WO2001032218A1 - Compositions and methods for controlled-release delivery and increased potency of pharmaceuticals via hydrophobic ion-pairing - Google Patents
Compositions and methods for controlled-release delivery and increased potency of pharmaceuticals via hydrophobic ion-pairing Download PDFInfo
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- WO2001032218A1 WO2001032218A1 PCT/US2000/030424 US0030424W WO0132218A1 WO 2001032218 A1 WO2001032218 A1 WO 2001032218A1 US 0030424 W US0030424 W US 0030424W WO 0132218 A1 WO0132218 A1 WO 0132218A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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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
- A61K47/541—Organic ions forming an ion pair complex with the pharmacologically or therapeutically active agent
Definitions
- the present invention relates to compositions and methods for improved efficacy of pharmaceutical compounds and controlled-release drug delivery systems. Antimicrobials having improved antimicrobial activity and anticancer agents having improved cytotoxic activity are provided using the process of hydrophobic ion pairing. The present invention also relates to the use poloxamer gels to deliver hydrophobic ion-paired pharmaceuticals.
- Many drug therapies require controlled and sustained release of the pharmaceutical product to a patient to optimize effectiveness of the pharmaceutical.
- These pharmaceutical products which include bioactive proteins or peptides, as well as small molecule drugs, are typically administered systemically. Administration of these drugs may be parenteral (e.g., intravenous, intramuscular or subcutaneous) or enteral (i.e., by mouth).
- parenteral e.g., intravenous, intramuscular or subcutaneous
- enteral i.e., by mouth.
- Many types of drug delivery are faced with a significant limitation in that there is an initially high level of drug provided to the patient immediately following administration, but there is no constant and controlled release of the drug (i.e., where the level of the drug remains constant over an extended period of time).
- compositions and methods for improving the longevity, potency and/or stability of pharmaceuticals in physiological conditions are provided.
- infections represent a significant health risk, which is acute in some groups of patients, including hospitalized individuals, as well as those with underlying disease conditions and/or immune defects. In particular, infections represent a serious risk to diabetic patients.
- Chemotherapy either for treating cancer or other diseases (e.g., diseases of the peritoneal cavity) is often accompanied by significant toxicity.
- cis-diamminedichloroplatinum (II) cisplatinum diammine dichloride; cisplatin
- II cisplatinum diammine dichloride
- cisplatin is a widely used chemotherapeutic agent for the treatment of many different cancers.
- due to the risk of neurotoxicity and nephrotoxicity its clinical application is limited to low-dose levels.
- compositions comprising cisplatin that are capable of being more bioavailable.
- methods and compositions are needed in the art are those which provide efficient, safe and effective treatment of disease.
- the present invention provides compositions and methods for improved efficacy of pharmaceutical compounds and controlled-release drug delivery systems by forming a hydrophobic ion-paired (HIP) surfactant complex.
- HIP hydrophobic ion-paired
- antimicrobials having improved antimicrobial activity and anticancer agents having improved cytotoxic activity are synthesized using the process of hydrophobic ion pairing (HIP).
- the present invention provides methods for metal-assisted hydrophobic ion pairing, the use of poloxamer gels to deliver hydrophobic ion-paired pharmaceuticals, and methods to treat a subject using the compositions provided by the present invention.
- the present invention provides methods for preparing pharmaceuticals with controlled-release properties, where the pharmaceutical is in a hydrophobic ion-paired complex with an amphiphilic counterion, where the complex has reduced aqueous- solubility, and further where the hydrophobic ion-paired complex is isolated.
- the hydrophobic ion-paired complex is dried to form a pharmaceutical powder.
- the invention provides the controlled- release pharmaceutical produced by the method.
- the present invention provided a method for preparing pharmaceuticals with controlled-release properties, where the pharmaceutical is an antimicrobial.
- the pharmaceutical used in the method is selected from vancomycin, tetracycline, tacrine, L-phenylephrine, cisplatin, isoniazid and isoniazid methanesulfonate.
- the present invention provides a method for preparing pharmaceuticals with controlled-release properties, where the pharmaceutical is in a hydrophobic ion-paired complex with an amphiphilic counterion, and where the amphiphilic counterion is selected from bis(2-ethylhexyl) sodium sulfosuccinate, sodium dodecyl sulfate (SDS), sodium octyl sulfate, sodium tetradecyl sulfate, sodium octadecyl sulfate, cholesterol sulfate, sodium laurate, cholesterol sulfate, sodium dodecyl sulfonate, sodium decyl sulfonate, sodium octyl sulfonate, sodium oleate, 1-heptanesulfonic acid, cetyl trimethyl ammonium bromide (CTAB), palmitoylcholine chloride, palmitoyl carnitine chloride (PCC),
- the invention provides a composition for pharmaceutical delivery comprising a hydrophobic ion-paired complex and a poloxamer solution having reverse thermal gelation properties.
- the poloxamer is poloxamer 407.
- the present invention provides a method for preparing a hydrophobic ion-paired complex comprising a neutral pharmaceutical or a zwitterionic pharmaceutical, and further where the hydrophobic ion-paired complex is formed by metal-assisted hydrophobic ion- pairing.
- the metal-assisted hydrophobic ion-paired complex is dried to form a pharmaceutical powder.
- the present invention provides a method for preparing pharmaceuticals with controlled-release properties, where the pharmaceutical is in a metal- assisted hydrophobic ion-paired complex with an amphiphilic counterion, and where the amphiphilic counterion is selected from bis(2-ethylhexyl) sodium sulfosuccinate, sodium dodecyl sulfate (SDS), sodium octyl sulfate, sodium tetradecyl sulfate, sodium octadecyl sulfate, cholesterol sulfate, sodium laurate, cholesterol sulfate, sodium dodecyl sulfonate, sodium decyl sulfonate, sodium octyl sulfonate, sodium oleate, 1-heptanesulfonic acid, cetyl trimethyl ammonium bromide (CTAB), palmitoylcholine chloride, palmitoyl carnitine chloride (CTAB),
- the pharmaceutical in the metal-assisted hydrophobic ion-paired complex is isoniazid. In another embodiment, the pharmaceutical in the metal-assisted hydrophobic ion-paired complex is a polypeptide. In other embodiments, the metal ion in the metal-assisted hydrophobic ion-paired complex is multivalent. In another embodiment, the metal ion is divalent. In another embodiment, the metal ion is calcium or zinc.
- the present invention provides a metal-assisted hydrophobic ion paired complex.
- the invention provides a composition for pharmaceutical delivery comprising a metal-assisted hydrophobic ion-paired complex and a poloxamer solution having reverse thermal gelation properties.
- the poloxamer is poloxamer 407.
- the present invention provides a hydrophobic ion-paired complex comprising an antimicrobial.
- the hydrophobic ion-paired complex is dried to form a pharmaceutical powder.
- the invention provides a hydrophobic ion-paired complex comprising an antimicrobial where the antimicrobial is selected from the group tetracycline, vancomycin, isoniazid and isoniazid methanesulfonate.
- the invention provides a composition for pharmaceutical delivery comprising a hydrophobic ion-paired complex comprising an antimicrobial and a poloxamer solution having reverse thermal gelation properties.
- the poloxamer is poloxamer 407.
- the present invention provides a method for preparing a hydrophobic ion-paired complex comprising a pharmaceutical with improved efficacy.
- the metal-assisted hydrophobic ion-paired complex is dried to form a pharmaceutical powder.
- the present invention provides a pharmaceutical with improved efficacy.
- the pharmaceutical in the hydrophobic ion-paired complex is an anti-cancer agent or an antimicrobial.
- the pharmaceutical is selected from vancomycin, tetracycline, tacrine, L-phenylephrine, cisplatin, isoniazid or isoniazid methanesulfonate.
- the present invention provides a method for preparing pharmaceuticals with improved efficacy, where the pharmaceutical is in a metal-assisted hydrophobic ion-paired complex with an amphiphilic counterion, and where the amphiphilic counterion is selected from bis(2-ethylhexyl) sodium sulfosuccinate, sodium dodecyl sulfate (SDS), sodium octyl sulfate, sodium tetradecyl sulfate, sodium octadecyl sulfate, cholesterol sulfate, sodium laurate, cholesterol sulfate, sodium dodecyl sulfonate, sodium decyl sulfonate, sodium octyl sulfonate, sodium oleate, 1-heptanesulfonic acid, cetyl trimethyl ammonium bromide (CTAB), palmitoylcholine chloride, palmitoyl caraitine chloride
- the invention provides a composition for improved pharmaceutical efficacy, comprising a hydrophobic ion-paired complex and a poloxamer solution having reverse thermal gelation properties.
- the poloxamer is poloxamer 407.
- the present invention provides a method for treating a subject, which comprises a hydrophobic ion-paired complex and a poloxamer solution.
- the hydrophobic ion-paired complex comprises an antimicrobial.
- the hydrophobic ion-paired complex comprises an antimicrobial selected from tetracycline, vancomycin, isoniazid and isoniazid methanesulfonate.
- the localized region of the subject for treatment is selected from a diabetic ulcer, a surgical incision site, a wound. In another embodiment, the localized region is selected from the group consisting of sites external to the body of said subject and sites internal to the body of said subject.
- antimicrobial is used in reference to any compound which inhibits the growth of, or kills microorganisms. It is intended that the term be used in its broadest sense, and includes, but is not limited to compounds such as antibiotics which are produced naturally or synthetically. It is also intended that the term includes compounds and elements that are useful for inhibiting the growth of, or killing microorganisms.
- drug refers to any molecule of any composition, including protein, peptide, nucleic acid, organic molecule, inorganic molecule, or combinations of molecules, biological or non-biological, which are capable of producing a physiological response and are suitable for administration to a subject to treat or prevent pathology (e.g., disease).
- pathology e.g., disease
- a drug or pharmaceutical provide at least one beneficial response in the cure, mitigation, treatment or prevention of a disease, condition or disorder (e.g., to treat a bacterial infection).
- drug and “pharmaceutical” are used interchangeably. It is intended that theses terms include, but not be limited to, such compounds as antimicrobials, anti-cancer compounds, and other compounds suitable for use in treating and/or preventing any type of pathology.
- controlled-release As used herein, the expressions "controlled-release,” “sustained-release,” “delayed- release,” “prolonged-release,” “extended-release,” and “depot-delivery” are all used synonymously. These expressions refer to the delivery of a pharmaceutical in which the delivery composition or method provides some aspect of prolonged or extended release of the pharmaceutical to the biological system as compared to the delivery of that pharmaceutical alone without incorporation into a "controlled-release” composition or method. For example, hydrophobic ion-pairing and poloxamer drug delivery methods both impart controlled-release features to a pharmaceutical.
- zwitterion is any molecule containing both positive and charges at physiological conditions (i.e., physiological ionic concentrations and pH).
- amphiphilic refers to any molecule having a water soluble polar head (hydrophilic) and a water insoluble organic tail (hydrophobic).
- systemically active drug and “systemically active agent” are used broadly to indicate a substance or composition which will produce a pharmacologic response at a site remote from the point of application.
- topically means application of a composition to the surface of the skin, mucosa, viscera, etc.
- topically active drug indicates a substance or composition which elicits a pharmacologic response at the site of application.
- PBS phosphate buffered saline solution, which contains 0.1 M NaCl and 20 mM phosphate buffer at pH 7.4.
- poly(oxyethylene) and poly(oxypropylene) refers to a copolymer of poly(oxyethylene) and poly(oxypropylene) (See, U.S. Pat. Nos. 4,100,271 and 5,457,093, both of which are herein incorporated by reference).
- the term "poloxamer gel” refers to a gel comprised of a copolymer of poly(oxyethylene) and poly(oxypropylene).
- the poloxamer composition of the present invention transitions from a gel to a fluid at and vice versa at temperatures about between 18-20°C. It is recognized that the transition temperature vary by the presence of other solutes in the poloxamer solution (e.g., salts [i.e., ions], carbohydrates or proteins).
- PLURONIC refers to poloxamers sold under the trade name PLURONIC.
- fluid refers to any composition capable of flowing.
- terapéutica amount refers an amount of a drug (e.g., an antimicrobial drug) that produces a beneficial effect (e.g., eradicates or inhibits a bacterial infection).
- the term "subject” refers to a human or other animal that is capable of being treated with a pharmaceutical composition.
- cancer refers to a malignant neoplasia characterized by the uncontrolled growth of anaplastic cells that tend to invade surrounding tissue and may develop the ability to metastasize to distant body sites.
- the term "suspected of having cancer” refers to a patient or subject who has been diagnosed as having cancer or tested positive in one or more tests diagnostic for cancer.
- the term “susceptible to cancer” refers to a subject or patient having one or more risk factors for cancer, is currently being treated for cancer, or is in remission from a previously diagnosed cancer.
- the term “post-operative for removal of a cancer” means the period of time after a tumor has been removed from a subject.
- the term "excision cavity” refers to an area of the body from which a tumor has been removed.
- a wound may constitute a variety of insults or damage to body tissues, for example a wound may involve a laceration, cut or scrape, surgical incision, sore, thermal burn, puncture, or decubitus ulcer (e.g., bedsores). Wounds can be classified in one of two general categories, namely either partial thickness or full thickness wounds.
- a partial thickness wound is limited to the epidermis and superficial dermis with no damage to the dermal blood vessels.
- a full thickness wound involves loss of the dermis and extends to deeper tissue layers and involves disruption of the dermal blood vessels.
- cytotoxic drug typically, but not exclusively, refers to a chemotherapeutic agent useful in treating cancer that is cytotoxic (i.e., detrimentally effects or kills cells), particularly to rapidly dividing cells.
- LD 50 refers to the concentration at which 50% cytotoxicity measured as lethality to cells is observed. Cytotoxicity can be measured by the method of Alley et al, Cancer Res. 48: 589-601 (1988) or Scudiero et al, Cancer Res., 48:4827 (1988). However, it is not intended that the present invention be limited to any particular method of measurement. In one embodiment, the LD 50 is based on the drug concentration at which a 50% reduction in the activity of mitochondrial enzymes is observed.
- sample is used in its broadest sense.
- Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases.
- Biological samples include, but are not limited to, blood products, such as plasma, serum and the like, and tissue samples, such as biopsy material.
- Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
- Figure 1 provides a graph showing the dissolution and release of tacrine from various hydrophobic ion-paired complexes in vitro in PBS as a function of time.
- P partitioning coefficients
- HBN hydrophilic-lypophilic balance number
- Figure 3 provides the chemical structures of isoniazid and isoniazid methanesulfonate, and the reaction scheme for metal-assisted hydrophobic ion-pairing using isoniazid.
- Figure 5 is a growth curve for E. coli exposed to tetracycline and tetracycline- AOT complex (as well as control cultures).
- Figure 6 is a growth curve for E. coli exposed to tetracycline and tetracycline- AOT complex (as well as control cultures).
- Figure 7 is a growth curve for E. coli exposed to tetracycline and tetracycline-AOT complex (as well as control cultures).
- Figure 8 is a growth curve for E. coli exposed to tetracycline and tetracycline-AOT complex (as well as control cultures).
- Figure 9 provides a histogram showing the intracellular platinum concentrations in
- Figure 10 provides a graph showing the cell survival rates of Chinese hamster ovary (CHO) cells exposed to either cisplatin or a cisplatin-AOT complex.
- Figure 11 provides a graph showing the vancomycin levels in mouse plasma following subcutaneous delivery of a poloxamer gel containing vancomycin or a vancomycin-AOT complex, and a control injection of water containing vancomycin-AOT.
- Figure 12 provides a graph showing the cumulative amount (%) of vancomycin released over time into PBS from a vancomycin-AOT complex.
- Figure 13 provides a graph showing the cumulative amount (%) of vancomycin released over time into PBS from a vancomycin-AOT complex.
- the present invention relates to the process of "hydrophobic ion pairing" (HIP), in which a drug (i.e., a pharmaceutical product or other biological agent) is non-covalently and reversibly modified to yield a drug species with advantageous properties.
- a drug i.e., a pharmaceutical product or other biological agent
- HIP hydrophobic ion pairing
- numerous drugs will find use with the HIP process of the present invention.
- the HIP process and applications of this process will find widespread use in the pharmaceutical industry, and in particular, will find use in improving or extending the usefulness of previously characterized pharmaceuticals.
- the HIP process is useful with any number of compounds, including small-molecule drugs as well as peptides and proteins.
- the present invention provides HIP-complexes containing a desired pharmaceutical that demonstrates improved drug delivery, controlled-release properties, and improvements in drug efficacy. Also, the present invention provides improved methods for HIP-complex formation.
- HIP complex formation involves the stoichiometric replacement of the small counterion(s) complexed with a drug in aqueous solution with an ionic detergent (i.e., an amphiphilic molecule or surfactant) of similar charge.
- an ionic detergent i.e., an amphiphilic molecule or surfactant
- anionic and cationic surfactants find use with the present invention to form HIP complexes with cationic and anionic drugs, respectively.
- HIP complexes can be formed by a variety of methods, including precipitation ion pairing, two-phase emulsion, and monophase processing, all of which are known in the art. In some embodiments in which both the surfactant and the drug are 5 water soluble, precipitation ion pairing is the most convenient of these three methods.
- HIP-drug complexes are formed by the stoichiometric replacement of the small, charged counter ions, such as chloride ions, which typically associate with larger charged molecules in an aqueous environment, with ionic surfactants at an appropriate molar ratio (Kendrick et al, Arch. Biochem. Biophys., 347:113-118 [1997]; Matsuura et al, JACS
- HIP-drug complexes display reduced aqueous solubility
- these same complexes demonstrate increased solubility in low-dielectric organic solvents, sometimes by orders of magnitude.
- This technique makes it possible to obtain true homogeneous 5 solutions of ionic compounds in neat organic solvents with the only requirement being an accessible charge on the drug molecule.
- HIP- complexed proteins retain their structural integrity (i.e., secondary and tertiary structure) and enzymatic activity while dissolved in organic solvents, and also retain activity following the dissociation from the surfactant and return to the aqueous phase (Bromberg 0 and Klibanov, Proc. Natl. Acad. Sci.
- HIP-complex solubility in organic solvents is an advantageous feature in the preparation of some pharmaceutical compositions provided by some embodiments of the present invention.
- HIP used in conjunction with precipitation by a compressed antisolvent technique has been shown to be effective in circumventing problems associated with microscale phase separation and consequent burst effects, and low incorporation yields in polymer [L-lactide] (PLA) microparticle processing (See e.g., Falk et al, J. Controlled Release 44:77-85 [1997]; Manning et al, Biotechnol Bioengin., 48:506-512 [1995]; U.S. Patent No. 5,770,559, and PCT WO 99/47543, herein incorporated by reference).
- PCA compressed antisolvent technique
- the anionic detergents sodium dodecyl sulfate (SDS) and sodium bis(2-ethylhexyl) sulfosuccinate (AOT; sodium dioctyl sulfosuccinate) are the most commonly used surfactants in HIP methods, but other surfactants have also been successfully utilized, including arginine octyl ester, arginine dodecyl ester, cholesterol sulfate, sodium laurate, sodium dodecyl sulfonate, sodium decyl sulfonate, sodium octyl sulfonate, sodium oleate, 1- heptanesulfonic acid, cetyl trimethyl ammonium bromide (CTAB), palmitoylcholine chloride, palmitoyl carnitine chloride (PCC), ZWITTERGENT 3-16, and polyethylene oxide polymer
- CTAB cetyl trimethyl ammonium bromide
- PCC
- the present invention provides HIP-complexed pharmaceuticals.
- these pharmaceuticals include antimicrobials (e.g., vancomycin, tetracycline and isoniazid), chemotherapy agents (e.g., cisplatin), drugs used in the treatment of Alzheimer's disease (e.g., tacrine), and decongestants (e.g., L-phenylephrine).
- antimicrobials e.g., vancomycin, tetracycline and isoniazid
- chemotherapy agents e.g., cisplatin
- drugs used in the treatment of Alzheimer's disease e.g., tacrine
- decongestants e.g., L-phenylephrine
- HIP-complexed drugs have improved efficacy and controlled (i.e., sustained or prolonged) release properties when used alone, as well as when they are incorporated into another delivery vehicle, such as poloxamer gel.
- the present invention also provides improved methods for hydrophobic ion pairing for the complexation of neutral or zwitterionic compounds, as well as HIP- complexes incorporating a covalently modified drug.
- the present invention be limited to the use of the drugs listed herein, as the invention finds use with numerous pharmaceuticals.
- the present invention provides compositions and methods for the non-covalent modification of pharmaceuticals to produce HIP complexes.
- the formation of HIP complexes involves the stoichiometric replacement of the small counter ion(s) associated with the pharmaceutical in physiological (or other ionic) conditions with an ionic surfactant of like charge.
- the formation of an HIP complex results in a reduction in aqueous solubility of the substrate (this reduction often reaches a factor of 50-100 fold), while at the same time significantly increasing its solubility in organic solvents.
- the pharmaceutical HIP complex precipitates when formed in aqueous solutions. In some embodiments, the resulting precipitate is collected, washed and dried to form a pharmaceutical powder.
- HIP complexes as well as compositions comprising the complexes of the present invention are useful in various treatment regimens.
- Some embodiments of present invention provide HIP-complexed antimicrobials, including tetracycline-AOT, vancomycin-AOT, isoniazid-AOT, and a covalently modified form of isoniazid, isoniazid methanesulfonate (IHMS), which is inco ⁇ orated into HIP complexes using a number of cationic surfactants.
- IHMS isoniazid methanesulfonate
- an HIP-complexed form of tetracycline, tetracycline-AOT is provided as described in Example 1.
- equimolar aqueous stock solutions were prepared for tetracycline hydrochloride (Sigma) and the anionic surfactant dioctylsulfosuccinate sodium salt, Aerosol OT (AOT; Sigma). Equal volumes of these solutions were combined, resulting in the precipitation of the corresponding ion paired complex (i.e., tetracycline-AOT). The mixture was centrifuged and the aqueous phase was discarded. The precipitate was washed with cold water to remove unpaired drug and surfactant.
- the complex was dissolved in methylene chloride (DCM, Fisher).
- DCM methylene chloride
- the tube containing the tetracycline-AOT complex was placed in a ventilation hood, and the DCM was allowed to evaporate. When all DCM was driven off, the dried complex was washed with cold water to remove any remaining salts or free tetracycline.
- the purified tetracycline-AOT complex was dissolved in water to a final concentration of approximately 0.25 mg/ml, as the tetracycline-AOT complex retains weak aqueous solubility.
- a hydrophobic ion-paired complex of vancomycin is provided.
- aqueous stock solutions of vancomycin and AOT were prepared, and equimolar quantities of vancomycin and AOT were combined from the stock solutions by slow dripping followed by thorough mixing.
- the combined solution turned into a milky white suspension due to the spontaneous precipitation of the vancomycin-HIP complex.
- This suspension was centrifuged, the supernatant was discarded, and the resulting pellet was washed with cold distilled water.
- the precipitate was dried overnight in a lyophilizer to form vancomycin- AOT powder.
- the solubility properties of HIP- vancomycin were determined, and are shown in
- uncomplexed vancomycin is soluble in water (>100 mg/ml) and has relatively poor solubility in methylene chloride (2.33 mg/ml).
- the AOT-complexed form of vancomycin has reduced aqueous solubility (2.47 mg/ml), while having significant solubility in a 75%> methylene chloride/25% methanol mixture (13 mg/ml).
- the present invention provides an HIP-complexed form of the anticancer agent cisplatin.
- a cisplatin-AOT complex was formed by first combining a 1 :2.2 molar ratio of aqueous solutions of cisplatin (3 mM; Aldrich) and hexafluorophosphate (Sigma; AgPF 6 ). After 2 hours, this mixture was centrifuged to remove precipitated silver chloride. Following the centrifugation, the supernatant (containing the cisplatin) was removed and combined with AOT (Sigma) at a 2:1 molar ratio. After thorough mixing, the solution was allowed to settle, then centrifuged to remove uncomplexed AOT from the solution phase.
- the present invention also provides HIP complexes comprising tacrine.
- the present invention provides three different HIP-tacrine complexes. These compounds were synthesized using the linear surfactants sodium dodecyl sulfate, sodium tetradecyl sulfate, and sodium octadecyl sulfate. Separate aqueous stock solutions of tacrine and the linear surfactants were prepared, all at identical molar concentrations. Equal volumes of the drug and surfactant solutions were combined and mixed vigorously, whereupon the ion-paired complexes precipitated out of solution. Reaction mixtures were centrifuged, the supernatant discarded, and the resulting pellet vacuum dried.
- the complexes were ground using a mortar and pestle to produce a fine powder and washed with water using a Millipore suction filter system in order to remove any un-paired tacrine and salts from the powdered HIP-complex. Following the washing, the ion-paired complexes were dried again under vacuum.
- the present invention also provides HIP-complexes comprising L-phenylephrine.
- an aqueous stock solution of L-phenylephrine hydrochloride (Sigma) was prepared, as well as aqueous stock solutions of the surfactants sodium octyl sulfate, sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium octadecyl sulfate (Aldrich), and AOT (Sigma) all in identical molar concentrations. Equal volumes of the drug and surfactant stock solutions were combined to form the aqueous-insoluble ion-paired complexes.
- HIP complexes with negatively charged pharmaceuticals using cationic surfactants, or conversely, HIP complexes can be formed comprising positively charged drugs (e.g., tetracycline or vancomycin) and anionic surfactants (e.g., AOT or SDS).
- positively charged drugs e.g., tetracycline or vancomycin
- anionic surfactants e.g., AOT or SDS
- HIP hydrophobic ion-pairing
- MHIP involves an initial complexation of the target pharmaceutical with a metal ion, preferably a multivalent metal ion, before proceeding with the HIP process.
- a metal ion preferably a multivalent metal ion
- the present invention provides various MHIP complexes involving diverse pharmaceuticals, thus demonstrating the wide applicability of this process.
- the present invention further provides MHIP complexes with a small neutral molecule and a polyionic zwitterionic compound (a protein).
- the group can be protonated prior to HIP complexing in order to generate a charged site to permit amphiphilic ion-pairing.
- the amine group acts as a Lewis base, and thus can act as a ligand to a metal ion.
- the amine- metal complex can interact with an anionic detergent to form a neutral complex which is significantly soluble in an organic solvent. This approach is useful in cases where the amine is unstable at certain pH values or when the pK ⁇ of the molecule is very low.
- isoniazid isonicotinic acid hydrazide; INH
- INH isonicotinic acid hydrazide
- isoniazid acts as a ligand to a divalent metal, which then forms an HIP complex, as shown in Figure 3.
- isoniazid (2 mmol) was dissolved in 0.5 ml of an aqueous solution of 0.10 M NaCl.
- Another solution was prepared containing 1 mmol of zinc chloride and 2 mmol of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in 0.5 ml of distilled water, which was added to the isoniazid solution.
- the resulting MHIP complex was partitioned into 1 ml of a water immiscible solvent (either 1-octanol or dichloromethane) by addition of the organic solvent, and vortexing the sample followed by centrifugation.
- the resulting solution contained two clear phases. The relative concentration of isoniazid in the two phases allowed the calculation of the partition coefficient to be made.
- the preparation of a metal-assisted hydrophobic ion-paired complex of isoniazid requires the addition of 2 molar equivalents of isoniazid to an aqueous solution containing one equivalent of zinc chloride.
- an anionic detergent e.g., AOT
- the resulting complex is readily partitioned into a water-immiscible solvent, such as 1-octanol.
- the AOT:metal ratio was not optimized in all of these experiments.
- Tables 2, 3 and 4 indicate that one equivalent of divalent zinc ion interacts with more than one equivalent of drug, and likely best carries two isoniazid molecules.
- Zwitterions such as proteins contain multiple charged groups, consisting of both cationic and anionic moieties.
- the rationale for applying the MHIP process to proteins is that calcium ions are able to bind negatively charged carboxylate groups in proteins. Following the binding of the calcium ion, it is contemplated that the carboxylate moiety is converted from having a single negative charge to one that has an overall charge of +1. The site can now bind, as opposed to repel, an anionic surfactant, such as SDS or AOT.
- This method to produce MHIP complexes comprising proteins was demonstrated using the protein ⁇ -chymotrypsin (CMT). Partitioning properties of MHIP complexes comprising this protein in organic solvents and water as a function of calcium ion concentration was determined, and is shown in Table 6. However, an understanding of the mechanism(s) is not necessary in order to use the present invention.
- MHIP- complexed pharmaceuticals in conjunction with precipitation with a compressed antisolvent (PC A).
- PC A compressed antisolvent
- the MHIP complexes appear to have sufficient solubility to allow them to be dissolved in a variety of organic solvents, but are not so hydrophobic that they cannot be processed using PCA technology to form fine powders and polymeric microspheres.
- the MHIP method of the present invention expands the range of ionic compounds that can be used with PCA technology. It is also contemplated that the solubility profile of some more hydrophobic compounds is modulated by forming metal complexes. In short, this technology facilitates the application of current HIP/PCA methodologies to be applied to new classes of compounds.
- HIP-complexed drugs have advantageous properties compared to the corresponding non-complexed drug forms.
- an HIP-drug complex is lipophihc
- the drug complex is more readily able to translocate across a cell membrane and/or cell wall (e.g., either a mammalian cell membrane or a bacterial cell wall) due to its increased lipophilicity, and thus, more drug is able to accumulate within the target cells of a patient than is possible using the corresponding chloride salt forms of a drug.
- this improved biotransport property also extends to microorganisms (e.g., bacteria), in the case where the drug is an antimicrobial.
- the present invention provides compositions and methods for improving the efficacy of pharmaceuticals.
- the present invention provides compositions and methods to modify pharmaceutical agents by forming an HIP complex to produce a pharmaceutical compound with improved efficacy.
- improved drug efficacy is the result of enhanced transport capability produced by increasing the biomolecule's lipophilicity.
- the present invention provides tetracycline-AOT complexes in which the efficacy (i.e., antimicrobial activity) of the tetracycline is improved, as compared to the activity of non-modified tetracycline (e.g., as determined in an E. coli growth rate assay).
- efficacy i.e., antimicrobial activity
- non-modified tetracycline e.g., as determined in an E. coli growth rate assay.
- cisplatin like other chemotherapy drugs, is associated with significant toxicity, in particular, neurotoxicity and nephrotoxicity. Thus, the clinical application of cisplatin is limited to low-dose levels.
- the present invention provides methods that reduce the aqueous solubility and increase the lipophilicity of cisplatin, increasing the intracellular delivery rate of the drug, thereby improving the efficacy of the drug. Thus, the concentration of drug required to produce a therapeutic benefit can be lowered or the number of required doses reduced, thereby minimizing toxic effects on a patient who receives the drug.
- a more lipophihc form of cisplatin is produced through hydrophobic ion pairing, resulting in enhanced intracellular bioavailability of the HIP-cisplatin complex relative to a non-complexed form of cisplatin.
- similar or higher therapeutic effects can be achieved using the ion-paired form compared to the parent form at a given dose level, reducing or avoiding the toxic effects of cisplatin through use of an ion-paired form of the drug.
- One embodiment for the synthesis of a cisplatin-AOT complex is described in Example 3.
- the intracellular availability and cytotoxic efficacy of the cisplatin-AOT complex were compared to those activities of non-complexed cisplatin in a tissue culture model system using Chinese hamster ovary (CHO) cells.
- the intracellular concentrations of platinum were measured following treatment of cultured mammalian cells with cisplatin or a cisplatin-AOT complex, and the cytotoxic activities of the cisplatin forms were compared using a cell culture cytotoxicity assay.
- CHO cells were plated into culture dishes and exposed to either cisplatin or cisplatin-AOT complex for one hour. Following this exposure, the intracellular levels of platinum were quantitated by harvesting the cells, normalizing for cell numbers, and analyzing by atomic abso ⁇ tion (AA) spectrometry. Two similar experiments were conducted, both yielding similar results, as described in Example 10 and Figure 9.
- AA atomic abso ⁇ tion
- the cell cultures exposed to cisplatin-AOT showed intracellular platinum concentration of lxl 0 "5 ⁇ M, while the culture exposed to 50 ⁇ M cisplatin-AOT showed an intracellular platinum concentration at least four-fold higher than cultures exposed to the parent cisplatin.
- this improved ability of cisplatin-AOT to accumulate within the cells results in greater cytotoxic activity of a given dose of cisplatin- AOT compared to non-complexed cisplatin.
- a plating efficiency/cell survival assay was used.
- CHO cell cultures were exposed to cisplatin or cisplatin-AOT at a range of concentrations for one hour. Following this exposure, the cells were harvested, counted, and replated at a final density of 100-500 cells per dish for each drug and exposure concentration. These cultures were allowed to grow an additional 7 days, after which time the percentage of originally plated cells which survived and grew on the plates was determined.
- the plating efficiency data for these cisplatin and cisplatin-AOT exposed cultures is shown in Figure 10.
- the LD 50 (50% of lethal dose) of cisplatin in this experimental system was approximately five-fold higher than the LD 50 for cisplatin-AOT. Furthermore, the plating efficiency of cultures treated with cisplatin-AOT was consistently and significantly lower than cultures treated with cisplatin at practically the full range of drug concentrations. From these data, it is concluded that the cisplatin-AOT drug complex has greater cytotoxic activity than the parent cisplatin.
- HIP-complexed pharmaceuticals e.g., tetracycline-AOT and cisplatin-AOT
- the HIP complexes of the present invention demonstrate advantageous activities at concentrations where the parent chloride salt has little or no effect.
- these complexes maintain their integrity in bacterial or mammalian cell culture media, allowing them to penetrate more easily into cells.
- the present invention provides methods and compositions for the development of pharmaceutical preparations with increased potency and efficacy. It is contemplated that the present invention will find use in the efficacious administration of commonly used (as well as less commonly used) drugs at concentrations lower than are currently used. In addition, the toxicity of the pharmaceutical compound is minimized.
- the HIP process is very adaptable, as it allows one to ion-pair essentially any ionic drug compound and change its partitioning, efficacy and release properties.
- the HIP process is used to form HIP complexes with negatively charged pharmaceuticals using cationic surfactants, or conversely, HIP complexes are formed comprising positively charged drugs and anionic surfactants.
- HIP process known in the art has a limitation, as it cannot form HIP complexes comprising neutral molecules.
- isoniazid isonicotinic acid hydrazide
- is a neutral molecule at physiological pH and is unable to form a traditional HIP complex.
- the present invention provides methods for the HIP complexing of isoniazid.
- a prodrug approach was used to introduce a negatively charged group onto the neutral isoniazid molecule, to yield sodium isoniazid methanesulfonate (IHMS) (See, U.S. Patent No. 2,759,944, herein inco ⁇ orated by reference), as described in detail in Example 8.
- the IHMS species has been demonstrated to retain anti-tuberculosis activity in vivo and in vitro. As described by the present invention involving the synthesis of the IHMS species permits ion-pair complex formation. Following synthesis of the covalently- modified IHMS, a number of HIP complexes were formed using different cationic surfactants, resulting in the formation of complexes that were relatively water-insoluble and organic-soluble. In contrast, no surfactant facilitated organic phase transfer of non-modified isoniazid. These results demonstrate the importance of the added negative charge on the isoniazid derivative in the formation of the HIP species.
- IHMS-HIP complexes numerous cationic surfactants were used to produce IHMS-HIP complexes, although three were chosen for further study. These three are tetraheptylammonium bromide, tetrapentylammonium bromide and tetraoctylammonuum bromide (Fluka).
- cationic lipids used to produce HIP complexes of IHMS using this method included: trimethylalkylammonium bromide, tetraalkylammonium bromide, l,2-diacyl-3-trimethylammonium-propane, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, tetraethylammonium bromide, tetrabutylammonium bromide, 1 ,2-dimyristoyl-3-trimethylammonium-propane,
- the present invention be limited to the covalent modification of isoniazid, as it is contemplated that covalent modification is applicable to numerous pharmaceuticals, so as to make such pharmaceuticals suitable for use in the HIP process. Furthermore, it is not intended that the present invention be limited to the methyl sulfonate reaction provided, as numerous other covalent modification reactions are equally effective at adding charged groups to molecules. It is also not intended that the present invention be limited to the surfactants used to produce the HIP complexes, described herein as numerous cationic surfactants find use with the invention.
- HIP-complexed drugs act as delayed-release systems, as the slow dissociation (i.e., reverse ion pairing) of a water-insoluble HIP-drug complex slowly releases free drug (i.e., water-soluble drug) into the biological system.
- a water-insoluble HIP-drug complex slowly releases free drug (i.e., water-soluble drug) into the biological system.
- free drug i.e., water-soluble drug
- such an HIP complex are viewed as a "depot delivery formulation.”
- the present invention provides "controlled-release" or “delayed-release" drug compositions comprising HIP-complexed pharmaceuticals, in which delivery ranges from hours to days rather than the short term delivery of transitional administration methods (i.e., minutes).
- the rate of HIP complex dissociation is a function of the nature of the amphiphilic surfactant in the complex. That is, the more lipophihc in nature is the surfactant, the more stable the HIP complex is likely to be, and thus, the rate of reverse ion-pairing in an ionic aqueous environment of that complex is likely to be slower that for a complex that inco ⁇ orates a less lipophihc surfactant.
- One method of monitoring the "strength" of the hydrophobic ion-pairing involves measuring the partitioning coefficient (P), which measures the equilibrium of drug concentration in the organic and aqueous phases in a solubility ratio, where:
- the Ion-paired surfactant-drug complexes disassociate because a lower chemical potential can be achieved by pairing with other ions in solution.
- the fluids of the human body contain salt ions, such as sodium, which may generate this behavior.
- salt ions such as sodium
- an HIP-complexed drug is slowly released into solution beyond the ion-paired drug complex's solubility limit as the drug is freed from the complex through an ion exchange process.
- the dissolution profile of several drug-surfactant complexes in continuously stirred PBS was measured.
- HIP complexes all inco ⁇ orated tacrine, and alternatively used the surfactants sodium dodecyl sulfate, sodium tetradecyl sulfate, or sodium octadecyl sulfate These HIP complexes were synthesized as described in Example 5.
- the tacrine- surfactant ion-paired complex rapidly dissolved into the aqueous ionic solution up to the complex's solubility limit. Once in solution, the presence of an aqueous salt begins the reverse ion exchange process eventually obtaining a chemical equilibrium between the dissolved drug and the dissolved drug- surfactant complex.
- the dissolution mechanism has two kinetic rate constants.
- the first rate constant governs the complex's phase change from a solid to liquid and can be derived from de-ionized water release values modeled by a single, non-zero 1 st order rate constant, while the other rate constant governs the ion exchange rate, and taking into account the salting in effects.
- Figure 1 shows the good agreement achieved between the predicted release profile and the experimental data.
- Table 2 shows the water and PBS solubilities of the ion-paired complexes, in addition to the HLB numbers for the corresponding surfactants.
- control of the ion exchange process through surfactant characteristics provides control over drug release into solution environments with free ions at concentrations typical of biological systems (e.g., the human body).
- decreasing the hydrophobic characteristic of the pairing surfactant increases the quantity of drug dissolved or becoming available for circulation within salt solutions compared to surfactants that exhibit higher hydrophobicity.
- the actual time duration for release depends upon the type of surfactant used and the circulation rate experienced at the administration site.
- the present invention provides numerous advantages over traditional drug delivery methods.
- the ability of the ion-paired complex to remain intact for many minutes or even hours or days under biological conditions was an unexpected finding that is in direct contrast with the common belief that these HIP complexes dissociate in biological media relatively quickly.
- the observation that increased hydrophobicity of the HIP-complex allows prolonged release of the complexed pharmaceutical was both a su ⁇ rising and advantageous observation.
- HIP complexes of the present invention Although vancomycin, tetracycline and isoniazid were used during the development of HIP complexes of the present invention, it is not intended that the present invention be limited to these antimicrobials or any subgroup of pharmaceuticals. Indeed, numerous other antimicrobials are suitable for use with the present invention.
- the present invention encompasses HIP complexes comprising various other bioactive compounds such as growth factors, hormones, cytokines, anti-inflammatories, etc., in order to provide improved therapeutic benefits with these agents.
- the prolonged release of antimicrobials, particularly at sites of infection or wounds that are refractory to treatment using traditional methods provides many significant advantages and facilitates the improvement in the quality of life for affected individuals.
- the present invention facilitates at-home care, reducing the necessity for hospitahzation and the associated risks of nosocomial infection.
- the delivery of antimicrobials in HIP complexes to wound sites and surgical sites provides advantages over current antimicrobial treatment regimens.
- the present invention provides approaches for the controlling the rate of release of a parenterally administered drug (e.g., an HIP-complexed pharmaceutical).
- a parenterally administered drug e.g., an HIP-complexed pharmaceutical.
- the drug is entrapped within a matrix, gel or bead, from which the drug slowly diffuses following administration or implantation.
- the drug is released from such a matrix via the slow biodegradation of the matrix material.
- poloxamer gels block co-polymers of poly[oxyethylene] and poly[oxypropylene]
- PLURONICS See, U.S. Pat. Nos. 4,100,271 and 5,457,092, both of which are herein inco ⁇ orated by reference).
- poloxamer 407 PLURONIC F-127
- PLURONIC F-127 PLURONIC F-127
- This unusual gelation property of poloxamer facilitates the synthesis of suitable poloxamer-drug mixtures, as well as simplifying administration to a patient.
- the poloxamer gels, and specifically poloxamer 407 also have the advantageous characteristics of providing a microenvironment that is compatible with protein stability, and preserves protein activity.
- the water-insoluble HlP-pharmaceutical complex is suspended in a suitable gel, or a liquid solution that will form a gel (e.g., poloxamer 407).
- a poloxamer/HIP-antimicrobial composition to a wound provides a reservoir drug delivery system capable of delivering an active level of antimicrobial over a clinically-useful period of many hours to days (e.g., up to 14 days).
- the water-insoluble HIP-antimicrobial complex undergoes a natural reverse ion pairing when exposed to physiological fluids.
- This reverse ion-pairing releases the soluble form of the antimicrobial to the surrounding tissue at a rate that is sufficiently slow to provide a slow and controlled release.
- the HIP-antimicrobial diffuses from the matrix material, or is released from the matrix material as the matrix undergoes degradation.
- the hydrophobic form of antimicrobial(s) penetrates the surrounding tissue more effectively than the corresponding hydrophilic salt form of the antimicrobial.
- poloxamer gels provide suitable gel matrices for HIP- pharmaceutical complexes.
- poloxamer gels it is not intended that the invention be limited to the use of poloxamer gels, as other appropriate gel compositions also find use with the present invention.
- the present invention provides a poloxamer gel containing a vancomycin-AOT ion-paired complex.
- Synthesis of the vancomycin-AOT is described in Example 2, and construction of the poloxamer-vancomycin-AOT composition is described in Example 12. Briefly, solutions of poloxamer 407 (22% w/w in water) containing 10 mg/ml (adjusted weight) vancomycin or vancomycin-AOT were prepared.
- plasma levels of the antimicrobial were assayed at time points following subcutaneous administration of the poloxamer.
- mice Thirty-six mice were divided into three groups of 12 mice each. These groups were treated as follows:
- Group I given a subcutaneous injection of non-complexed vancomycin in a poloxamer solution into the mouse flank fat pad;
- Group II given a subcutaneous injection of HIP-complexed vancomycin suspended in DI water
- Group III given a subcutaneous injection of HIP-complexed vancomycin in poloxamer solution.
- the dose level of vancomycin delivered to each mouse in the poloxamer gel was
- the gel HIP-antimicrobial composition of the present invention find particular use in the treatment and prevention of infections in which controlled drug release is desirable.
- the present invention provides improved methods and compositions for treating diabetic patients presenting with ulcers of the extremities (e.g., foot and hand ulcers).
- the compositions and methods of the present invention will find use in the prevention and/or treatment of infections associated with surgical incision sites.
- present invention it is not intended that present invention be limited to treating or preventing any particular type of patient, disease or wound. Indeed, the present invention finds use in numerous settings and treatment regimens.
- the present invention provides compositions and methods for the prolonged delivery of antimicrobial(s) to a site to be treated (e.g., a lesion) over a period of days.
- the compositions of the present invention comprise at least one modified antimicrobial of choice suspended in a suitable gel.
- the antimicrobial modification results in a form of the antimicrobial that is less water-soluble than the unmodified form.
- This gel/antimicrobial suspension is applied to a site to be treated (e.g., a diabetic ulcer), or on, in or around a surgical incision site following a surgical procedure, or any other type of wound.
- a site to be treated e.g., a diabetic ulcer
- the present invention be limited to any particular type of lesion, wound, or treatment site.
- the present invention finds use in the treatment of any appropriate pathological process, including but not limited to wounds, ulcers, surgical incisions, abrasions, burns, etc., present either on the external body sites (e.g, skin and mucous membranes) or internal sites (e.g., within organs, cavities, vessels, etc.).
- the present invention provides methods and compositions that are suitable for the treatment in a wide variety of settings.
- Aldrich Sigma-Aldrich Co., Milwaukee, WI
- BASF BASF Co ⁇ ., Mt. Olive, NJ
- Becton Dickinson/BBL Becton Dickinson-BD Biosciences, Sparks, MD
- Life Science Products Life Science Products, Inc., Denver, CO
- Sigma Sigma Chemical Co., St. Louis, MO
- Fisher Fisher Scientific, Pittsburgh, PA
- Fluka Fluka (Fluka Chemika-BioChemika, Switzerland); Gibco/BRL/Life Technologies (GIBCO BRL Life Technologies, Gaithersburg, MD); and Scientific Adsorbant (Scientific Adsorbant, Inc., Atlanta, GA)
- This Example describes the formation of a hydrophobic ion-paired vancomycin complex.
- An aqueous stock solution of vancomycin HC1 (MW 1486, 16.2 mM) was prepared. Concentration of the vancomycin was confirmed by UV absorbance measurement, where the extinction coefficient is 4 [(mg/ml)(cm) "1 ] measured at 282 nm.
- An aqueous stock solution of AOT was also prepared (MW 444.55; 29 mM; 1.29 g AOT in 100 ml water).
- a volume of 15 ml of the vancomycin stock solution (equivalent to 0.243 mmols) was added to a 50 ml Falcon polypropylene conical tube.
- 8.38 ml of AOT stock solution (equivalent to 0.243 mmols) was added by slowly dripping with a transfer pipet, and mixed by slow swirling.
- the brownish-colored vancomycin solution turned milky white due to spontaneous precipitation of the vancomycin-AOT complex following addition of the AOT. Following precipitation, the tubes were spun at room temperature for 10 min at 4000 x g.
- This Example describes the formation of a cisplatin-HIP complex.
- Cisplatin Aldrich; cis-diamminedichloroplatinum (II); cisplatinum diammine chloride) and hexafluorophosphate (Sigma; AgPF 6 ) were combined in a 1:2.2 molar ratio in aqueous solution with a cisplatin concentration of 3 mM. Two hours later, the mixture was spun twice at 1000 x g for 5 minutes to remove precipitated silver chloride. Following centrifugation, the supernatant was collected and treated with 30 mM AOT surfactant (Sigma) at a 2:1 molar ratio with the cisplatin solution.
- the solution was allowed to settle for 2 hours at room temperature, then centrifuged at 1000 x g for 5 minutes. This centrifugation allowed the removal of uncomplexed AOT from the solution phase. Following the centrifugation, the supernatant was discarded, and the pellet was washed three times with deionized water and centrifuged again as above. Following the wash steps, the precipitate was dried in a lyophilizing centrifuge to yield a white greasy solid consisting of the cisplatin-AOT complex. Determination of the final concentration of the cisplatin-AOT complex was made by dissolving a portion of the dried solid in dimethyl sulfoxide (DMSO) for use in atomic abso ⁇ tion (AA) spectrometry.
- DMSO dimethyl sulfoxide
- This Example describes the synthesis of HIP-complexes inco ⁇ orating tacrine.
- Three different HIP-tacrine complexes were synthesized using the linear surfactants sodium dodecyl sulfate, sodium tetradecyl sulfate, and sodium octadecyl sulfate.
- Aqueous stock solutions of 15 mg/ml tacrine and equimolar aqueous stock solutions of each of the three surfactants were prepared. Occasionally, the processing temperature was increased to facilitate surfactant dissolution into water. Equal volumes of the drug and surfactant solutions were combined and mixed vigorously for 5 minutes, whereupon the ion-paired complexes precipitated out of solution. Reaction mixtures were centrifuged at 2300 ⁇ m for 25 minutes, and the supernatant was discarded. The resulting pellet was vacuum dried. After drying, the complexes were ground using a mortar and pestle to produce a fine powder and washed using a Millipore suction filter system and 150-ml of de-ionized water. Washing removed any un-paired tacrine and salts from the powdered HIP-complex. Following the washing, the ion-paired complexes were dried again under vacuum. EXAMPLE 5
- This Example describes the dissolution properties of HIP complexes of tacrine in an aqueous ionic solution. The following procedure was repeated for all three surfactant-drug complexes (i.e., HIP complexes containing tacrine, and either sodium dodecyl sulfate, sodium tetradecyl sulfate, or sodium octadecyl sulfate).
- a 20 mg sample of the ion-paired tacrine complex was placed in a continuously stirred, tapered flask containing 50 ml of PBS. At designated times, 2.5 ml samples were collected using 0.2 mm pore size syringe filters, and then replaced with fresh 2.5 ml solution. The fresh solution was flushed back through the syringe filter into the flask, thereby limiting the amount of solid sample lost during this procedure.
- the concentration of tacrine at each time point was measured using UV-spectrophotometry at 324 nm.
- This Example describes the formation of HIP-complexes using L-phenylephrine and a series of surfactants.
- aqueous stock solution of 2.0 mg/ml L-phenylephrine hydrochloride (Sigma) was prepared.
- aqueous stock solutions of the surfactants sodium octyl sulfate, sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium octadecyl sulfate (Aldrich) and sodium dioctyl sulfosuccinate (AOT; Sigma) were prepared at molarities equal to that of the stock drug solution.
- AOT sodium dioctyl sulfosuccinate
- the partitioning number, P is a ratio of the HIP-tacrine in the organic phase versus the aqueous phase
- the hydrophilic-lypophilic balance number (HLB) is a measure of the hydrophilic nature of the surfactant, where the greater the HLB number, the more hydrophilic the surfactant.
- HLB hydrophilic-lypophilic balance number
- This Example describes the formation of an isoniazid HIP-complex using metal- assisted hydrophobic ion pairing.
- Isoniazid (2 mmol) was dissolved in 0.5 ml of an aqueous solution of 0.10 M NaCl.
- Another solution was prepared containing 1 mmol of zinc chloride and 2 mmol of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in 0.5 ml of distilled water, which was added to the isoniazid solution.
- the resulting MHIP complex was partitioned into 1 ml of a water immiscible solvent (either 1-octanol or dichloromethane) by addition of the organic solvent, vortexing the sample for 30 seconds, and then centrifuging at 5000 ⁇ m for 5 minutes.
- the resulting solution contained two clear phases.
- the relative concentration of isoniazid in the two phases allowed the calculation of the partition coefficient to be made. If there was an appreciable emulsion- type interface after centrifugation, addition of more NaCl and a repeat of the vortexing and centrifugation steps produced a clear interface.
- Isoniazid isonicotinic acid hydrazide; INH
- IHMS sodium isoniazid methanesulfonate
- the IHMS species has been demonstrated to have anti-tuberculosis activity in vivo and in vitro (Kitamoto et al, Japanese Journal of Tuberculosis, 1:92 [1953]; and Orlowski et al, Arzneim.-Forsch., 26:409 [1976]).
- the synthesis of the IHMS species permits ion-pair complex formation.
- a number of HIP complexes were formed using different cationic surfactants, resulting in relatively water insoluble and organic soluble species.
- no surfactant facilitated organic phase transfer of non-modified isoniazid (data not shown).
- the neutral isoniazid (Sigma) was used as the starting material in the synthesis of IHMS. This chemical synthesis is described in U.S. Patent No. 2,759,944 (herein inco ⁇ orated by reference).
- An aqueous solution of sodium bisulfite (100 ml, 52 g [0.5 mol] sodium bisulfite) was added to an equimolar mixture of isoniazid (68.5 g, 0.5 mol) in formaldehyde (39%, 38 ml). The mixture was heated to 100°C for 8 hours and was then evaporated under reduced pressure to give a yellow solid. Recrystallization from an ethanol water mixture yielded white, solid sodium IHMS. The derivative was characterized by both 'H and 13 C NMR spectra (data not shown).
- IHMS hydrophobic ion-paired complexes a 1.0 mM aqueous solution of sodium IHMS was mixed with 2.1 volumes of methanol. One volume of 1.0 mM cationic surfactant solution in dichloromethane was added and a homogeneous mixture was formed. The mixture was placed at room temperature for at least 15 min to allow full interaction between the drug and the cationic surfactant. One additional volume of water and one additional volume of dichloromethane were then added. Phases were separated by centrifugation at 2,000 ⁇ m for 5 min. The drug concentrations in the aqueous and organic layers were estimated by UV spectrophotometry, and the partition coefficient of the IHMS ion-paired complex was calculated.
- IHMS-HIP complexes Numerous cationic surfactants were used to produce IHMS-HIP complexes, although three were chosen for further study. These three are tetraheptylammonium bromide, tetrapentylammonium bromide and tetraoctylammonuum bromide (Fluka).
- cationic lipids used to produce HIP complexes of IHMS using this method included: trimethylalkylammonium bromide, tetraalkylammonium bromide, l,2-diacyl-3-trimethylammonium-propane, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, tetraethylammonium bromide, tetrabutylammonium bromide, 1 ,2-dimyristoyl-3-trimethylammonium-propane, l,2-dipalmitoyl-3-trimethylammonium-propane, 1 ,2-distearoyl-3-trimethylammonium-propane, and l,2-dioleoyl-3-trimethylammonium-propane.
- the complexes were purified by flash chromatography.
- the IHMS -tetraheptylammonium complex was purified using a 200 ml flash-chromatography assembly (Sigma-Aldrich).
- the "flash" silica gel (“Flash,” 32-63 ⁇ m, 60 A; Scientific Adsorbant, Inc.) was packed into the column to a height of 15-22 cm.
- the solvent consisted of 60% 2-propanol and 40% ethanol. The samples were applied as a 20-30% solution in the solvent. Medical grade compressed nitrogen was applied to the column to maintain a flow rate of 1.5-2.5 ml min.
- the eluents were collected as 5-ml aliquots and were subject to UV spectrophotometry and thin layer chromatography to identify the elution fraction containing the HIP complex, as well as estimate the drug concentration and purity.
- the aliquots containing pure ion-pairing complexes were saved and dried by centrifugation under vacuum.
- three HIP complexes were chosen for further study.
- the water/dichloromethane partition coefficients for IHMS-tetrapentylammonium, IHMS-tetraheptylammonium and IHMS-tetraoctylammonium HIP complexes were determined. The results of this analysis are shown in Figure 4, as log P, where:
- EXAMPLE 9 Improved Efficacy of Hydrophobic Ion Paired Tetracycline
- the efficacy i.e., antimicrobial activity
- HIP-modified tetracycline was tested and compared to the activity of non-modified tetracycline, as determined in an E. coli growth rate assay.
- a first set of experiments was designed to determine the appropriate concentration of tetracycline that would inhibit the growth of E. coli without completely killing the bacterial culture.
- the analysis of E. coli culture growth rates in response to free tetracycline and tetracycline-AOT complex also included control cultures that received no drug treatment and cultures that received treatment with AOT alone.
- E. coli ATCC 25922 The bacterial strain used in these experiments, E. coli ATCC 25922, was chosen for use in this study because of its known susceptibility to tetracycline and a variety of other antimicrobials. Tetracycline hydrochloride (Sigma), AOT (Sigma), and methylene chloride (DCM; Fisher) were used as they were received from the manufacturer.
- the E. coli was cultured as a suspension in trypticase soy broth (Becton Dickinson, BBL 11768) using a shaking water bath at 37°C and a shaker speed of 200 ⁇ m. The suspension cultures were initiated by inoculating 1-2 colonies from an agar plate to 5 ml of liquid culture media, and allowed to grow overnight.
- the resulting starter culture was then used to inoculate a 250 ml shaker flask containing 95 ml of culture media.
- the culture was grown until it reached an optical density (OD, 600 nm) of approximately 0.75 (the cells were still in the exponential growth phase), at which time each of ten flasks were inoculated with 5 ml of the cell suspension.
- the growth rate was again monitored by OD readings every 20-30 minutes.
- the susceptibility of E. coli to varying concentrations of tetracycline, the tetracycline-AOT complex, and AOT alone was determined by quantitating culture growth rates.
- the antimicrobial agents for testing were added to the cultures when the cell suspensions were at an OD of approximately 0.2 (i.e., typically 60-75 minutes after the inoculation). After reaching this concentration, 1.0 ml of the tetracycline solution were added to the culture flasks, to produce a final concentration of tetracycline in the range of 0-24 ⁇ g/ml.
- a second E. coli growth rate experiment was run using 5.0 ⁇ g/ml concentrations of the free tetracycline and the tetracycline-AOT complex. This concentration was also still high enough to completely stop all growth of E. coli.
- a third E. coli growth rate experiment was run using 0.6 ⁇ g/ml concentrations of the free tetracycline and the tetracycline-AOT complex. As shown in this Figure, the ion-paired tetracycline inhibited the growth rate of the E. coli culture 45% more effectively than the free tetracycline.
- AOT solution was added to a final concentration of 1.0 ⁇ g/ml.
- Another control culture received the same volume of water.
- the growth rate of the AOT-treated and free tetracycline-treated cultures were not significantly different from the water-treated control culture.
- Drug was added to these cultures after 60 minutes of incubation. Almost immediately following the addition of the tetracycline-AOT complex, a reduction in growth rate was observed for this culture.
- an HIP-complexed form of cisplatin was shown to be more effective at inhibiting target cells in an in vitro culture system than a non-complexed form of the drug.
- This Example describes the improved efficacy of the cisplatin-AOT complex compared to non-complexed cisplatin, as determined using a tissue culture model system.
- the intracellular concentrations of platinum were measured following treatment of cultured mammalian cells with cisplatin or cisplatin-AOT, and the cytotoxic activities of the cisplatin forms were compared using a cell culture cytotoxicity assay.
- the mammalian cells used in this Example were cultured using standard culture techniques and conditions well known in the art (e.g., cells were grown in ⁇ -MEM culture medium containing 10% fetal bovine serum at 37°C in 5% CO 2 ). Mammalian cell culture reagents are widely available from numerous manufacturers, including, but not limited to, GibcoBRL and Sigma.
- the AOT-cisplatin complex was synthesized as described in Example 3.
- CHO cells Chinese hamster ovary (CHO) cells were plated into culture dishes at a density of lxl 0 6 cells/ml, where each dish received 3 ml of the cell suspension. Following establishment of the adherent culture, the cells were either exposed to cisplatin at a concentration of 200 ⁇ M or exposed to the cisplatin-AOT complex at a concentration of 50 ⁇ M for one hour. To quantitate the intracellular levels of platinum, the CHO cells were harvested and normalized to a density of lxl 0 7 cells/ml before analysis by atomic abso ⁇ tion (AA) spectrometry.
- AA atomic abso ⁇ tion
- the cell culture exposed to 200 ⁇ M cisplatin showed an intracellular platinum concentration of lxl 0 '5 ⁇ M, while the culture exposed to 50 ⁇ M AOT-cisplatin showed an intracellular platinum concentration of 5x10 ⁇ 5 ⁇ M.
- the cisplatin-AOT complex demonstrates at least a four-fold enhancement in the ability to accumulate in the intracellular environment of cultured mammalian cells compared to the non-complexed form of cisplatin.
- a plating efficiency/cell survival assay was used.
- CHO cell cultures were exposed to cisplatin or AOT-cisplatin at concentrations of 1, 5, 10, 20, 50, 75 or 100 ⁇ M for one hour.
- cisplatin and cisplatin-AOT were dissolved in DMSO, and the concentrations were determined based on the correction of platinum concentration, as detected by atomic abso ⁇ tion spectrometry.
- the cells were harvested, counted, and replated at a final density of 100-500 cells per dish for each drug and exposure concentration. These cultures were allowed to grow an additional 7 days, after which time they were stained with crystal violet (2% crystal violet in ethanol).
- Colonies in each dish were counted and the plating efficiency for each different exposure was calculated by comparing the colony number after 7 days to the number of cells used to initiate the culture. Cell survival rate in each exposure group was then calculated based on the plating efficiency of cisplatin-AOT exposed cells compared to that obtained from the control group (i.e., the cisplatin exposed cells).
- the plating efficiency data for the cisplatin and cisplatin-AOT exposed cultures is shown graphically in Figure 10. Based on these plating efficiency assays, the LD 50 (50% of lethal dose) for cisplatin in this experimental system was 22 ⁇ M, while the LD 50 for cisplatin-AOT was 4.5 ⁇ M. Furthermore, the plating efficiency of cultures treated with cisplatin-AOT was consistently and significantly lower than cultures treated with cisplatin at practically the full range of drug concentrations. From these data, it is concluded that the cisplatin-AOT drug has greater cytotoxic activity than the parent cisplatin.
- EXAMPLE 11 Efficacy Study of Hydrophobic Ion Paired Cisplatin Delivered in a Poloxamer Gel in vivo
- This Example provides a protocol for the inco ⁇ oration of HIP-complexed cisplatin described in Example 3 into a poloxamer gel delivery vehicle.
- this Example provides a protocol for analyzing the efficacy of the HIP-cisplatin complex in vivo compared to non-HIP-complexed cisplatin, using a mouse tumor model.
- Poloxamer 407 (BASF) is dissolved in DI water (22%, w/w) at 4°C and filter sterilized through a 0.20 ⁇ m cellulose acetate membrane (Life Science Products). Cisplatin (Aldrich) is then suspended in the poloxamer solution at a final concentration of 100 ⁇ M. Cisplatin-AOT (described in Example 3) is suspended into dimethyl sulfoxide (DMSO) and then suspended into the poloxamer solution at a final cisplatin concentration of 100 ⁇ M.
- DMSO dimethyl sulfoxide
- mice Female C3H-HeJ mice aged 6-8 weeks are injected with a mouse mammary carcinoma cell line, Gollin-B at a concentration of 0.5 million cells per mouse into the flank fat pad. When a tumor arising from the injected cells reaches 7 mm in any direction, the tumor is surgically excised.
- Gollin-B a mouse mammary carcinoma cell line
- mice receives no treatment; Group II is injected with 0.2 ml poloxamer solution only (with no inco ⁇ orated drug) at the surgical site; Group III is injected with 0.2 ml poloxamer solution containing cisplatin at the surgical site; and Group IV is injected with 0.2 ml poloxamer solution containing the cisplatin-AOT complex at the surgical site.
- Regrowth of the tumors is monitored in the Group I control mice.
- the animals in all the groups are sacrificed, and tissue from the liver, kidney, small intestine, brain, and the original surgical site is collected for histopathology examination.
- the tumor reoccurrence rate and metastasis are assessed for each treatment group.
- This Example describes the formation of a poloxamer gel containing the vancomycin HIP-complex described in Example 2, and the drug release characteristics from the poloxamer gel in an in vivo mouse model system.
- MW vancomycin-HIP complex 1872.11 g/mol
- weight percentage of vancomycin pharmacophore within the HIP complex is: (1450.55)/(1872.11), or 77.5%
- poloxamer 407 BASF; 22% w/w in water
- Group I given a subcutaneous injection of non-complexed vancomycin in a poloxamer solution into the mouse flank fat pad;
- Group II given a subcutaneous injection of HIP-complexed vancomycin suspended in DI water;
- Group III given a subcutaneous injection of HIP-complexed vancomycin in poloxamer solution.
- the dose level of vancomycin delivered to each mouse was 100 mg vancomycin per kg of mouse weight. Calculation of the vancomycin to be administered to each mouse was corrected for the difference in molecular weights between the complexed and non- complexed vancomycin, such that each mouse received the same concentration of "parent" vancomycin per kg of mouse weight using the calculation described above.
- the vancomycin levels in the plasma were assayed using a TDx clinical analyzer (Abbott Laboratories).
- the TDx instrument detects fluorescence polarization of a fluorescently- tagged, polyclonal antibody specific for the analyte in question (i.e., an anti-vancomycin antibody). The larger the signal, the greater the concentration of vancomycin.
- This Example provides protocols for the analysis of vancomycin distribution in mouse tissues following delivery of vancomycin or HIP-vancomycin from a poloxamer gel.
- HIP- vancomycin is synthesized as described in Example 2. Vancomycin and HIP-vancomycin are inco ⁇ orated into poloxamer solutions, which are delivered subcutaneously or intraperitoneally to mice. Following a suitable time period, the mice are sacrificed, tissues are harvested, and slides suitable for analysis by immunochemistry are prepared. Tissue samples from muscle, liver, lung and kidney, as well as other tissues, are collected for analysis.
- the slides are incubated with a commercially available anti-vancomycin primary antibody for one hour at 37°C, followed by two washes with PBS. Slides are then incubated with a suitable secondary antibody (preferably gold conjugated) specific to the primary antibody. The slides are then visualized using a silver-enhancing kit (Sigma). Slides are observed under light microscopy. Light microscopy images are also be quantitated for vancomycin concentration.
Abstract
Description
Claims
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EP00975580A EP1225916A1 (en) | 1999-11-01 | 2000-11-01 | Compositions and methods for controlled-release delivery and increased potency of pharmaceuticals via hydrophobic ion-pairing |
CA002389304A CA2389304A1 (en) | 1999-11-01 | 2000-11-01 | Compositions and methods for controlled-release delivery and increased potency of pharmaceuticals via hydrophobic ion-pairing |
AU13609/01A AU1360901A (en) | 1999-11-01 | 2000-11-01 | Compositions and methods for controlled-release delivery and increased potency of pharmaceuticals via hydrophobic ion-pairing |
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US16282299P | 1999-11-01 | 1999-11-01 | |
US60/162,822 | 1999-11-01 | ||
US23125700P | 2000-09-08 | 2000-09-08 | |
US23137400P | 2000-09-08 | 2000-09-08 | |
US60/231,257 | 2000-09-08 | ||
US60/231,374 | 2000-09-08 |
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Cited By (14)
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JP2005291958A (en) * | 2004-03-31 | 2005-10-20 | Shionogi & Co Ltd | Sugar chain labeling reagent |
WO2006069133A2 (en) * | 2004-12-21 | 2006-06-29 | Alza Corporation | Ionic drug-detergent complexes |
WO2006118948A2 (en) * | 2005-04-29 | 2006-11-09 | Cubist Pharmaceuticals, Inc. | Therapeutic compositions |
US7323169B2 (en) | 2004-04-23 | 2008-01-29 | Amgen Inc. | Sustained release formulations |
WO2008031440A2 (en) * | 2006-09-14 | 2008-03-20 | Pharma 2100 | Isoniazid mediated healing of wounds and ulcers |
EP1915138A2 (en) * | 2005-08-17 | 2008-04-30 | The Board Of Trustees Of The University Of Arkansas | Drug-surfactant complexes for sustained release |
US7527807B2 (en) | 2000-06-21 | 2009-05-05 | Cubist Pharmaceuticals, Inc. | Compositions and methods for increasing the oral absorption of antimicrobials |
CN103772215A (en) * | 2014-02-19 | 2014-05-07 | 赤峰艾克制药科技股份有限公司 | Method for preparing L-phenylephrine |
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DE102014013751A1 (en) * | 2014-09-22 | 2016-03-24 | Justus-Liebig-Universität Giessen | Pulmonary drug formulation with delayed release |
GB2544843A (en) * | 2015-07-14 | 2017-05-31 | Professional Compounding Centers Of America (Pcca) | Poloxamer-based intralesional injections for the delivery of chemotherapeutic agents |
US10322191B2 (en) | 2014-06-30 | 2019-06-18 | Tarveda Therapeutics, Inc. | Targeted conjugates and particles and formulations thereof |
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US5770559A (en) * | 1992-10-14 | 1998-06-23 | The Regents Of The University Of Colorado | Solubilization of pharmaceutical substances in an organic solvent and preparation of pharmaceutical powders using the same |
-
2000
- 2000-11-01 AU AU13609/01A patent/AU1360901A/en not_active Abandoned
- 2000-11-01 EP EP00975580A patent/EP1225916A1/en not_active Withdrawn
- 2000-11-01 CA CA002389304A patent/CA2389304A1/en not_active Abandoned
- 2000-11-01 WO PCT/US2000/030424 patent/WO2001032218A1/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5770559A (en) * | 1992-10-14 | 1998-06-23 | The Regents Of The University Of Colorado | Solubilization of pharmaceutical substances in an organic solvent and preparation of pharmaceutical powders using the same |
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Also Published As
Publication number | Publication date |
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AU1360901A (en) | 2001-05-14 |
CA2389304A1 (en) | 2001-05-10 |
EP1225916A1 (en) | 2002-07-31 |
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