged with an Axiovert SlOO fluorescent microscope (Zeiss) and the same fields were imaged with phase contrast illumination and in the rhodamine fluorescence channel using identical settings (Panels a-d).
  • Panel g - Flow Cytometry was conducted on Jurkat cells following treatment with PI (Control runs 1-4, no OS, no mixing chamber), and on a suspension of Jurkat cells in ITG, treated with PI (PI runs 1-4) or II (BDMODS-PI runs 1-4) through the mixing chamber. The results represent a histogram of the relative PI intensity of all single cell events.
  • FIG. 5 Images of SK-OV-3 cells treated with: la&b - unmodified propidium iodide (PI); 2a&b - BDMODS-PI; 3a&b - C12PMMA-PI; or 4a&b - pre-hydrolyzed BDMODS-PI.
  • Ia- 4a images under phase contrast illumination.
  • lb-4b images of the same fields under fluorescent illumination with rhodamine filter.
  • B Images of Jurkat cells treated with (i) propidium iodide or (ii) Cl 2PMM A-PI. Top panels show cells under phase contrast illumination. Bottom panels show the same field of cells under fluorescent illumination with rhodamine filter.
  • FIG. 6 Bar graph illustrating antiproliferative/cytotoxic effect of prodrugs on B16 murine melanoma cells as measured by CellTiter-Glo luminescent cell viability assay.
  • FIG. 7 Confocal images illustrating propidium iodide delivery to cells in vivo following treatment with BDMODS-PI. Targeting of cells exposed in peritoneal cavity in normal ICR mice.
  • A, B, C intraperitoneal application RRH-PI to normal peritoneal organs.
  • A Fallopian tube;
  • B Jejunum;
  • C Small monocyte infiltrate in visceral mesentery.
  • D Application of unmodified-Pi on jejunum.
  • Upper left panels - fluorescence of DNA- intercalated PI, Upper right panels - actin stained with Phalloidin Alexa 488, Lower left panels - nuclear stain with ToPro-3, Lower right panels - composite images. Frozen sections, LSM 510 confocal microscopy, bar 100 microns.
  • FIG. 8. First-pass targeting of peritoneal disseminated ovarian cancer in mouse with RRH-
  • PI Peritoneal targeting was performed via peritoneal perfusion with aspiration.
  • A Targeting of multiple cell layers in large ovarian tumor.
  • B Targeting of tumor tissue growing on colon wall.
  • C Targeting of mesenteric micrometastasis.
  • D Targeting of tumor cell cluster growing on and invading large bowel.
  • E-F Heart & lung tissues of animal that received RRH-PI via intraperitoneal perfusion. Upper left panels - fluorescence of DNA-intercalated PI, Upper right panels - actin stained with Phalloidin Alexa 488, Lower left panels - nuclear stain with ToPro-3,
  • FIG. 9 Pathological features of mouse model of disseminated peritoneal ovarian cancer, 5 wks after nude-Foxnlnu mice inoculation with human SK-OV-3 cancer cells.
  • A Micro tumor growth on duodenal mesentery (xlOO).
  • B Loose cell organization of mesentery tumor (x630).
  • C Loose tumor cell growth on duodenal wall and pancreas.
  • D Tumor cell growth on mesenteric lymph node.
  • E Tumor cell growth on abdominal surfaces of liver.
  • F Tumor cell growth on the diaphragm with invasion, all x200. Paraffin sections, H&E stain.
  • FIG. 10 Confocal images following IPPC of C12PMMA-PI: (A) Surface of a large peritoneal tumor, and (B) a micro-ovarian tumor on the surface of the colon, x630, 5 weeks post
  • SK-OV-3 cell inoculation Propidium iodide (upper left panels of A and B), ToPro-3 nuclear stain (lower left panels of A and B); Actin stained with Phalloidin Alexa 488 (upper right panel of A and B).
  • FIG. 11 Fluorescent images of liver sections following injection of modified propidium iodide (BDMODS-PI; A,B & D) or unmodified propidium iodide (C).
  • BDMODS-PI modified propidium iodide
  • A,B & D modified propidium iodide
  • C unmodified propidium iodide
  • FIG. 12 Delivery of BDMODS-PI to mouse liver with colon metastases. Left - *400 confocal image of liver with colon carcinoma tumors, arrow indicates portal tract with artery labeled, arrowheads indicate liver metastasis with vast majority of cells labeled.
  • FIG. 13 Days of survival following LABI delivery of Cl 2PMM A-PI (III) or hydrolyzed
  • C12PMMA-PI to C57BL mice. Following three weeks of MC38 tumor development C12PMMA-PI or Hydrolyzed C12PMMA-PI (0.150 ⁇ mol in 20 ⁇ l DMSO, 200 ⁇ l ITG) were delivered by LABI. The mouse abdomen was closed 4 min after drug treatment and the animals were monitored for survival time.
  • FIG. 14 First-pass delivery of labile hydrophobic drugs to: (A) hepatic artery endothelial and smooth muscle cells; (B) Gall bladder vascular and epithelial cells; (C) bile duct epithelia and nearby hepatocytes; (D) hepatocytes; (E) endothelial cells and neurons; (F) mouse liver containing metastises following injection of modified propidium iodide into the portal vein; (G) hepatic artery endothelia, smooth-muscle cells and tumors cells staining with modified propidium iodide; (H) ureter transitional epithelia; (I) renal pelvis transitional epithelia; (J) beginning renal pelvis epithelia; (K) collecting tubules; and, (L) cornea epithelia.
  • A hepatic artery endothelial and smooth muscle cells
  • B Gall bladder vascular and epithelial cells
  • C bile duct epithelia and nearby he
  • INOVA 400 spectrometer Mass analysis was conducted on a PE Sciex API 150EX mass spectrometer. Cells were purchased from ATCC (Manassas, VA), unless otherwise noted, and cultured according to the distributor's instructions.
  • Example 1 Labile (rapidly reversible) and non-labile hydrophobic modifications of propidium iodide.
  • Propidium iodide was utilized as a model reporter-drug. This membrane impermeable reporter drug is routinely used as a fluorescent agent to visually identify cells possessing compromised membranes. Cells with intact cellular membranes effectively exclude propidium iodide.
  • Propidium iodide exhibits a 20-30-fold enhanced fluorescence upon intercalation into DNA, facilitating detection of propidium iodide positive (PI + ) cells.
  • PI + propidium iodide positive
  • IH NMR analysis 300 MHz, N,N-dimethylformamide-d7
  • a trisilylated PI was observed (based on integration), arising from two silylation reactions taking place on one of the PI amino groups.
  • the second minor component was identified as the phenanthridinium salt of BDMODS-PI (verified by independent synthesis, from the reaction of PI and chloro(dimethyl)octadecylsilane in dichloromethane in the absence of a base).
  • a second modification utilizes an amidation reaction between PI and the disubstituted maleic anhydride derivative N-dodecyl-3-(4-methyl- 2,5-dioxo-2,5-dihydro-furan-3-yl)-propionamide (C12PMMA), to form the bis-maleamic acid
  • Maleamic acids are known to be labile under acidic pH, reverting to the amine and the cyclic anhydride, with derivatives of disubstituted maleic anhydride showing the most rapid rate of hydrolysis (ti/ 2 ⁇ 5 min at pH 5)
  • Example 3 Hydrophobic modification of cisplatin chemotherapeutic.
  • silylation of cisplatin cis-diamminedichloro-platinum(II), FIG. 2
  • DMODSiCl chloro(dimethyl)octadecylsilane
  • DMODS-Doxorubicin (FIG. 2).
  • Doxorubicin HCl (2.00 mg, 0.00345 mmol, Aldrich) was taken up in 200 ⁇ L of DMSO.
  • molecular sieves (3A 5 20 mg), K 2 CO 3 (4.8, 0.035 mmol), and chloro(dimethyl)-octadecylsilane (2.4 mg, 0.0069 mmol, Aldrich).
  • the reaction was stirred at ambient temperature. After 16 h, the resulting blue solution was diluted with DMSO (200 ⁇ L) and centrifuged to remove solids to afford DMODS-Doxorubicin.
  • Example 5 Development of a Mixing Chamber. Due to the instability of the RRH-PI prodrugs in water, it was necessary to dissolve them in a small amount of pharmaceutically acceptable organic solvent (OS) and mix them with an aqueous solution immediately prior to delivery. Rapid and efficient mixing was critical for optimum delivery and minimalization of toxicity due to the organic solvent.
  • a passive mixing chamber with colliding flows was utilized to mix the drug / RRH-prodrug (dissolved in a carrier solvent such as an organic solvent) with an aqueous solution immediately prior use.
  • Two syringe pumps (Harvard Pumps, PHD 2000) were utilized to deliver the solutions to the mixing chamber.
  • mixtures were 1 :10 by volume with 0.67 ⁇ l of PI or RRH-PI/OS solution (7.48 mM) diluted with 6.7 ⁇ l of isotonic glucose (ITG), buffer, or media per sec, for a total delivery volume of 220 ⁇ l (0.150 ⁇ mol of PI or RRH- PI) over 30 sec (final organic solvent concentration of 9.1% by vol.).
  • ITG isotonic glucose
  • the mixing chamber (FIG. 3) was constructed from 18 G stainless steel tubing (38 mm in length) by drilling a 0.2 mm hole at a 45 degree angle in the tubing wall and then inserting 30 G stainless steel tubing. The 30 G stainless tubing was angled and advanced into the 18 G tubing so as to be centered within the larger tubing and then soldered in place. Tubing connectors were attached to the chamber inlets by successively soldering 23 G and 27 G stainless steel tubing in place. Polyethylene tubing (PElO Intramedic tubing, Becton Dickinson and Company) was attached to the chamber inlets for connection to the syringes. Drug or prodrug in organic solvent and aqueous solution (1 : 10 by volume) were pumped in a colliding direction, creating a turbulent mixing flow
  • Example 6 Analysis of prodrug lability.
  • the prodrugs were tested for their rate of hydrolysis at pH 6.0, 7.2 and 8.5 using fluorescent spectroscopy.
  • Lability studies for C12CON- PI and RRH-PI (rapidly reversible hydrophobic-PI) derivatives were conducted on a Cary Eclipse Fluorescence Spectrophotometer (Varian Inc.).
  • Example 7 In Vitro cellular uptake of PI, RRH-PI compounds, and IV evaluated by fluorescence measurements. PI and the RRH-prodrugs were tested for cellular uptake on several cell lines which included B16 (murine melanoma), Hepa 1-6 (mouse hepatoma), SK-OV-3 (also SKO V-3, human ovarian carcinoma), OVCAR-3 (human ovarian carcinoma), Jurkat (human T- lymphocyte), 293 (human embryonic kidney), and MC38 (mouse colon carcinoma) cells. Adherent cells were plated at 2.5 ⁇ l O 5 cells / well on 6-well plates 24 h prior to testing.
  • the growth media was removed from individual wells and the drug / prodrug was immediately added to the well with the mixing chamber.
  • Final drug / prodrug concentrations were 0.150 ⁇ mol in 220 ⁇ l total volume (20 ⁇ l OS, 200 ⁇ l ITG). After 30 sec the drug solution was removed (aspirated) and 2 ml complete media was added to the cells.
  • prodrug that was hydrolyzed prior to application to the cells followed a similar protocol, however the combined solution obtained after passing through the mixing chamber was collected and allowed to sit at ambient temperature for 5 min prior to application on the cells.
  • Calcein AM Invitrogen, 100 ⁇ l of 20 ⁇ M solution in complete media
  • SYTOX Green (Invitrogen) followed a similar protocol for drug treatment.
  • SYTOX Green (20 ⁇ l of 5 ⁇ M solution in 20 mM Hepes, pH 7.4) was added to the wells and the cells were examined. Each condition was repeated in 2 or 3 wells, and the cells were immediately examined by microscopy.
  • IV was also mixed with PI and then delivered to SK-OV-3 cells, and the cells were imaged as before.
  • equal amounts of IV (15.0 mM in DMSO) and PI (15.0 mM in DMSO) were combined and the resulting solution was added to cells via the mixing chamber.
  • PI was taken up in the ITG solution (0.748 mM) and mixed with IV (7.48 mM in DMSO) in the mixing chamber.
  • V (15 mM and 30 mM in DMSO) was mixed with PI and delivered to Hepa 1-6 and SK-OV-3 cells using the second method. The cells were imaged as before following 30 sec - 4 min exposure to the drug solution.
  • the cells were isolated by centrifugation and resuspended in ITG at a density of 4.0 ⁇ 10 6 cells / ml. Exposure to the drug / prodrug was conducted by passing the cells in suspension in ITG (200 ⁇ l) through the mixing chamber. After 30 sec the cells were centrifuged and the drug solution was removed (aspirated), the cells were resuspended in complete media (2 ml) and plated in 6-well plates for imaging. Passing the cells through the mixing chamber (no drug / prodrug treatment) had no effect on cell viability when compared to cells plated without passing through the mixing chamber.
  • Reported data represents a histogram of PI intensity for single cell events from four independent preparations. Cells were considered PI positive at a value of 100 on the PI intensity axis of the histogram for the cell percentages described.
  • PI, RRH-PI prodrugs, and IV were tested for the ability to stain viable cells (both tumor cells and lymphocytes) which included B16 (murine melanoma), Hepa 1-6 (mouse hepatoma), SK-OV-3 (human ovarian carcinoma), OVCAR-3 (human ovarian carcinoma), Jurkat (human T-lymphocyte), 293 (human embryonic kidney), and MC38 (mouse colon carcinoma) cells. Stained cells is indicative of successful PI-prodrug uptake, intracellular release of functional PI, and DNA intercalation.
  • FIG. 4a-b show representative results following the application of PI and II to Hepa 1-6 cells. Unmodified PI stained very few cells (FIG.
  • FIG. 5A-B show additional results from the addition of PI, RRH-PI prodrugs, or hydrolyzed PI-prodrug to SK-OV-3 (FIG. 5A) or Jurkat (FIG. 5B) cells.
  • SK-OV-3 cells unmodified propidium iodide stained very few cells (FIG. 5 A, panel 1), representing normally occurring dead cells in the population.
  • BDMODS-PI and C12PMMA-PI stained 60-80% of the cells (FIG. 5A, panels 2-3), demonstrating that the hydrophobically modified prodrugs efficiently enter viable human ovarian cancer cells with successful intracellular formation of active free propidium iodide.
  • Flow cytometry was used to quantitate prodrug delivery to Jurkat cells.
  • Flow cytometry of cell suspensions that were treated with PI in aqueous solution indicated very low PI uptake in most cells, with very few displaying a signal above 100 (0.3 ⁇ 0.1 %, control run 1-4, FIG. 4g).
  • Flow cytometry on cells that were suspended in ITG, and passed through the mixing chamber with II in DMSO indicated that nearly all cells were PI positive (99.5 ⁇ 0.1%, BDMODS-PI run 1-4).
  • the results from the flow cytometry correlated well with our observations using fluorescent microscopy for cells treated with RRH-PI prodrugs.
  • the stable PI derivative, IV was also tested for cellular uptake in SK-OV-3 cells (FIG. 4e- f). Immediately following application of IV, a diffuse signal was observed along the cell membrane in the fluorescein channel (FIG. 4e). After one hour incubation, a more defined punctate signal was observed (FIG. 4f), likely due to endocytosis of the membrane-bound derivative. Thus, in the absence of lability, the hydrophobic prodrug was sequestered and retained in the membrane.
  • Calcein AM was used as a live cell marker in Hepa 1-6 cells following treatment with II (FIG. 4h). Calcein AM is a cell permeable non- fluorescent dye that is converted to fluorescent calcein by intracellular esterases. Following cellular treatment with II, approximately 86 % of the cells were both PI and calcein positive, indicating viable cells. In another experiment, SYTOX Green, a dead cell indicator, was added to cells following treatment with II.
  • Example 8 Antiproliferative/Cytotoxic in vitro studies on PI, RRH-PI prodrugs, melphalan, and Cl 2PMMA-melphalan.
  • In vitro cytotoxicity testing was conducted on Hepa 1-6 (mouse hepatoma), SK-OV-3 (human ovarian carcinoma), and MC38 (mouse colon carcinoma) cells using a tetrazolium based assay (WST, Dojindo Molecular Technologies).
  • Hepa 1-6 (2.OxIO 5 cells well), SK-OV-3 (7.5xlO 5 cells well), and MC38 (8.5xlO 5 cells well) were seeded in 1000 L media into 12-well plates 24 h prior to testing (starting confluency -50%).
  • drug / prodrug solution was added drop-wise to quadruplicate wells, using the mixing chamber. Effective drug concentrations were calculated from the amount of drug added in the total volume of OS and aqueous solution. Following 4 min of drug solution exposure, the solution was removed, and 1000 1 fresh complete media was added to each well.
  • WST-I (20 ⁇ l of a 5 mM solution in PBS) and N-methylphenazonium methyl sulfate (PMS, 20 ⁇ l 0.2 mM solution in PBS) were added and the cells were incubated for 1-4 h.
  • PMS N-methylphenazonium methyl sulfate
  • the amounts of WST- 1 and PMS were doubled.
  • 100 ⁇ l of sample was transferred to quadruplicate wells on a 96-well plate, and the absorbance (438 nm) values were measured on a SPECTRAmax Plus 384 microplate spectrophotometer (Molecular Devices Corporation).
  • Data represents the mean A438 values of 16 wells with standard deviation, corrected for media contribution, and normalized against cells in media with no drug / prodrug treatment (reported as % cell viability ⁇ standard deviation).
  • concentration of drug ( ⁇ M) that is required for 50% inhibition in vitro is reported as the IC50.
  • the reported values were calculated from the best fit line (Excel) for a plot of effective drug concentration against % cell viability, and include the r 2 value.
  • the reported IC50 values for multiple RRH-PI plots are reported as IC50 ( ⁇ M) ⁇ standard deviation.
  • the WST-I cell viability assay at 48 h post treatment indicated an IC 50 of 334 ⁇ M ( ⁇ 72 ⁇ M) for II, while PI again had little effect on cell viability at the highest concentration tested (93 ⁇ 5.7% cell viability at 1.7 mM).
  • II that was premixed with ITG (10 min) before application cells resulted in levels of cell viability that were similar to the PI treated cells (108 ⁇ 1.3% at 833 ⁇ M, the highest concentration tested).
  • the stable PI derivative IV was also tested for toxicity in Hepa 1-6 cells at 24 and 48 h post treatment, resulting in IC 50 's of 585 ( ⁇ 279 ⁇ M) and 429 ⁇ M ( ⁇ 37 ⁇ M) respectively.
  • the toxicity resulting from IV may have been the result of endocytosis of the prodrug (hydrolysis of the amide would then yield PI), or its cationic amphipathic property. Regardless, the rapidly reversible prodrug II was more cytotoxic than IV, and would be less likely to cause cellular toxicity of non-targeted cells. Similar results were obtained with SK-OV-3 cells (Table 3).
  • MC38 (150) (94) a- Cell viability was -80% of no treatment control at these maximum concentrations. b- Cell viability was > 90% of no treatment control at these maximum concentrations. c- Cell viability was -70% of no treatment control at these maximum concentrations. Cell viability was >90% in cultures exposed to the vehicle alone (DMSO/ITG).
  • Example 9 Enhanced antiproliferative/cytotoxic effect of RRH-PI and RRH-cisplatin prodrugs on B 16 murine melanoma cells.
  • hydrophobically modified drugs demonstrate enhanced antitumor activity.
  • BDMODS-CP reduced RLU levels to those observed with blank wells (media only wells without B16 cells), indicating complete cytotoxic effect against the B16 tumor cells. Similar trends were observed for PI and BDMODS-PI. These results clearly indicate that hydrophobic modifications of PI, cisplatin, and melphalan facilitates enhanced antitumor effects against melanoma and colon carcinoma cells in vitro.
  • Example 11 BDMODS-PI and C12PMMA-PI show enhanced drug uptake by surface tissue following IP application.
  • IP application of propidium iodide, BDMSODS-PI, and Cl 2PMM A-PI to both normal mice and in a mouse model of disseminated peritoneal ovarian cancer. All procedures were executed under Isoflurane inhalation anesthesia. In normal mice either the abdominal cavity was opened and the drug mixture was directly applied on abdominal organs (220 ⁇ L of drug-OS/ITG over 30 sec), or the drug mixture was injected through the abdominal wall (1 mL of drug-OS/ITG over 1 min). For both delivery protocols, the dual pump mixing chamber was used.
  • Example 12 BDMODS-PI and CI2PMMA-PI show enhanced drug uptake by surface tissue and microtumors following IP and intraperitoneal perfusion chemotherapy (IPPC) application in a mouse tumor model.
  • IPPC intraperitoneal perfusion chemotherapy
  • 2x10 6 SK-OV-3 cells were injected IP into nude mice.
  • the mice were examined at two weeks following cell inoculation, or at the first manifestation of ascites (about 4-5 wks).
  • Tissue samples were fixed in 10% NBF, routinely processed, stained with H&E stain, and subjected to histopatho logical analysis.
  • tumor lesions appeared to be more susceptible to prodrug uptake, showing greater tissue penetration and intense PI + -staining as compared to non-malignant tissues.
  • Extensive sectioning and analysis indicated that tumors of any size were effectively targeted (up to 500 ⁇ m from the tumor surface). Tumors without propidium iodide-staining were not observed.
  • similar analyses indicated near-exclusive PI + -staining of the outer cells exposed to the peritoneal cavity. Cells situated deeper in the tissues were labeled at a much lower intensity or not at all.
  • peritoneal fluid was again aspirated, and the peritoneal cavity was perfused with 10 mL of PBS via the top catheter, together with simultaneous aspiration via the lower two catheters. Special care was taken to avoid elevated abdominal pressure during the procedure. All animals survived the perfusion well and were sacrificed 3-5 h later. Confocal microscopy indicated a similar staining pattern as observed previously, with strong propidium iodide nuclear labeling of all of the outer cells exposed to peritoneal cavity, including ovarian tumors. Large tumors (5-7 mm) were labeled to a depth of about 500 microns (FIG.
  • FIG. 10A shows that all tumor cells in microtumors (0.1-1 mm) were heavily labeled throughout the tumor (FIG. 10B). All cells exposed to peritoneal cavity and thereby to the prodrug solution indicated prodrug uptake and staining, including: mesentery cells, outer layer of cells of abdominal organs, and disseminated peritoneal ovarian tumors. The tumor lesions still appeared to be more susceptible to drug uptake, indicating both greater tissue penetration and intense Pi -staining as compared to normal abdominal tissue, with the exception of the mesentery which was also was heavily PI + .
  • tissue penetration of small molecular weight drugs is difficult to accomplish.
  • tissue penetration depths many tumor types
  • anti- neoplasties on the order of hours to days for 50-500 ⁇ m penetration.
  • our prodrugs resulted in a 500 ⁇ m penetration depth within 10 min. This could be a result a more effective interaction of the hydrophobic prodrug with the cell membrane, similar to what is described in the literature as lateral diffusion.
  • Example 13 Enhanced prodrug uptake by hepatic metastases following single bolus injection. All animal experiments were performed in accordance with Institutional Animal Care and Use Committee protocols. All surgical procedures were performed under Isoflurane anesthesia. Liver tumors models were established in C57BL mice using MC38 (colon carcinoma) and Hepa 1-6 (hepatoma), in BALB/c mice using B16 (melanoma), and in A/J mice using NXS2 (neuroblastoma) cell lines. Mice were inoculated via the portal vein with IxIO 4 MC38 cells or 0.5-1.25xlO 6 Hepa 1-6 cells, or via the tail vein with 0.5-1.25xlO 6 B16 or NXS2 cells. Tumor formation was allowed to progress for 2-3 weeks with periodic examination for tumor growth prior to drug / prodrug treatment. Microscopically, all tumors were arterialized with minimal necrosis or apoptosis.
  • Drug / prodrug solutions were delivered via a liver arterial bolus injection (LABI) to the right gastro-duodenal artery for retrograde delivery to the hepatic artery similar to procedures used clinically.
  • the right gastro-duodenal artery was freed from surrounding tissue and the common hepatic artery was clamped occluding blood flow.
  • the distal part of the right gastro duodenal artery was sutured, a 35 G needle was inserted into the gastro duodenal artery, and secured during the single bolus injection. Following the injection, the needle was retracted and the proximal part of gastro duodenal artery was sutured, and hepatic artery flow was restored.
  • the celiac trunk was clamped close to the aorta and a 35 G needle was inserted above the clamp.
  • the left gastric, splenic, and gastro-duodenal arteries were clamped in order to direct all of the drug solution to the liver. This latter approach was advantageous in the C57BL mouse model because of anatomical variations involving the hepatic artery.
  • LABI delivery of drug / prodrug (0.150 ⁇ mol, 220 ⁇ l total injection volume) and controls were performed using the mixing chamber and syringe pumps as previously described.
  • the livers were harvested 5 min after LABI, snap frozen in O. C. T.
  • both the portal vein and the celiac artery were clamped to occlude blood flow into the liver.
  • the portal vein and the celiac artery were not clamped, preserving full portal blood flow during the experiment.
  • the vena cava was not clamped in order to avoid increased pressure within the liver during the injection procedure.
  • the blood in the liver was flushed out with 1 ml of ITG delivered through the portal vein (1 min). Then 0.300 ⁇ mol of PI or II in 40 ⁇ l of DMF was delivered together with 400 ⁇ l of ITG via the mixing chamber. Five min after injection the livers were perfused with 3 ml of ITG to flush any drug remaining in the vasculature, and livers were sectioned as above or the hepatocytes were isolated.
  • mice In order to test in vivo tumor utility, a tumor model was established in mice with MC38 (mouse colon carcinoma) cells. All animals were examined for tumor development prior to testing with drug or RRH-prodrug, to insure that the developed tumors were large enough to insure hepatic arterialization (generally 3-5 mm, 2 to 3 weeks post inoculation). Following three weeks of tumor development PI and RRH-PI prodrug solutions were delivered using the mixing chamber via a liver arterial bolus injection (LABI). As shown in FIG. 1 IA-B, left panel, delivery of BDMODS-PI resulted in intense, near-exclusive Pi-staining of MC38 liver metastases, while normal parenchyma appeared relatively free of PI staining.
  • a liver arterial bolus injection As shown in FIG. 1 IA-B, left panel, delivery of BDMODS-PI resulted in intense, near-exclusive Pi-staining of MC38 liver metastases, while normal parenchyma appeared
  • Confocal imaging was also utilized to approximate the numbers of PI positive cells following LABI in three different regions of the liver: a) MC38 metastases, b) portal vein areas of the liver, and c) liver parenchyma representing zone 2 of the hepatic unit.
  • Five images (0.262 mm 2 each) were obtained for each compartment from a lobe with MC38 tumors and the total number of nuclei (ToPro-3-positive staining) and PI positive nuclei were determined.
  • LABI with II resulted in an average of 94% of cells in MC38 metastases being PI positive. In the portal vein region, an average of 23% of the cells were PI positive.
  • PI positive cells in this region were comprised of arterial cells, some adjoining hepatocytes, and bile duct cells (all of which are exposed to II during the injection). In the liver parenchyma region, an average of 1% of cells were PI positive. These data were used to estimate the overall percentage of non tumor cells in the liver that were PI positive. As the portal vein region comprises less than 5-7% of the liver (in humans, similar in rodent), the total number of PI positive non tumor cells in the liver can be estimated to be 2-3%. To demonstrate the arterial supply difference between tumor and hepatocytes, RRH-PI prodrug was delivered into the portal vein of normal and tumor-bearing mice with no clamping of liver outflow.
  • Example 14 Uptake of BDMODS-PI by hepatocytes in vivo. Intraportal injections were conducted in order to target hepatocytes. For the injections, both the portal vein and the celiac artery were clamped to occlude blood flow into the liver. The vena cava was not clamped in order to avoid increased pressure within the liver during the injection procedure. The blood in the liver was flushed out with 1 ml of ITG delivered through the portal vein (1 min). 0.300 ⁇ mol PI or II in 40 ⁇ l of DMF was then delivered together with 400 ⁇ l ITG via the mixing chamber.
  • livers were perfused with 3 ml of ITG to flush any drug remaining in the vasculature, and livers were sectioned as above or the hepatocytes were isolated.
  • the amount of PI retained in hepatocytes was also quantitated following intraportal injections of PI and II.
  • the isolated hepatocyte cell suspensions were dissolved in 0.5% octyl glycoside in 10 mM HEPES buffer, pH 7.5. Nucleic acid was then isolated from cell suspensions (0.5 ml) using phenol extraction.
  • PI fluorescence spectra were monitored (Shimadzu RF 1501 Spectrophotometer) using an excitation wavelength of 530 nm and an emission wavelength of 617 nm. All spectra were background subtracted using the fluorescence of cell suspensions from untreated animals.
  • PI calibration curves were generated by mixing increasing amounts of PI in each of the samples.
  • the obtained curves were linear, indicating that PI binding to nucleic acid in the samples was not saturated, and the fluorescence was a result of nucleic acid bound PI and not free PI, thus allowing for determination of the amount of PI present in the samples.
  • Example 15 Delivery of RRH-PI to rapidly dividing cells results in a decrease in the number of cells in mitosis.
  • the effect of drug / prodrug treatment was also evaluated on rapidly dividing cells following a 70% partial hepatectomy on normal ICR mice. Following the hepatectomy, PI or III (0.150 ⁇ mol, 220 ⁇ l total injection volume) were delivered to the liver via the portal vein using the mixing chamber. The abdominal cavity was closed in two layers with 4- 0 Braunamid suture. After 48 h, the animals were sacrificed, the livers were harvested, formalin fixed, paraffin imbedded, sectioned, and H&E stained. The sections were examined by microscopy on an Axioplan2 fluorescent microscope (Zeiss).
  • metaphase hepatocyte mitotic figures
  • PI and III were intraportally injected (occluded blood flow) into normal mice immediately after being subjected to a 70% hepatectomy in order to evaluate their effect on rapidly dividing cells. At 2 days after treatment with PI an average of 7.0% ( ⁇ 2.2) of the hepatocytes were in metaphase. In contrast, after treatment with III an average of 2.3% ( ⁇ 2.9) of the hepatocytes were in metaphase. These results are indicative of an antiproliferative effect for III.
  • Example 16 In vivo antitumor effect following a liver arterial bolus injection with a RRH-
  • Example 17 Propidium iodide delivery to a variety of target cells. Propidium iodide and RRH-PI prodrugs were delivered via injection or topical administration using the mixing chamber and as detailed below.
  • Injection into the bile duct of normal mouse liver Injection of II into bile duct of normal mouse liver resulted in strong nuclear staining of all bile duct epithelial cells as well as staining in hepatocytes near the bile duct (FIG. 14C).
  • mice were inoculated with MC38 colon carcinoma cells. After three weeks, mice were injected with modified PI. Injection of 400 ⁇ l propidium iodide into the portal vein of mouse liver with cancer metastases did not result in nuclear staining of any structures (FIG. 14F, 200 ⁇ , top panel - metastisis, bottom panel - portal triad). However, injection of 350 ⁇ l II into hepatic artery of mouse liver with cancer metastases resulted in strong nuclear staining of hepatic artery endothelial and smooth-muscle cells, and in strong nuclear staining of the metastases
  • FIG. 14G top panel - 10Ox, bottom panel - 20Ox).
  • Injection into the ureter and bladder of normal mice Injection of II into the ureter of normal mice resulted in strong staining of ureter transitional epithelium nuclei (FIG. 14H), renal pelvis transitional epithelium nuclei (FIG. 141), including beginning renal pelvis (FIG. 14J), and a majority of collecting tubules (FIG. 14K). Injection was performed using similar 35 G needle into right ureter close to the bladder. Injection into emptied bladder resulted strong staining of bladder transitional epithelium.

Abstract

L'invention porte sur un médicament anti-tumoral rendu hydrophobe rapidement réversible, en vue d'une utilisation dans un procédé pour traiter une tumeur. Le groupe hydrophobe est lié de manière covalente au médicament anti-tumoral par l'intermédiaire d'une liaison rapidement réversible. Une modification hydrophobe augmente l'administration de médicament, tandis que la labilité rend minimale l'entrée du médicament à l'intérieur de cellules non cibles.
PCT/EP2008/064971 2007-11-06 2008-11-05 Hydrophobisation réversible pour une administration de médicament de premier passage WO2009059984A2 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US6590071B1 (en) * 1998-12-10 2003-07-08 University Of Southern California Reversible aqueous pH sensitive lipidizing reagents, compositions and methods of use
WO2003100081A2 (fr) * 2002-05-24 2003-12-04 Mirus Corporation Modification reversible de l'interaction membranaire
US20040151766A1 (en) * 2003-01-30 2004-08-05 Monahan Sean D. Protein and peptide delivery to mammalian cells in vitro
WO2005026083A2 (fr) * 2003-09-08 2005-03-24 Mirus Bio Corporation Transfert ameliore de medicaments par des modifications hydrophobes instables

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US5936092A (en) * 1995-01-25 1999-08-10 The University Of Southern California Methods and compositions for lipidization of hydrophilic molecules
US6590071B1 (en) * 1998-12-10 2003-07-08 University Of Southern California Reversible aqueous pH sensitive lipidizing reagents, compositions and methods of use
WO2003100081A2 (fr) * 2002-05-24 2003-12-04 Mirus Corporation Modification reversible de l'interaction membranaire
US20040151766A1 (en) * 2003-01-30 2004-08-05 Monahan Sean D. Protein and peptide delivery to mammalian cells in vitro
WO2005026083A2 (fr) * 2003-09-08 2005-03-24 Mirus Bio Corporation Transfert ameliore de medicaments par des modifications hydrophobes instables

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MONAHAN SEAN D ET AL: "Rapidly reversible hydrophobization: an approach to high first-pass drug extraction." CHEMISTRY & BIOLOGY SEP 2007, vol. 14, no. 9, September 2007 (2007-09), pages 1065-1077, XP002524450 ISSN: 1074-5521 *

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