Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
  • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
• • 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
• • 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
  • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6957—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a device or a kit, e.g. stents or microdevices
• • 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
  • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
  • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• the present invention relates generally to methods for improving the transport and delivery of pharmaceutical agents across membranes. More particularly, the invention relates to methods for enhancing the transport and delivery of pharmaceutical agents through the addition of one or more chemical modifiers to the pharmaceutical agent.
• the therapeutic efficacy of pharmaceutical or therapeutic agents relies on the delivery of adequate doses of a pharmaceutical agent to the site of action.
• Many modes of delivery have been developed, including, for example, enteral (oral), parenteral (intramuscular, intravenous, subcutaneous), and topical administration. In most instances the administration system is chosen for reliable dosage delivery and convenience.
• parenteral administration is the most reliable means of delivering a pharmaceutical to a patient. See, Goodman et al . , Goodman and Gilman's Pharmacological Basis of Therapeutics, Pergamon Press, Elmsford, New York (1990) and Pratt et al . Principles of Drug Action: The Basis of Pharmacology, Churchill Livingstone, New York, New York (1990). Each parenteral mechanism insures that a prescribed dosage of the pharmaceutical agent is inserted into the fluid compartment of the body where it can be transported. The disadvantage of these modes of delivery is that they require an invasive procedure. The invasive nature of administration is inconvenient, painful and subject to infectious contamination.
• Enteral and topical administration are more convenient, generally non-painful, and do not predispose to infection; however, both are limited.
• the gastrointestinal and dermal surfaces present formidable barriers to transport and therefore, some pharmaceutical agents are not absorbed across these surfaces.
• Another drawback to patient directed modes of administration is compliance. Pharmaceutical agents that have a short half-life require multiple daily doses. As the number of doses increases, patient compliance and therapeutic efficacy decrease. Simplified and/or less frequent administration schedules can aid in optimizing patient compliance.
• the skin is an efficient barrier to the penetration of water soluble substances, and the rate of transdermal pharmaceutical agent absorption is primarily determined by the agent's lipid solubility, water solubility, and polarity. Highly polar or water soluble pharmaceutical agents are effectively blocked by the skin. Even very lipophilic pharmaceutical agents penetrate the dermis very slowly compared with the rate of penetration across cell membranes. See Pratt et al . supra .
• transdermal delivery systems Efforts to develop more effective and convenient modes of pharmaceutical administration have led to the development of transdermal delivery systems. Many current transdermal pharmaceutical agent delivery systems rely upon pharmaceutical agents that are absorbed when admixed with inert carriers. See Cooper et al . (1987) "Penetration Enhancers", in Transdermal Delivery of Drugs. Vol. II,
• Nonionic drugs may also be delivered iontophoretically provided that a charge can be induced on them, for example, by adsorption of drug onto an ionic carrier or entrapment in an ionic micelle. See Banga (1988) J. Controlled Release, 7:1-24.
• the rate of drug delivery in iontophoresis is directly proportional to the system current; the higher the current, the greater the driving force and pharmaceutical agent delivery.
• Ionic strength also affects the iontophoretic drug delivery rate. See Banga supra . Ionic strength is related to the concentration of various ions present in the solution of the pharmaceutical agent in the reservoir. Other factors that may affect the delivery rate include pH, concentration, extraneous ions, conductivity, and electronic factors.
• This invention provides for methods of modifying a pharmaceutical agent comprising covalently bonding a charged chemical modifier, via a physiologically cleavable bond, to the pharmaceutical agent, such that the transport and/or delivery of the agent through membranes, or other biological or physical property of the agent, are enhanced.
• the chemical modifier covalently bonded to the pharmaceutical agent forms a pharmaceutical agent-chemical modifier complex which is administered to a patient, wherein the chemical modifier is cleaved from the complex by a physiological process once the complex has been delivered, and the pharmaceutical agent is released within the patient in an active form.
• One or more chemical modifiers is covalently bonded to the agent and the modifiers can be positively or negatively charged.
• a functionality modifier will also be covalently bonded to the pharmaceutical agent-chemical modifier complex, optionally via a spacer group.
• This invention provides for chemical modifiers which are bound to pharmaceutical agents to enhance the transport, delivery, or other biological or physical properties of the pharmaceutical agent.
• Representative pharmaceutical agents include digitalis drugs; steroidal compounds; nonsteroidal anti-inflammatories; protein and peptide drugs; nucleotide- based drugs; and nitrogen heterocycles, including yohimbine, morphine, methotrexate, lorazepam, 6-mercaptopurine, and 5-fluorouracil.
• modifiers include taurine, betaine, carnitine, oligomeric carnitine, choline, lysine, polylysine, e-methylated lysine, oligomeric methylated lysine, other methylated amino acids, trigonelline, stachydrine, betonicine, histones, protamines, histones, nucleotide-based chemical modifiers, cytochrome c, squalamine, chonemorphine, conessine, and other bi- or multi-functional quaternary ammonium salts.
• This invention provides for methods for the delivery of a pharmaceutical agent, comprising the steps of: a) binding one or more chemical modifiers to the pharmaceutical agent through physiologically cleavable covalent bonds thereby forming a pharmaceutical agent-chemical modifier complex; b) contacting the a patient's membrane or skin with a therapeutically effective amount of the complex.
• the pharmaceutical agent- chemical modifier complex is administered to the skin by applying an electric field to the interface of the complex and the skin, such that the electric field transdermally delivers the complex.
• the complex has an enhanced delivery or transport rate through membranes over the pharmaceutical agent alone.
• an iontophoretic drug delivery device is utilized and the complex is delivered through skin by applying an electric field.
• an iontophoretic drug delivery device is utilized to delivery the complex through skin via application of an electric field.
• compositions comprising a pharmaceutical agent covalently bonded to one or more charged chemical modifiers through physiologically cleavable bonds to form a pharmaceutical agent-chemical modifier complex, wherein the modifiers enhance the transport and/or delivery of the pharmaceutical agent through membranes, or other biological or physical property of the agent.
• This invention also provides a pharmaceutical formulation which comprises a pharmaceutically effective amount of a pharmaceutical agent- chemical modifier composition and acceptable physiological carriers or excipients thereof.
• the invention also provides pharmaceutical agent compositions containing a pharmaceutical agent in a complex with one or more chemical modifiers such as taurine, betaine, carnitine, oligomeric carnitine, choline, lysine, polylysine, e-methylated lysine, oligomeric methylated lysine, other methylated amino acids, histones, protamines, nucleotide-based chemical modifiers, cytochrome c, squalamine, chonemorphine, conessine, and quaternary ammonium salts which are useful in enhancing pharmaceutical agent transport and delivery.
• chemical modifiers such as taurine, betaine, carnitine, oligomeric carnitine, choline, lysine, polylysine, e-methylated lysine, oligomeric methylated lysine, other methylated amino acids, histones, protamines, nucleotide-based chemical modifiers, cytochrome c, squal
• “Pharmaceutical agent or drug” refers to any chemical or biological material, compound, or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Some drugs are sold in an inactive form that is converted in vivo into a metabolite with pharmaceutical activity. For purposes of the present invention, the terms “pharmaceutical agent” and “drug” encompass both the inactive drug and the active metabolite.
• Transmembrane transport and transmembrane delivery refers to the transport and delivery, respectively, of a substance across the skin (i.e., transdermal), including the epidermis and dermis, or across a mucosal membrane (i.e., sublingual, buccal and vaginal), where the substance can contact, and be absorbed into, the capillaries. In certain instances, the transport and/or delivery of the substance across other membranes will be effected.
• “Buccal delivery” refers to any system or device for the oral administration of a drug to a patient that is held in the mouth and is used to deliver a drug through the buccal mucosa and into the patient's body.
• Enhanced delivery refers both to the facilitation of the delivery of a pharmaceutical agent and an absolute increase in the molar volume of the pharmaceutical agent transported per unit time through a constant surface area utilizing an equimolar pool of transported material as compared to unenhanced delivery.
• Iontophoresis or “iontophoretic” refers to the introduction of an ionizable chemical through skin or mucosal membranes by the application of an electric field to the interface between the ionizable chemical compound and the skin or mucosal membrane.
• Permeability refers to the ability 'of an agent or substance to penetrate, pervade, or diffuse through a barrier, membrane, or a skin layer, for example, between the cells of the stratum corneum and/or through the shunt pathways, including the sweat ducts and the hair follicles and/or intracellularly.
• Chemical modifier refers to a charged molecule capable of covalently bonding to a pharmaceutical agent, another modifier, and/or a spacer group.
• the term “chemical modifier” is meant to include both the charged molecule and its counterions, if any.
• the covalent bond between the modifier and agent is preferably reversible and can be cleaved in vivo by biological or physiological processes which release the agent in an active form.
• Space group refers to a molecule capable of covalently bonding simultaneously to both a pharmaceutical agent and a chemical modifier, optionally via another spacer group(s), and which does not necessarily carry a charge.
• “Functionality modifier” refers to a molecule capable of covalently bonding to a pharmaceutical agent, a chemical modifier, or a spacer group, optionally via a spacer group(s), which does not necessarily carry a charge, and which serves to affect or modify a chemical, physical, or biological property of a pharmaceutical agent-chemical modifier complex, for example, providing a means for detection, for modifying the excretion half-life, for targeting, for increasing avidity, for decreasing aggregation, for decreasing the inflammation and/or irritation accompanying the delivery of the pharmaceutical agent across membranes, and for facilitating receptor crosslinking.
• the functionality modifier will carry a charge and thus, is also a chemical modifier.
• “Pharmaceutical agent-modifier”, “pharmaceutical agent-modifier complex”, “pharmaceutical agent-chemical modifier”, or “pharmaceutical agent-chemical modifier complex” refers to at least one chemical modifier covalently bound, optionally via a spacer group, to a pharmaceutical agent.
• Single or multiple chemical modifiers and according to some embodiments, single or multiple functionality modifiers may be bound to a single pharmaceutical agent molecule to enhance the transport and delivery of the pharmaceutical agent.
• the chemical modifiers may be bound to different sites on the pharmaceutical agent molecule, bound to other chemical modifiers which in turn are bound to the pharmaceutical agent, or a combination of both.
• pharmaceutical agent-chemical modifier complex is meant to include both the charged molecule and any counterions. In other words, the net charge on the pharmaceutical agent-chemical modifier complex is zero.
• Pharmaceutical agent-chemical modifier complexes may be schematically represented as pharmaceutical agent-[(spacer) x -(chemical modifier) y ] z or A-(S x -M y ) z wherein x is 0-10, y is 1-10 and z is 1-10 and A is the pharmaceutical agent, S is the spacer group, and M is the chemical modifier.
• the pharmaceutical agent-chemical modifier complex will further comprise at least one functionality modifier.
• Model compound refers to a compound which is relatively easy to synthesize and which can be used to validate or assess the methods described herein. More specifically, a model compound will comprise a "pharmaceutical agent”-chemical modifier complex in which the "pharmaceutical agent” is a substance not generally considered to be therapeutically active.
• “Chemical functionality” refers to a chemically reactive moiety or group on the pharmaceutical agent, spacer group, functionality modifier, and/or chemical modifier through which the covalent bonding occurs to form the pharmaceutical agent-modifier complex.
• Physiologically cleavable bond refers to a chemical bond which can be cleaved by physiological processes in a cell, an organ, the skin, a membrane, or the body of the patient. Cleavage can occur by enzymatic and nonenzymatic processes, such as by proteases, chemical hydrolysis, and the like.
• Penetration enhancer refers to a substance which is used to increase the transdermal or transmembrane flux of a compound. A penetration enhancer is typically applied to the skin or mucous membrane in combination with the compound. Enhancers are believed to function by disrupting the skin or mucous membrane barrier or changing the partitioning behavior of the drug in the skin or mucous membrane.
• Protecting group refers to a chemical group which generally exhibits the following characteristics: 1) the group must react selectively with the desired functionality in good yield to give a protected substrate that is stable to a future projected reaction; 2) the protecting group must be selectively removable from the protected substrate to yield the desired functionality; and 3) the protecting group must be removable in good yield by reagents that do not attack one or more of the other functional group (s) generated or present in the projected reaction. Examples of protecting groups can be found in Greene et al . Protective Groups in Organic Synthesis. 2nd Ed., John Wiley & Sons, New York (1991). "Acyloxyalkyl amine” refers to the group
• R may be a chemical modifier.
• R and R' may be chemical modifiers.
• "Acyloxyalkyl carbamate" refers to the group -NR-
• R may be a chemical modifier.
• R may be a chemical modifier.
• Acyloxyalkyl sulfonamide refers to the group -SO 2 -
• R and R' may be chemical modifiers.
• Aldehyde refers to the group -CHO.
• R' or R may be a chemical modifier.
• Alkyl refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen. This term is further exemplified by groups such as methyl, heptyl, (CH 2 ) 2 -, and adamantyl. Alkyl groups can either be unsubstituted or substituted with one or more non-interfering substituents, e.g., halogen, alkoxy, acyloxy, hydroxy, mercapto, carboxy, benzyloxy, phenyl, benzyl, or other functionality which has been suitably blocked with a protecting group so as to render the functionality non-interfering.
• substituents e.g., halogen, alkoxy, acyloxy, hydroxy, mercapto, carboxy, benzyloxy, phenyl, benzyl, or other functionality which has been suitably blocked with a protecting group so as to render the functionality non-interfering.
• Each substituent may be optionally substituted with additional non-interfering substituents.
• non-interfering characterizes the substituents as not adversely affecting any reactions to be performed in accordance with the process of this invention.
• Aminal ester refers to the group -N-(CRR')-O-(CO)-
• R where R, R', and R" are independently hydrogen, alkyl, aryl, heteroaryl, or arylalkyl.
• Amino refers to the group -NR'R", where R' and R" are independently hydrogen, alkyl, or aryl. If an amino group is to serve as a chemical functionality according to the present invention, either R' or R" must be hydrogen.
• Anhydride refers to the group -(CO)-O-(CO)-.
• Aryl or “Ar” refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can optionally be unsubstituted or substituted with hydroxy, lower alkyl, alkoxy, chloro, halo, mercapto, and other non-interfering substituents.
• Arylalkyl refers to the groups -R-Ar and -R-HetAr, where Ar is an aryl group, HetAr is a heteroaryl group, and R is straight-chain or branched-chain aliphatic group. Examples of arylalkyl groups include benzyl and furfuryl.
• Carbonate refers to the group -O(CO)O-.
• Carboxy or “carboxylic acid” refers to the group -COOH.
• Disulfide refers to the group -SS-.
• Ester refers to the group -(CO)O-R where R is alkyl, aryl, arylalkyl, or heteroaryl.
• Heteroaryl or “HetAr” refers to a monovalent aromatic carbocyclic group having a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzo[b]thienyl) and having at least one hetero atom, such as N, O or S, within the ring, which can optionally be unsubstituted or substituted with hydroxy, alkyl, alkoxy, halo, mercapto, and other non-interfering substituents.
• Heterocycle or “heterocyclic” refers to a monovalent saturated, unsaturated, or aromatic carbocyclic group having a single ring or multiple condensed ring and having at least one hetero atom, such as N, O, or S, within the ring, which can optionally be unsubstituted or substituted with hdyroxy, alkyl, alkoxy, halo, mercapto, and other non- interfering substituents.
• nitrogen heterocycles include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, and indoline.
• Hydroxy or hydroxyl refer to the group -OH.
• Hydrophilicitymethyl ketone ester refers to the group -(CO)-CH 2 -O-(CO)R where R is alkyl, aryl, arylalkyl or heteroaryl. R may be a chemical modifier.
• Ketone or keto refers to the group
• N-Mannich base refers to compounds formed through the reaction of amines with formaldehyde and certain reactive amide compounds. N-Mannich bases have moderate stability at acidic pH, but rapidly hydrolyse at physiological pH to liberate the free amino species. N-Mannich bases possess the group -NCH 2 N-.
• “Mercapto”, “sulphydryl”, or “thiol” refers to the group -SH.
• N-acylamide refers to the group - (CO)-NR-(CO)- where R is hydrogen, alkyl, aryl, arylalkyl, or heteroaryl. R may be a chemical modifier.
• "Nucleotide” refers to a phosphoric acid ester of a N-glycoside of a heterocyclic nitrogenous base and is meant to encompass both non-cyclic and cyclic derivatives. The phosphate can be present on position 2', 3', and/or 5". Generally, the glycoside component will be a pentose, however, in some embodiments, hexoses will be employed.
• the nitrogenous base typically will be selected from the group consisting of adenine, guanine, hypoxanthine, uraeil, cytosine, and thymine, and analogs or chemical modifications thereof.
• Nucleotide-based pharmaceutical agent or “nucleotide-based drug” refer to a pharmaceutical agent or drug comprising a nucleotide, an oligonucleotide or a nucleic acid.
• Nucleotide-based chemical modifier refers to a chemical modifier comprising a nucleotide, an oligonucleotide, or a nucleic acid.
• Nucleic acid refers to either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), whether single-stranded or double-stranded, and any chemical modifications thereof. Such modifications include, but are not limited to, alternate linkages other than the conventional phosphodiester linkage, modifications at cytosine exocyclic amines, substitution of 5-bromouracil, backbone modifications, base analogs, methylations, unusual base-pairing combinations, and the like.
• alternate linkages include the methylphosphonates wherein one of the phosphorous-linked oxygens has been replaced by methyl; phosphorothioates, wherein sulfur replaces one of the oxygens; various amidates, wherein NH 2 or organic amine derivatives, such as morpholidates or piperazidates, replace an oxygen; carbonate and carbamate linkages; and linkages involving sulfur rather than oxygen as the linking substituent.
• Oligonucleotide generally refers to linear sequences of nucleotides, joined by phosphodiester bonds, typically prepared by synthetic means. Position 3' of each nucleotide unit is linked via a phosphate group to position 5' of the next unit. In the terminal units, the respective 3' and 5' positions can be free (i.e., free hydroxyl groups) or phosphorylated. Those oligonucleotides employed in the present invention will vary widely in length.
• Phosphate ester refers to a compound having the general formula RO(PO) (OR') (OR”), where R, R' and R" are independently selected from hydrogen, alkyl, aryl, arylalkyl, and heteroaryl.
• Phosphodiester refers to a phosphate ester in which two hydroxyl groups of the phosphoric acid are esterified with organic residues: R'O-PO 2 H-OR" where R' and R" are independently selected from the group consisting of hydrogen, alkyl, aryl, arylalkyl, or heteroaryl. Oligonucleotides and nucleic acids are typically phosphodiesters in which the 3' and 5' positions of neighboring pentose units are linked by esterification with a phosphate residue.
• Phosphoramidate refers to a phosphodiester in which one or more of the hydroxyl groups is replaced with an amino group.
• Quaternary ammonium salt refers to the positively charged group -N + R'R"R"', where R', R", and R'" are independently alkyl or aryl.
• Sulfate refers to the group -OSO 3 -.
• R may be a chemical modifier.
• Thioester refers to the group -(CO)S-R where R is alkyl, aryl, arylalkyl, or heteroaryl.
• “Pharmaceutically or therapeutically effective dose or amount” refers to a dosage level sufficient to induce a desired biological result. That result may be the delivery of a pharmaceutical agent, alleviation of the signs, symptoms or causes of a disease or any other desired alteration of a biological system.
• novel methods for enhancing the transport and delivery of pharmaceutical agents in a controlled fashion are provided through the use of chemical modifiers. More generally, however, the present invention relates to methods of derivatizing pharmaceutical agents so as to improve a usefulbiological property of the pharmaceutical agent.
• properties of the pharmaceutical agent that may be enhanced or altered using the methods of the present invention include its membrane transport rate, its delivery rate, its serum half-life, and its biodistribution, including the enhancement of its pharmacokinetic and pharmacodynamic properties, such as its lipophilicity and/or its solubility, and its partition coefficient.
• the methods described herein also will find use for decreasing the inflammation and/or irritation accompanying the delivery of. the pharmaceutical agent across membranes.
• the methods of the present invention can be utilized to modify a pharmaceutical agent's ability to pass through the blood-brain barrier.
• the agent's ability to transport through the placental barrier can also be altered using the compositions and methods described herein.
• chemical modifiers that safely and reversibly add charge to pharmaceutical agents.
• Preferred pharmaceutical agents that best can be modified for transmembrane transport are those effective at low concentrations, for example, less than 50 milligrams per day, or are topically administered. Marked improvements in pharmaceutical agent bioavailability can be expected for those pharmaceutical agents that are poorly absorbed enterally or undergo extensive first pass hepatic inactivation.
• Pharmaceutical agents, according to the present invention should possess (or be capable of being modified to possess) at least one chemical functionality that can be altered with chemical modifiers or that can be substituted with or otherwise covalently coupled to a charged group.
• Preferred forms of chemical functionality include hydroxy, carboxy, amino, ketone, mercapto, sulfonamide, and amide groups.
• Exemplary pharmaceutical agents or drugs that may be delivered by the system of the present invention include analgesics, anesthetics, antifungals, antibiotics, antiinflammatories, anthelmintics, antidotes, antiemetics, antihistamines, antihypertensives, antimalarials, antimicrobials, antipsychotics, antipyretics, antiseptics, antiarthritics, antituberculotics, antitussives, antivirals, cardioactive drugs, cathartics, chemotherapeutic agents, corticoids (steroids), antidepressants, depressants, diagnostic aids, diuretics, enzymes, expectorants, hormones, hypnotics, minerals, nutritional supplements, parasympathomimetics, potassium supplements, sedatives, sulfonamides, stimulants, sympathomimetics, tranquilizers, urinary antiinfectives, vasoconstrictors, vasodilators, vitamins, xanthine derivatives, and the like.
• Examples of pharmaceutical agents with recommended dosage levels of less than 50 mg/day and thus, preferred for modification with chemical modifiers according to the present invention so as to improve the delivery and/or transport of the agents through membranes include the compounds set forth in Table 1.
• Preferred examples of pharmaceutical agents include the digitalis drugs, such as digoxin, digitoxin, digoxigenin, and digitoxigenin. These drugs are all primarily used as cardiac agents. However, they differ widely in their pharmacokinetics properties.
• Digoxin (3 ⁇ , 5 ⁇ , 12 ⁇ -3-[(O-2,6-dideoxy- ⁇ -D-ribo-hexopyranosyl(1 ⁇ 4)-O-2,6-dideoxy- ⁇ -D-ribo-hexopyranosyl-(1 ⁇ 4)-2,6-dideoxy- ⁇ -D-ribo-hexopyranosyl)oxy]-12,14-dihydroxycard-20(22)-enolide) and digitoxin (3 ⁇ ,5 ⁇ -3-[(O-2,6-dideoxy- ⁇ -D-ribo-hexopyranosyl(1 ⁇ 4)-O-2,6-dideoxy- ⁇ -D-ribo-hexopyranosyl-(1 ⁇ 4)-2,6-dideoxy- ⁇ -D-ribo-hexopyranosyl)oxy]-14-hydroxycard-20(22)-enolide) are two digitalis compounds available as pharmaceutical agents.
• Both digoxin and digitoxin are glycosides with three digitoxose residues attached.
• the amount of each compound absorbed depends largely on its polarity, which is a function of the net electronic charge on the molecule.
• the nonpolar, lipophilic digitoxin is completely absorbed.
• Other digitalis glycosides are not as well absorbed.
• nonpolar digoxin is over 90% bound to tissue proteins. Digoxin also crosses both the blood-brain barrier and the placenta.
• Digitoxin differs from the other commonly used digitalis glycosides not only in its firm binding to protein but also because it is metabolized in the liver, with the only active metabolite being digoxin.
• the portion of digitoxin that is bound to protein is in equilibrium with free digitoxin in the serum.
• Digitoxin is the most slowly secreted of the digitalis compounds.
• Digoxin possesses five secondary hydroxyl and one tertiary hydroxyls, whereas digitoxin has four secondary and one tertiary hydroxyl groups.
• the tertiary hydroxyl is not readily esterified.
• one or more of the secondary hydroxyls may serve as a chemical functionality to form linkages to chemical modifiers or spacer groups. Cleavage of the tridigitoxose moieties from digoxin and digitoxin yields digoxigenin and digitoxigenin, respectively.
• Digoxigenin (3,12,14-trihydroxycard-20(22)-enolide) contains two secondary hydroxyls and digitoxigenin (3,14-dihydroxycard-20(22)-enolide) contains one secondary hydroxyl which may be used to form linkages with chemical modifiers or spacer groups.
• Steroidal compounds form another preferred class of pharmaceutical agent.
• An example of a steroidal pharmaceutical agent is testosterone (17 ⁇ -hydroxyandrost-4-en-3-one), the principal male steroid. Its main therapeutic use is in the treatment of deficient endocrine function of the testes.
• Testosterone contains a single hydroxyl group, the 17 ⁇ -hydroxyl group, which may be used to form linkages with chemical modifiers or spacer groups.
• Estradiol (estra-1,3,5(10)-triene-3,17 ⁇ -di1l) is also a preferred steroidal pharmaceutical agent. Estradiol and its ester derivatives are indicated for the treatment of symptoms of menopause and other conditions that cause a deficiency of endogenous estrogen production.
• the two hydroxyl groups of estradiol serve as chemical functionalities for forming bonds with chemical modifiers or spacer groups.
• Progesterone is also a preferred steroidal pharmaceutical agent. Progesterone is used primarily to suppress or synchronize estrus as well as to control habitual abortion and diagnose and treat menstrual disorders.
• the enone and ketone groups may be utilized to form bonds with chemical modifiers or spacer groups.
• Additional preferred steroidal pharmaceutical agents include 3-hydroxy-5 ⁇ -pregnan-20-one (with a hydroxyl group and a ketone as chemical functionalities), 3- ⁇ -hydroxy-pregn-5-ene-20-one (with a hydroxyl and a ketone group as chemical functionalities), and related compounds.
• Piroxicam (4-hydroxy-2-methyl-N-2-pyridinyl-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide) and indomethacin (1- (4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid) are nonsteroidal anti-inflammatory, analgesic, and antipyretic drugs used in the treatment of osteoarthritis and rheumatoid arthritis.
• the phenolic hydroxyl group of piroxicam serves as a chemical functionality and can be linked to a chemical modifier or spacer group.
• Indomethacin possesses a carboxy group as its chemical functionality.
• Protein and peptide drugs may also be used as pharmaceutical agents according to the present invention.
• the problems associated with conventional delivery strategies for protein and peptide drugs are widely appreciated. Oral administration of these drugs is generally impractical due to degradation and non-absorption in the gastrointestinal tract. Thus, the parenteral route remains the principal delivery route.
• Protein and peptide drugs are capable of either covalently binding to a chemical modifier and so can serve as pharmaceutical agents according to the present invention.
• most protein and peptide drugs possess either an amino, carboxy, hydroxy, or mercapto group that can be used to bind covalently to a chemical modifier or spacer group.
• particularly preferred as protein and peptide drugs include those which contain either a cysteine or a lysine residue.
• the mercapto group of cysteine and the e-amino group of lysine can be used as chemical functionality to bind to a chemical modifier or spacer group. See, also copending application Serial No. (Attorney docket No. 11509-92), filed May 24, 1993.
• protein and peptide drugs which are not amenable to coupling with a chemical modifier can be modified using recombinant DNA techniques. These modifications could entail the replacement of an existing amino acid of the drug with an amino acid or other group which can be easily coupled to a chemical modifier or the addition of amino acid(s) or other group(s) which can be easily coupled to a chemical modifier. In some instances, the net charge, charge distribution, or charge localization of the protein itself is modified using recombinant techniques (infra) thus, eliminating the necessity of additional bonding to a chemical modifier.
• Amino acid-based drugs such as the cephalosporins, will typically have a molecular weight less than about 5000, and preferably, less than about 2500, and more preferably, less than about 1000.
• Protein and peptide drugs typically have a molecule weight of at least about 100 daltons, and more typically a molecular weight in the range of about 200 to 40,000 daltons. Specific examples of peptides and proteins in this size range include, but are not limited to, those found in Table 2.
• Calcitonin e.g., eel, Bradykinin potentiator B salmon, human
• Calcitonin e.g., eel, Bradykinin potentiator B salmon, human
• Bradykinin potentiator C Calcitonin gene related Brain-derived neurotrophic peptide factor Endorphin (alpha, beta, and Cystic fibrosis transmembrane gamma) conduct regulator (CFTR)
• Thyrotropin-releasing Chorionic gonadotropin hormone Ciliary neurotrophic factor NT-36 (N-[[(s)-4-oxo-2- (CNTF) azetidinyl]carbonyl]-L- Corticotropin-releasing histidyl-L-prolinamide) factor (CRF)
• NT-36 N-[[(s)-4-oxo-2- (CNTF) azetidinyl]carbonyl]-L- Corticotropin-releasing histidyl-L-prolinamide) factor (CRF)
• NT-36 N-[[(s)-4-oxo-2- (CNTF) azetidinyl]carbonyl]-L- Corticotropin-releasing histidyl-L-prolinamide) factor (CRF)
• CBF Thyrotropin-releasing Chorionic gonadotropin hormone
• G-CSF Granulocyte macrophage colony Follicle luteoids stim. factor. aANF growth factor releasing sargramostrim.
• GM-CSF GM-CSF factor
• GFRF Multilineage colony Melanocyte-stimulating stimulating factor (CSF) hormone (alpha, beta, and Macrophage-specific colony gamma) stimulating factor (CSF-1)
• CSF-4 Bradykinin
• EGF Somatotropin Epidermal growth factor
• Interferons e.g., alpha, Heparin binding neurotrophic beta, and gamma factor (HBNF) Interleukins, e.g., IL-1 Fibroblast growth factor receptor antagonist; (FGF) IL-10, CSIF (cytokine Hirudin synthesis inhibitory HIV inhibitor peptide factor); Inhibin-like peptide IL-11; IL-6; IL-4; and IL-2 Insulinotropin Menotropins Lipotropin Urofollitropin (Follicle Macrophage-derived neutrophil stimulating hormone, FSH) chemotaxis factor Leutinizing hormone (LH) Magainin I/II LH-releasing hormone (LHRH) Melatonin, tryptophan Gonadotropin releasing hydroxylase hormone Midkine (MK) Oxytocin Neurophysin Streptokinase Neurotrophin-3 Tissue plasminogen activator Nerve growth factor (NGF) Urokinase Oxytocin Vasopressin Phospho
• TK Thymidine kinaise
• the protein or peptide drug is one of the highly related sequences of alpha interferon 2 (IFN- ⁇ 2).
• IFN- ⁇ 2a (sold by Roche as Roferon A)
• INF- ⁇ 2b (sold by Schering as Intron A) are 19 kilodalton (kDa) proteins of 165 amino acids. These proteins contain two pairs of disulfide linked cysteines and include 20 basic and 22 acidic residues for a net positive charge of approximately -1.69 at neutral pH.
• IFN has been used as an antiproliferative agent in the treatment of renal cell carcinoma, hairy cell leukemia, Kaposi's sarcoma, melanoma, and T-cell lymphoma, as well as an antiviral agent in the treatment of non-A,B-hepatitis, genital warts, Epstein-Barr virus, CMV, AIDS, and rhinovirus.
• parathyroid hormone is a linear polypeptide consisting of 84 amino acids. See Harper et al ., Eds., Review of Physiological Chemistry, 16th Ed., Lange Medical Publications, Los Altos, California (1977) p. 468. However, a fragment consisting of about 34 amino acid residues from the N-terminal has been isolated and found to display the full biological activity of PTH. See Potts et al ., in
• PTH Parathyroid Hormone and Thyrocalcitonin (Calcitonin), R. V. Talmage, et al . , Eds. Excerpta Medica, New York (1968).
• the sequence of the polypeptide varies slightly among mammalian species.
• PTH is meant to include human parathyroid hormone, as well as the other variants and the 34 amino acid fragment.
• PTH contains a variety of chemical functionalities (e.g., hydroxyl groups from serine residues, carboxylic acid groups from aspartic acid residues, and amino groups from glutamine, lysine, and arginine residues) which may potentially be used to bind to chemical modifiers.
• PTH serves as a regulatory factor in the homeostatic control of calcium and phosphate metabolism. See, e.g., Parsons, et al . "Physiology and Chemistry of Parathyroid Hormone” in Clinics in Endocrinology and Metabolism, I. Maclntyre, Ed. Saunders, Philadelphia (1972) pp. 33-78.
• the main therapeutic use for PTH is in the treatment of osteoporosis.
• PTH has also been used as a blood calcium regulator.
• Calcitonin is also a preferred peptide pharmaceutical agent. Calcitonin is a polypeptide containing 32 amino acid residues. See Harper et al . , Eds., Review of
• calcitonin is meant to include all calcitonin, including that of humans, mammals, and fish, as well as other variants.
• Calcitonin contains several chemical functionalities (e.g., hydroxly groups from serine residues, mercapto groups from cysteine residues, amino groups from lysine and amide groups from glutamine, and asparagine, and guanidino groups from arginine residues) which may potentially be used to bind to chemical modifiers.
• Calcitonin is a calcium regulating hormone and has been used in the treatment of osteoporosis, hypercalcemia, and Paget's disease.
• An additional preferred protein drug is the cytokine IL-10.
• 11-10 is produced by the TH2 helper subset, B cell subsets and LPs-activated monocytes.
• IL-10 inhibits several immune functions that are relevant to the skin immune response and thus, the development of the irritation and inflammation that is sometimes associated with the transdermal or iontophoretic delivery of drugs. More specifically, the release of IFN- ⁇ , which initiates the cascade of cellular activation leading to the skin's immune response, is inhibited by IL-10.
• IL-10 also suppresses the synthesis of numerous proinflammatory cytokines by macrophages, as well as the proliferation of antigen-specific T cell proliferation by down regulating class II MHC expression.
• the present invention also contemplates the simultaneous or sequential transdermal, either passively or iontophoretically, delivery of a drug that elicits inflammation or irritation with a pharmaceutical agent-chemical modifier complex wherein the pharmaceutical agent is IL-10, or an analog thereof.
• the complex is delivered in an amount sufficient to prevent the inflammation and irritation generally associated with delivery of the other drug.
• G-CSF a colony stimulating factor that stimulates production of granulocytes, particularly neutrophils
• GM-CSF a colony stimulating factor that stimulates production of granulocytes/macrophages/ monocytes
• human growth factor insulin, a hormone (protein) naturally secreted by the ⁇ cells of the pancreas (when stimulated by glucose and the parasympathetic nervous system); antibodies (subfragments); EPO, a glycoprotein hormone produced in the kidneys which stimulates the bone marrow to produce red blood cells; the interleukins; interferon-gamma, a cytokine protein produced by vertebrate cells following a virus infection and possessing potent antiviral effects; Vasotec ® , a antihypertensive (Enalapril maleate, Merck, Sharp & Dohme, West Point, PA); Capoten ® , a antihypertensive (Captopril, E.
• Rocephin ® an antiinfective (ceftriaxone sodium, Roche); Augmentin ® , an antiinfective (Smith Kline & French Laboratories, Philadelphia, PA); Ceclor ® , an antiinfective (Cefaclor, 3-chloro-7-D-(2-phenylglycinamido)-3-cephem-4-carboxylic acid, Eli Lilly and Company, Indianapolis, IN); Sandimmune ® , an immunosuppressive (cyclosporine, a cyclic polypeptide consisting of 11 amino acids, Sandoz Pharmaceuticals Corporation, East Hanover, NJ); Premaxin ® , an antiinfective (Imipenem-cilastatin sodium, Merck); Fortaz ® , an antiinfective (ceftazidime, Glaxo Pharmaceuticals, Research Triangle Park, NC); Amoxil, an antiinfective (amoxicillin, Beecham Labor).
• Aptamers are single- or double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets.
• aptamers function by inhibiting the actions of the molecular target, e.g., proteins, by binding to the pool of the target circulating in the blood.
• Aptamers possess chemical functionality and thus, can covalently bond to chemical modifiers and/or serve as chemical modifiers, according to the methods described herein.
• molecular targets will be capable of forming non-covalent but specific associations with aptamers, including small molecules drugs, metabolites, cofactors, toxins, saccharide-based drugs, nucleotide-based drugs, glycoproteins, and the like
• the molecular target will comprise a protein or peptide, including serum proteins, kinins, eicosanoids, cell surface molecules, and the like.
• aptamers include Gilead's antithrombin inhibitor GS 522 and its derivatives (Gilead Science, Foster City, CA). See also Macaya et al . (1993) Proc. Natl. Acad. Sci. USA 90:3745-9; Bock et al . (1992) Nature (London) 355:564-566 and Wang et al . (1993) Biochem. 32:1899-904.
• Aptamers specific for a given biomolecule can be identified by using techniques known in the art. See, e . g . , Toole et al . (1992) PCT Publication No. WO 92/14843; Tuerk and Gold (1991) PCT Publication No. WO 91/19813; Weintraub and Hutchinson (1992) PCT Publication No. 92/05285; and Ellington and Szostak (1990) Nature 346:818. Briefly, these techniques typically involve the complexation of the molecular target with a random mixture of oligonucleotides. The aptamermolecular target complex is separated from the uncomplexed oligonucleotides. The aptamer is recovered from the separated complex and amplified. This cycle is repeated to identify those aptamer sequences with the highest affinity for the molecular target.
• Antisense compounds are single stranded DNA or RNA oligonucleotides that are designed to bind and disable or prevent the production of the mRNA responsible for generating a particular protein.
• Antisense compounds complementary to one or more sequences are employed to inhibit transcription, RNA processing, and/or translation of the cognate mRNA species and thereby effect a reduction in the amount of the respective encoded polypeptide.
• Antisense compounds can provide a therapeutic function by inhibiting in vivo the formation of one or more proteins that cause or are involved with disease.
• Antisense compounds complementary to certain gene messenger RNA or viral sequences have been reported to inhibit the spread of disease related to viral and retroviral infectious agents (See, for example, Matsukura et al . (1987) Proc. Natl. Acad. Sci. USA 84:7706, and references cited therein).
• Triple helix compounds are oligonucleotides that bind to sequences of double-stranded DNA and are intended to inhibit selectively the transcription of disease-causing genes, such as viral genes, e.g., HIV and herpes simplex virus, and oncogenes.
• nucleotide-based drugs have had limited success as therapeutic agents, in part, because of problems associated with their stability and delivery.
• Nucleotide-based pharmaceutical agents contain a phosphodiester bond which is sensitive to degradation by nucleases. Such degradation would be a significant impediment to the use of an oligonucleotide or nucleic acid as a pharmaceutical agent that depends upon the integrity of the sequence for its recognition specificity.
• Naturally occurring oligonucleotides and nucleic acids often must be chemically modified to render them resistant to nucleases which would degrade them in vivo , or even in vitro unless care is taken to choose appropriate conditions.
• nucleotide-based drugs of the present invention will have a molecular weight greater than about 350.
• nucleotide-based drugs include di- and trinucleotides, such as GS 375, a dinucleotide analog with potential therapeutic activity against the influenza virus (Gilead Sciences, Inc., Foster City, CA), antisense compounds, and triple helix drugs.
• Antisense compounds include antisense RNA or DNA, single or double stranded, oligonucleotides, or their analogs, which can hybridize specifically to individual mRNA species and prevent transcription and/or RNA processing of the mRNA species and/or translation of the encoded polypeptide (Ching et al . Proc. Natl. Acad. Sci. U.S.A. 86:10006-10010 (1989); Broder et al . Ann. Int. Med. 113:604-618 (1990); Loreau et al . FEBS Letters 274:53-56 (1990); Holcenberg et al . W091/11535; U.S.S.N. 07/530,165 ("New human CRIPTO gene"); WO91/09865; WO91/04753; WO90/13641; WO 91/13080, WO 91/06629, and EP 386563).
• Triple helix drugs are oligonucleotides that stop protein production at the cell nucleus. These drugs bind directly to the double stranded DNA in the cell's genome to form a triple helix and thus, prevents the cell from making a target protein. See, e.g., PCT publications Nos. WO 92/10590, WO 92/09705, WO91/06626, and U.S. Patent No. 5,176,996.
• oligonucleotides e.g., antisense compounds and triple helix drugs
• the site specificity of oligonucleotides is not significantly affected by modification of the phosphodiester linkage or by chemical modification of the oligonucleotide terminus. Consequently, these oligonucleotides can be chemically modified; enhancing the overall binding stability, increasing the stability with respect to chemical degradation, increasing the rate at which the oligonucleotides are transported into cells, and conferring chemical reactivity to the molecules.
• the general approach to constructing various oligonucleotides useful in antisense therapy has been reviewed by vander Krol et al . (1988) Biotechnigues 6 : 958-976 and Stein et al . (1988) Cancer Res. 48:2659-2668.
• antisense compounds and triple helix drugs also can include nucleotide substitutions, additions, deletions, or transpositions, so long as specific hybridization to the relevant target sequence is retained as a functional property of the oligonucleotide.
• some embodiments will employ phosphorothioate analogs which are more resistant to degradation by nucleases than their naturally occurring phosphate diester counterparts and are thus expected to have a higher persistence in vivo and greater potency (see, e.g., Campbell et al . (1990) J. Biochem. Biophvs. Methods 20: 259-267).
• Phosphoramidate derivatives of oligonucleotides also are known to bind to complementary polynucleotides and have the additional capability of. accommodating covalently attached ligand species and will be amenable to the methods of the present invention. See, for example, Froehler et al . (1988) Nucleic Acids Res. 16(11):4831.
• the antisense compounds and triple helix drugs will comprise O-methylribonucleotides (EP Publication No. 360609). Chimeric oligonucleotides may also be used (Dagle et al . (1990) Nucleic Acids Res. 18: 4751).
• antisense oligonucleotides and triple helix may comprise polyamide nucleic acids (Nielsen et al . (1991) Science 254:1497 and PCT publication No. WO 90/15065) or other cationic derivatives (Letsinger et al . (1988) J. Am. Chem. Soc. 110:4470-4471).
• oligonucleotides wherein one or more of the phosphodiester linkages has been substituted with an isosteric group, such as a 2-4 atom long internucleoside linkage as described in PCT publication Nos. WO 92/05186 and 91/06556, or a formacetal group (Matteucci et al . (1991) J. Am. Chem. Soc. 113:7767-7768) or an amide group (Nielsen et al . (1991) Science 254: 1497-1500).
• an isosteric group such as a 2-4 atom long internucleoside linkage as described in PCT publication Nos. WO 92/05186 and 91/06556, or a formacetal group (Matteucci et al . (1991) J. Am. Chem. Soc. 113:7767-7768) or an amide group (Nielsen et al . (1991) Science 254: 1497-1500
• nucleotide analogs for example wherein the sugar or base is chemically modified, can be employed in the present invention.
• "Analogous" forms of purines and pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents.
• An exemplary but not exhaustive list includes 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uraeil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N 6 -isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil,
• the conventional bases by halogenated bases.
• the 2'-furanose position on the base can have a non-charged bulky group substitution.
• non-charged bulky groups include branched alkyls, sugars and branched sugars.
• Terminal modification also provides a useful procedure to modify cell type specificity, pharmacokinetics, nuclear permeability, and absolute cell uptake rate for oligonucleotide pharmaceutical agents.
• substitutions at the 5' and 3' ends include reactive groups which allow covalent crosslinking of the nucleotide-based pharmaceutical agent to other species and bulky groups which improve cellular uptake. See, e . g .
• Oligodeoxynucleotides Antisense Inhibitors of Gene Expression, (1989) Cohen, Ed., CRC Press; Prospects for Antisense Nucleic Acid Therapeutics for Cancer and AIDS, (1991), Wickstrom, Ed., Wiley-Liss; Gene Regulation: Biology of Antisense RNA and DNA, (1992) Erickson and Izant, Eds., Raven Press; and Antisense RNA and DNA, (1992), Murray, Ed., Wiley-Liss.
• Antisense RNA and DNA (1988), DNA. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
• Antisense polynucleotides of various lengths may be delivered, although such antisense polynucleotides typically comprise a sequence of at least about 15 consecutive nucleotides.
• antisense compounds include G 1128 (Genta, Inc., San Diego, CA), OL(1)p53 (Lynx Pharmaceuticals), Ampligen (Hemm Pharmaceuticals), Isis 1082 and Isis 2105 (Isis Pharmaceuticals, Carlsbad, CA).
• the triple helix drug will comprise a DNA oligonucleotide in the range of about 20 to 40 bases.
• Heterocyclic drugs and particularly those containing at least one nitrogen heterocyclic ring can be employed as pharmaceutical agents in the methods described herein.
• yohimbine is an indole alkaloid that blocks ⁇ -2-adrenergic receptors. Its peripheral effects are to increase cholinergic activity at the same time that it decreases adrenergic activity. This combination has led to the use of yohimbine in the treatment and diagnostic classification of certain types of male erectile impotence.
• Yohimbine the methyl ester of yohimbic acid, possesses a free hydroxyl group which may serve as the necessary chemical functionality for binding to a chemical modifier or spacer group.
• Yohimbic acid which contains both a free hydroxyl group and a carboxy group as potential chemical functionalities, is also a preferred pharmaceutical agent.
• Morphine (7,8-didehydro-4,5-epoxy-17-methyl-(5 ⁇ ,6 ⁇ )-morphinan-3,6-diol sulfate (2:1), pentahydrate) is the most important alkaloid of opium. Morphine exerts its primary effect on the central nervous systems and organs containing smooth muscle. Morphine, as other opiods, acts as an agonist interacting with stereospecific and saturable binding sites/receptors in the brain, spinal cord and other tissues.
• the central nervous system effects of intravenously administered morphine sulfate are influenced by its ability to cross the blood-brain barrier.
• the delay in the onset of analgesia following epidural or intrathecal injection may be attributed to its relatively poor lipid solubility (i.e., an oil/water partition coefficient of 1.42) and its slow access to the receptor sites.
• the hydrophilic character of morphine may also explains its retention in the central nervous system and its slow release into the systemic circulation.
• the introduction of a charged chemical modifier should effectively prevent morphine from crossing the blood-brain barrier.
• the morphine-chemical modifier complex should serve as a peripheral acting analog.
• Morphine possesses two hydroxyl groups which can be coupled to chemical modifiers.
• Methotrexate (formerly Amethopterin, N-[4-[[(2,4-diamino-6-pteridinyl)-methyl]methylamino]benzoyl]-L-glutamic acid) is an antimetabolite used in the treatment of certain neoplastic diseases, severe psoriasis, and adult rheumatoid arthritis.
• Methotrexate inhibits dihydrofolic acid reductase and therefore interferes with DNA synthesis, repair, and cellular replication.
• Methotrexate possesses several chemical functionalities, including carboxy groups and amino groups, which can be bound to chemical modifiers according to the present invention.
• Lorazepam (7-chloro-5-(o-chlorophenyl)-1,3-dihydro- 3-hydroxy-2H-1,4-benzodiazepin-2-one) is a benzodiazepine with antianxiety and sedative effects.
• the mean half-life for lorazepam is about 16 hours when given intravenously or intramuscularly.
• Lorazepam is rapidly conjugated at the 3-hydroxyl group into its major metabolite, lorazepam glucuronide, which is excreted in the urine. This metabolite has no demonstrable central nervous system activity in animals. Lorazepam possesses a free hydroxyl group and an amino group which can be derivatized according to the methods described herein.
• 6-Mercaptopurine (1,7-dihydro-6H-purine-6-thionemonohydrate) is one of large series of purine analogues which interfere with nucleic acid biosynthesis and has been found active against human leukemias. See, e . g. , Hitchings and Elion (1954) Ann. NY Acad. Sci. 60:195-199. Clinical studies have shown that the absorption of an oral dose of mercaptopurine in man is incomplete and variable averaging approximately 50% of the administered dose. 6-Mercaptopurine contains a free mercapto group and an amino group for bonding to chemical modifiers.
• 5-Fluorouracil is another preferred pharmaceutical agent.
• 5-Fluorouracil (5-fluoro-2,4 (1H, 3H) -pyrimidinedione) is an antineoplastic antimetabolite.
• fluorouracil interferes with the synthesis of DNA and to a lesser extent inhibits the formation of RNA.
• Fluorouracil distributes into tumors, intestinal mucosa, bone marrow, liver, and other tissues throughout the body. In spite of its limited lipid solubility, fluorouracil diffuses readily across the blood-brain barrier. Fluorouracil is effective in the palliative management of carcinoma of the colon, rectum, breast, stomach, and pancreas. Fluorouracil possesses two amino groups which can bond to chemical modifiers according to the present invention.
• Another preferred heterocyclic drug is theophylline, a xanthine bronchodilator, pulmonary vasodilator, and smooth muscle relaxant.
• Theophylline is used for the symptomatic relief and/or prevention of asthma and the reversible bronchospasm associated with chronic bronchitis and emphysema.
• Theophylline possesses an amino functionality for coupling with a chemical modifier.
• Nalidixic acid is also a heterocyclic drug which can be delivered using the methods described herein.
• Nalidixic acid is an antibacterial agent with activity against gram negative bacteria and is rapidly absorbed from the gastrointestinal tract.
• Nalidixic acid possesses an amino group, a keto group, and a carboxy group as chemical functionality.
• Melatonin a tryptamine derivative, is a hormone of the pineal gland and is also produced by extrapineal tissues. melatonin has been postulated as the mediator of photic-induced antigonadotrophic activity in photoperiodic mammals. Melatonin has been used in the treatment of psychiatric disorders. Melatonin possesses amino functionality for coupling with chemical modifiers. Nicotinic acid or niacin functions in the body as a component of two hydrogen transporting coenzymes. In addition to its functions as a vitamin, nicotinic acid exerts several distinctive pharmacological effects which vary according to the dosage level employed. Nicotinic acid, in large doses, causes a reduction in serum lipids. Nicotinic acid is a nitrogen heterocycle having a hydroxyl group.
• the chemical modifier is preferably susceptible to in vivo cleavage from the pharmaceutical agent-chemical modifier complex, such that the agent is in an active form and the modifier becomes a non-toxic compound or is rapidly degraded.
• the modifier will be a naturally occurring substance.
• the modifier is a biologically-active molecule or a second pharmaceutical agent.
• Chemical modifiers, according to the present invention comprise either permanently charged organic compounds or organic compounds which carry an ionic charge by virtue of the conditions of pH which exist during transmembrane or transdermal delivery. According to some embodiments, the net ionic charge of a chemical modifier
• chemical modifiers function primarily to alter the charge characteristics of a pharmaceutical agent (e.g., by the addition, deletion, or redistribution of charge), they also can serve to modify the solubility parameters of the pharmaceutical agent.
• more than one chemical modifiers can be coupled to a charged pharmaceutical agent to produce a complex having the same net charge as the pharmaceutical agent, but exhibiting different water or lipid solubility due to the introduction of the additional hydrophilic or lipophilic groups of the chemical modifiers.
• Chemical modifiers possess at least one chemical functionality which can be covalently bonded to a pharmaceutical agent, optionally via a spacer group. Examples of chemical functionality include hydroxy, carboxy, amino, ketone, mercapto, sulfonamide, amide groups and the like.
• the chemical modifier carries a positive charge.
• the positive charge typically arises by virtue of covalent bonds, such as in a quaternary ammonium group.
• the charged complex is prepared by the covalent attachment of an uncharged moiety in such a manner as to generate a charge.
• the complex can be dissociated under physiological conditions. For example, an ⁇ -haloalkyl carbonates or esters can be reacted with a pharmaceutical agent having a tertiary amino group to provide a pharmaceutical agent-chemical modifier complex having a quaternary ammonium group. Chemical or enzymatic hydrolysis of the carbonate or ester bond is followed by spontaneous elimination of the alkyl aldehyde and regeneration of the active pharmaceutical agent.
• the pharmaceutical agent comprises deprenyl, as illustrated below:
• the chemical modifier acquires a positive charge only after the modifier is complexed with the pharmaceutical agent.
• the pharmaceutical agent-chemical modifier complex may obtain a positive charge via protonation in the delivery buffer or formulation due to the pH conditions which exist during drug delivery.
• choline (2-hydroxy-N,N,N-trimethylethanaminium, available from Aldrich Chemical Co., Milwaukee, Wisconsin).
• Choline carries a positive charge and possesses a hydroxyl group as a chemical functionality.
• the hydroxyl group may be utilized, for example, to form ester or carbonate linkages with carboxy or hydroxyl groups, respectively, of pharmaceutical agents or to form carbamates with amine groups of pharmaceutical agents.
• Homologs of choline such as 3-hydroxy-N,N,N-trimethylpropaminium chloride, may also serve as chemical modifiers.
• Carnitine 3-carboxy-2-hydroxy-N,N,N-trimethyl-1-propanaminium hydroxide, inner salt, available from Aldrich Chemical Co.
• Carnitine is a nontoxic, naturally occurring amino acid. See Fix et al . (1986) Am. J. Physiology 251:G332-G340.
• the N,N,N-trimethylammonium group of carnitine serves to impart a positive charge to the molecule.
• Carnitine possesses two types of chemical functionality with which it can bond to a pharmaceutical agent. For example, the hydroxyl of carnitine may be reacted with a carboxy group of a pharmaceutical agent.
• the carboxy group of carnitine may be utilized to form, for example, either ester, thioester, or amide linkages with hydroxy, mercapto, or amino groups of a pharmaceutical agent.
• Homologs of carnitine such as 4 ,4-dimethylamino-3-hydroxybutyric acid or 6, 6-dimethylamino-3-hydroxyhexanoic acid, may also serve as chemical modifiers.
• the dimethylamino group of these chemical modifiers can be alkylated to form a quaternary ammonium salt either before or after the chemical modifier is coupled to a pharmaceutical agent.
• These homologs of carnitine differ from carnitine in their number of carbon atoms. Like carnitine, homologs of carnitine possess both a hydroxyl group and a carboxy group with which they can form bonds to pharmaceutical agents.
• Oligomers of carnitine may also be employed as chemical modifiers.
• the molecular weight of the oligomer is usually between 100-2000. Typically 2 to 10 carnitine molecules are used to construct the polyester.
• a further carnitine derivative which may function as a chemical modifier is thiocarnitine.
• Thiocarnitine is the mercapto analog to carnitine and thus, possesses a mercapto group and a carboxylic acid group as chemical functionality.
• Lysine (2,6-diaminohexanoic acid, available from Aldrich Chemical Co.) and particularly its N ⁇ -methylated homologs, especially N ⁇ -trimethyllysine, may also be used as chemical modifiers.
• lysine shall be understood to refer to lysine and to its N ⁇ -methylated homologs, including N ⁇ -trimethyllysine and its ⁇ -hydroxyl derivative.
• lysine possesses two functionalities with which it can bind to a pharmaceutical agent.
• the amino or carboxy groups of lysine may be exploited to form linkages with pharmaceutical agents.
• the amino group for example, may be converted to an amide through reaction with a carboxy group of the pharmaceutical agent or to a carbamate by reaction with the chloroformate derivative of a hydroxy-containing pharmaceutical agent.
• the carboxy group of lysine may be transformed into either an ester, thioester or amide through reaction with a hydroxy, mercapto, or amino group of a pharmaceutical agent.
• derivatives of lysine may be used as chemical modifiers.
• the ⁇ -hydroxyl derivative which provides a third functionality for selective modification may be used as a chemical modifier.
• the chemical modifier exhibits therapeutic effects apart from its carrier function.
• An example of a therapeutic chemical modifier is oligomeric or polymeric lysine (polylysine). Polylysine possesses antiviral and antibacterial activities, as well as a specific affinity for tumor cells in cancerous tissue. Ryser, H.J.-P. and Shen, W.-C. (1986) in "Targeting of Drugs with Synthetic Systems," G. Gregoriadis, J. Senior and G. Poste, Eds., pp. 103-121, Plenum Publishing Corp. New York.
• Polylysine has also been shown to enhance the uptake into cells of conjugated proteins such as albumin and peroxidase. Shen, W.-C. and Ryser, H.J.- P, (1978) Proc. Natl. Acad. Sci. USA 75:1872-1876.
• An additional desirable chemotherapeutic property of poly(L-lysine) is its facile degradation by intercellular trypsin.
• Polylysine contains amino and carboxy groups. Typically, 2 to 10 lysines are used to form the polyamide. The molecular weight of the oligomer is usually between 200-2000. The polymerization of lysine may be accomplished by methods well known by those skilled in the art.
• polylysine derivatives containing specific protease cleavage sites may serve as chemical modifiers.
• An example of such a derivative is (Lys) n -Phe-Pro-Arg, where "Lys n " represents polylysine; “Phe” refers to phenylalanine; “Pro” refers to proline, and “Arg” refers to arginine.
• This polylysine derivative may be cleaved by the serine proteinase thrombin.
• amino acids such as ornithine (2,5-diaminopentanoic acid, available from Aldrich Chemical Co.) and its N ⁇ -methylated homologs, serine, threonine, and tyrosine may also be used as chemical modifiers in an analogous manner.
• folate or folic acid N-[4-[[2(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid
• folic acid N-[4-[[2(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid
• Folate Is internalized by cells by receptor-mediated endocytosis and it has been reported that covalently conjugating folic acid to a macromolecule, such as BSA, bovine IgG, bovine RNase, and horseradish peroxidase, allows for the delivery of the conjugates into many living cells by the cellular uptake system for folate.
• a macromolecule such as BSA, bovine IgG, bovine RNase, and horseradish peroxidase
• betaine (1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt, available from Aldrich Chemical Co.).
• Betaine possess a carboxylic acid as chemical functionality and carries a permanent positive charge from the quaternary ammonium group.
• Homologs of betaine such as N,N,N-trimethyl-4-aminobutyric acid may also serve as chemical modifiers.
• Other preferred chemical modifiers include betonicine (trans-2-carboxy-4-hydroxy-1,1-dimethylpyrrolidinium hydroxide, inner salt), stachydrine (2-carboxy-1, 1-dimethylpyrrolidinium hyroxide inner salt), and trigonelline (3-carboxy-1-methylpyridinium hyroxide inner salt).
• betonicine trans-2-carboxy-4-hydroxy-1,1-dimethylpyrrolidinium hydroxide, inner salt
• stachydrine (2-carboxy-1, 1-dimethylpyrrolidinium hyroxide inner salt
• trigonelline 3-carboxy-1-methylpyridinium hyroxide inner salt
• histones include histones.
• H4 The smallest histone is H4 with 103 amino acids and a net charge of approximately +18 at neutral pH.
• the largest histone H1 carries a charge of approximately +46 at neutral pH over approximately 207 residues.
• Histone H1 is rich in lysine groups. These lysine groups contain amino groups which may be used to bind to pharmaceutical agents or spacer groups.
• Histones H2A and H2B also contain lysine groups, and thus, amino groups as chemical functionality.
• Histones H3 and H4 are rich in arginine. The arginine residue provides an amino group which may be bonded to spacer groups or pharmaceutical agents.
• one variant of the histone H3 contains a free cysteine residue that may be used to couple with spacer groups or pharmaceutical agents.
• the protamines are a group of proteins that contain basic amino acids and can serve as chemical modifiers.
• Anther lysine rich protein which can be employed as chemical modifiers according to the methods described herein is cytochrome c, a heme protein in which the active prosthetic group is a derivative of iron protoporphyrin IX.
• Cytochrome c occurs in the cells of all aerobic organisms and can be found in animal cells in the mitochondrial protein-lipid complex. See Margoliash and Schejter (1966) Advan. Protein Chem. 21:113, Structure and Function of Cytochromes, Okunuki et al . (eds.), University Park Press: Baltimore (1968). Cytochrome c can be iontophoretically transported readily across human or mouse skin. See (1993) "Electrotransport: A Technology whose Time Has Come".
• proteins that complex metal ions, and particularly iron, such as siderophores, enterobactin, HBED (N,N'-bis(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid), and ferrioxamines see, e . g . , Kirk-Othmer Encyclopedia of Chemical Technology, 3rd Ed., Vol. 13, pages 782-786; proteins with covalently attached heme or porphyrin groups; and other compounds covalently attached to heme or porphyrin groups will also find use as chemical modifiers in the methods described herein.
• Aminosteroids also can be employed as chemical modifiers.
• squalamine an antimicrobial about as potent as ampicillin, possesses three quaternary ammonium groups, a sulfate group, as well as a hydroxyl group.
• the mechanism of squalamine's activity may at least partially be due to a disruption of the lipid bilayer in membranes.
• squalamine could serve as a penetration enhancer, as well as a chemical modifier and/or a pharmaceutical agent.
• chonemorphine ( (3 ⁇ ,5 ⁇ ,2OS)-N 20 ,N 20 -dimethylpregnane-3,2-diamine) possesses two amino groups, one of which can be derivatized to produce the corresponding ammonium salt, while the other can be coupled to a pharmaceutical agent.
• conessine (3 ⁇ -(dimethylamino) con-5-enine) is a naturally occurring plant steroid having two amino groups and can find use as a chemical modifier according to the methods described herein.
• chemical modifiers carry negative charges.
• the negative charge typically arises from a sulfate group, a phosphate group, or a carboxy group.
• the sulfate and carboxy groups will provide a single negative charge, whereas the phosphate group may impart either a single or double negative charge.
• Taurine is a particularly versatile chemical modifier in that it can provide either a negative or positive charge.
• Taurine (2-aminoethanesulfonic acid, available from Aldrich Chemical Co.) acquires a negative charge by deprotonation of its sulfonic acid group to form the corresponding sulfonate salt.
• Taurine also possesses an amino group as chemical functionality. This amino group may be converted to the corresponding ammonium salt to provide a positive charge.
• cysteic acid (3-sulfoalanine, available from Aldrich Chemical Co.) which possesses both amino and carboxy functionalities in addition to the sulfonic acid group, may provide either positive or negative charge.
• sulfate salts may be produced through the reaction of a hydroxyl containing compound with sulfur trioxide and its amine and ether adducts, chlorosulfonic acid, sulfonic acid, or sulfuric acid. See, e.g., Barton and Ollis, Comprehensive Organic Chemistry. Pergamon Press, New York (1979).
• Phosphates may be produced through the reaction of a hydroxyl containing pharmaceutical agent, chemical modifier or spacer group with phosphonyl halides, orthophosphoric acid, or phosphorus pentoxide. See, e.g., Barton and Ollis, Comprehensive Organic Chemistry.
• Preferred examples of negatively charged pharmaceutical agent-modifier complexes include digitoxigenin-3-sulfate, triethylammonium salt; digitoxin-4'''-sulfate, triethylammonium salt; and the like.
• Nucleotides are phosphate esters of glycosides of heterocyclic bases and are the structural units of both oligonucleotides and nucleic acids. Each phosphate functionality found in a nucleotide, oligonucleotide, or nucleic acid potentially is capable of imparting a negative charge to the molecule.
• a hydroxyl group of a sugar residue, an amino group from a base residue, or a phosphate oxygen of the nucleotide will be utilized as the needed chemical functionality to couple the nucleotide-based chemical modifier to the pharmaceutical agent.
• chemical modifiers with other chemical functionalities can be prepared by conventional techniques.
• the hydroxyl group of the sugar residue can be converted to a mercapto or amino group using techniques well known in the art.
• the chemical modifier exhibits therapeutic effects apart from its carrier function and comprises an antisense compound or triple helix drug.
• the nucleotide-based chemical modifier will be covalently coupled to the pharmaceutical agent via a spacer group.
• An illustrative embodiment calls for the use of the spacer group 3-maleimidobenzoic acid, as shown below. See, generally, Tung et al . (1991) Bioconj. Chem. 2 : 464.
• the 5'-terminal phosphate group of the oligonucleotide is modified to produce the corresponding amin ⁇ alkyl phosphate ester.
• the spacer group, 3-maleimidobenzoic acid is then introduced via reaction of the amino group of the phosphate ester with 3-maleimidobenzoic acid N-hydroxysuccinimide ester.
• Pharmaceutical agents containing a mercapto group or a hydroxyl group can be covalently coupled to the spacer group through this functionality.
• This coupling motif is particularly preferred for peptide and protein drugs containing cysteine residues which can be coupled to the spacer group through the mercapto functionality of the cysteine residue. See, e.g., Tung et al (1991) Bioconj. Chem. 2:464.
• Aminoalkyl phosphate derivatives of nucleotide-based chemical modifiers also can be covalently coupled to pharmaceutical agents containing carboxy groups or hydroxyl groups.
• the chemical functionality of the pharmaceutical agent is activated prior to coupling with the chemical modifier (e.g., an activated ester is produced from the carboxy group or a chloroformate is formed from the hydroxyl group) as shown below.
• the chemical modifier e.g., an activated ester is produced from the carboxy group or a chloroformate is formed from the hydroxyl group
• the 5'-terminal phosphate group of the oligonucleotide is modified to produce the corresponding mercaptoalkyl phosphate ester, as shown below.
• the mercapto group can be coupled directly to another mercapto group of a pharmaceutical agent (e.g., a mercapto group from a cysteine residue in a protein/peptide drug) via a disulfide linkage or the mercapto group can be coupled to a spacer group, such as 3-maleimidobenzoic acid, which has been previously been linked to a pharmaceutical agent (e.g., via a hydroxy, mercapto, or amino group of the pharmaceutical agent). See, generally, Eritja et al. (1991) Tetrahedron 47:4113.
• the chemical functionality of the pharmaceutical agent is activated prior to coupling with the chemical modifier (e.g., an activated ester is produced from the carboxy group or a chloroformate is formed from the hydroxyl group) as shown below.
• the chemical modifier e.g., an activated ester is produced from the carboxy group or a chloroformate is formed from the hydroxyl group
• One protecting group of the precursor is removed to yield a reactive group that can be exploited for synthesis of the peptide/protein drug.
• the second protecting group is then removed to allow for oligonucleotide synthesis.
• Cleavage from the substrate yields a pharmaceutical agent-nucleotide-based chemical modifier complex wherein the pharmaceutical agent is a peptide/protein drug and the spacer comprises a negatively charged phosphate group.
• a pharmaceutical agent- chemical modifier complex can be produced wherein the pharmaceutical agent is a nucleotide-based drug.
• PCR The most commonly used in vitro DNA amplification method is PCR.
• Alternate amplification methods include, for example, nucleic acid sequence-based amplification (Comptom (1991) Nature 350:91-92) and amplified antisense RNA (Van).
• the optimal chemical modifier component for covalently binding to a given pharmaceutical agent can be identified using a variety of screening techniques, including the screening procedures set forth in copending application Serial Nos. 07/762,522, 07/946,239, 07/778,223, 07/876,288, 07/946,239, and 07/963,321, Fodor et al. (1991) Science 251:767-773, PCT publication No. WO 92/10092, and PCT publication No. 92/05285, 92/12842, 92/12843, 91/19813, 91/17271, 91/19818, and 90/15070, each of which is expressly incorporated herein by reference.
• a library of covalently linked pharmaceutical agent-chemical modifier complexes can beproduced, generally a library comprising chemical modifiers will first be synthesized to screen for transport activity. Typically, this library will be synthesized in a solid-state format with each modifier bound to a substrate via a cleavable linker. The compound are then cleaved from the substrate and screened in vitro as to their transport characteristics.
• the optimal modifiers are amplified, for example, in the case of nucleotide-based chemical modifiers, by PCR or other means well known to those skilled in the art, to provide sufficient chemical modifier to be accurately sequenced. Once the optimal chemical modifier is identified, the screening procedure can be repeated to further optimize the composition of the pharmaceutical agent.
• the chemical modifier array will be bound to a solid substrate.
• This format allows for the rapid and efficient synthesis and screening of the modifiers.
• solid phase substrates include synthesis resins (see, e.g., Merrifield (1963) J. Am. Chem. Soc. 85: 2149-54; U.S. patent application Serial No.
• the solid substrate can be composed of any of a wide variety of materials, for example, polymers, plastics, resins, polysaccharides, silicon or silica-based materials, carbon, metals, inorganic glasses, and membranes.
• Substrate-bound oligomer collections having a random monomer composition can be produced using the techniques described in copending application Serial No. 07/946,239.
• advanced techniques for synthesizing polymer arrays are utilized such as those described in copending application Serial No. 07/796,243, or light-directed, spatially-addressable techniques disclosed in Pirrung et al . , U.S. Patent No. 5,143,854, such techniques being referred to herein for purposes of brevity as VLSIPSTM (Very Large Scale Immobilized Polymer Synthesis) techniques.
• Some embodiments will exploit a soluble array of chemical modifiers or pharmaceutical agent-chemical modifier complexes. These soluble collections can be prepared directly or, in some embodiments, a solid support is used to synthesize a library or array of chemical modifiers or complexes of diverse length and composition. The members of the library are cleaved from the support prior to use.
• the pharmaceutical agent prior to forming the linkage between the chemical modifier, the pharmaceutical agent and optionally, the spacer group, at least one of the chemical functionalities will be activated.
• chemical functionalities including hydroxy, amino, and carboxy groups
• a hydroxyl group of the chemical modifier or pharmaceutical agent can be activated through treatment with phosgene to form the corresponding chloroformate.
• the hydroxyl functionality is part of a sugar residue, then the hydroxyl group can be activated through reaction with di-(n-butyl) tin oxide to form a tin complex.
• Carboxy groups may be activated by conversion to the corresponding acyl halide. This reaction may be performed under a variety of conditions as illustrated in March, supra pp. 388-89. In a preferred embodiment, the acyl halide is prepared through the reaction of the carboxy containing group with oxalyl chloride. D. Methods of Linking
• the pharmaceutical agent is linked covalently to a chemical modifier using standard chemical techniques through their respective chemical functionalities.
• the chemical modifier or pharmaceutical agent can be coupled to the pharmaceutical agent through one or more spacer groups.
• the spacer groups can be equivalent or different when used in combination.
• the chemical modifiers can be equivalent or different.
• the pharmaceutical agent-modifier complex is prepared by linking a pharmaceutical agent to a chemical modifier (or optionally to a spacer group which has been or will be attached to a chemical modifier) via their respective chemical functionalities.
• a pharmaceutical agent e.g., chemical functionality l
• the pharmaceutical agent is joined to the chemical modifier, optionally via a spacer group, (e.g., chemical functionality 2) via the linkages shown in Table 3.
• spacer group e.g., chemical functionality 2
• the chemical functionalities shown in Table 3 can be present on the pharmaceutical agent, spacer, or chemical modifier, depending on the synthesis scheme employed.
• Ketal type linkages that may be produced in the pharmaceutical agent-chemical modifier complexes of the present invention include, but are not limited to, imidazolidin-4-ones, see Prodrugs, supra ; oxazolin-5-ones, see Greene et al . supra at 358; dioxolan-4-one, see Schwenker et al . (1991) Arch. Pharm. (Weinheim) 324:439; spirothiazolidines, see Bodor et al . (1982) Int. J. Pharm., 10:307 and Greene et al . supra at 219 and 292; and oxazolidines, see March supra at 87 and Greene et al. supra at 217-218 and 266-267.
• nucleic acid construction principles can be exploited using various restriction enzymes which make sequence specific cuts in the nucleic acid to produce blunt ends or cohesive ends, DNA ligases, enzymatic addition of single-stranded ends to blunt-ended DNA, and construction of synthetic DNAs by assembly of short oligonucleotides.
• a preferred linkage for peptide and protein pharmaceutical agents may be formed by the reaction of an aldehyde containing chemical modifier or spacer group with the terminal amino acid of the pharmaceutical agent.
• This cyclic derivative serves to stabilize the pharmaceutical agent towards enzymatic action, yet is easily hydrolyzed to release the peptide drug.
• This linkage is shown below wherein R 1 , R 2 , and R 3 are independently hydrogen, alkyl, arylalkyl, aryl, and heteroaryl. H. Bundgaard (1991) Drugs of the Future. 16(5):443-458.
• Preferred examples of pharmaceutical agent-chemical modifier complexes linked via an ester include: 3 ⁇ -hydroxy-5 ⁇ -pregnan-20-one 3 ⁇ -(O-palmityl)-L-carnitine ester; 3 ⁇ -hydroxy-5 ⁇ -pregnan-20-one 3 ⁇ -(O-acetyl)-L-carnitine ester; 3 ⁇ -hydroxy-5 ⁇ -pregnan-20-one 3 ⁇ -(O-acetyl)-L-carnitine ester; 3 ⁇ -hydroxy-5 ⁇ -pregnan-20-one 3 ⁇ -(O-acetyl)-D,L-carnitine ester; digitoxigenin-3-(O-acetyl-D,L-carnitine) ester, chloride salt; digitoxigenin-3-(O-acetyl-L-carnitine) ester, chloride salt; digitoxigenin-3-(O-palmityl-L-carnitine) ester, chloride salt; 2-(4-nitrophenyl)ethanol O-acety
• a preferred example of a pharmaceutical agent-chemical modifier complex with a carbamate linkage is 17 ⁇ - hydroxy-estra-1,3,5(10)- trien-3-yl 2-(N,N,N-trimethyl-amino)ethoxycarbonylmethyl carbamate, iodide salt.
• Preferred examples of pharmaceutical agent-chemical modifier complexes with a carbonate linkage include: 3,17 ⁇ -estradiol-3-choline carbonate, iodide salt; 3 ,17 ⁇ -estradiol-17 ⁇ -choline carbonate, iodide salt; digitoxigenin-3-(O-cholinechloride carbonate) ester; and digitoxin-4'''-(choline chloride) carbonate ester.
• Preferred examples of pharmaceutical agent-chemical modifier complexes using a spacer group include: 3-hydroxy-estra-l,3,5(10)-trien-17 ⁇ -yl 2-(N,N,N-triethylamino) ethoxycarbonylmethyl carbamate, iodide salt; indomethacin 2-[N-(6'-N',N'-dimethylamino)hexanamido]-ethyl ester; indomethacin 2-[N-methyl-N-(6'-N',N'-dimethylamino)-hexanamido]ethyl ester; indomethacin 2-[N-(6'-N',N',N'-trimethylamino)-hexanamido]ethyl ester, iodide salt; indomethacin 2-[N-methyl-N-(6'-N',N',N'-trimethyl-amino)hexanamido
• model compounds useful in evaluating the methods described herein include 2-(4-nitrophenyl)ethylamine3-(O-acetyl)-L-carnitine amide, chloride salt; 2-(4-nitrophenyl)ethylamine choline carbamate, chloride salt; 2-(4-nitrophenyl)ethanol choline carbonate ester, chloride salt; 4-(4-nitrophenyl)cyclohexanol choline carbonate ester, iodide salt; 2-[4-(4-methoxyphenyl)butyramido]ethyl O-acetyl-L-carnitinate, chloride salt; 2-[4-(4-methoxyphenyl)butyramido]ethyl O-acetyl-L-carnitinthioate, chloride salt; 2-(4-nitrophenyl)ethanol 6-(O-acetyl-L-carnitinamido))-hexanoateest
• Spacer groups optionally may be introduced between the chemical modifier and the pharmaceutical agent.
• Spacer groups contain at least two chemical functionalities and, as opposed to chemical modifiers, do not carry a charge.
• one chemical functionality of the spacer group bonds to a chemical functionality of the chemical modifier, while the other chemical functionality of the spacer group is used to bond to a chemical functionality of the pharmaceutical agent.
• Examples of chemical functionalities of spacer groups include hydroxy, mercapto, carbonyl, carboxy, amino, ketone, and mercapto groups.
• Spacer groups may also be used in combination. When a combination of spacer groups is used, the spacer groups may be different or equivalent.
• Preferred spacer groups include 6-aminohexanol, 6- mercaptohexanol, 10-hydroxydecanoic acid, glycine and other amino acids, 1,6-hexanediol, ⁇ -alanine, 2-aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic acid, 6-aminohexanoic acid, 3-maleimidobenzoic acid, phthalide, ⁇ -substituted phthalides, the carbonyl group, aminal esters, and the like.
• the spacer can serve to introduce additional molecular mass and chemical functionality into the pharmaceutical agent-chemical modifier complex. Generally, the additional mass and functionality will affect the serum half-life and other properties of the pharmaceutical agent-chemical modifier complex. Thus, through careful selection of spacer groups, pharmaceutical agent-chemical modifier complexes with a range of serum half-lives can be produced.
• Table 4 lists the serum half-lives for several model compounds and two representative pharmaceutical agent-chemical modifier complexes using an in vitro assay system described in the Experimental section below.
• the serum half-lives will vary in vivo depending upon the age and health condition of the patient.
• Each complex comprises a model compound or a pharmaceutical agent, a spacer group, and a chemical modifier.
• Many of the model compounds utilize 4-nitrophenyl(ethanol, propanol, or cyclohexanol) or 4-nitrophenylethylamine as the "model" pharmaceutical agent.
• a more complete listing of serum half-lives may be found in the Experimental section below. As can be seen in Table 4, the number of carbons in a spacer group has an effect on the serum half-life of the complex.
• 2-(4-nitrophenyl) ethanol 6- (O-acetyl-L-carnitinamido) hexanoate ester, chloride salt, which utilizes 6-aminohexanoic acid as its spacer group has a serum half-life of 7.9 seconds.
• the spacer group By changing the spacer group to ⁇ -alanine to produce 2- (4-nitrophenyl) ethyl 3-(O-acetyl-L-carnitinamido)-propanoate, one can extend the half-life to 5.5 minutes.
• 1-(4-nitrophenyl)-2-propanol 6-(O-acetyl-L-carnitinamido)-hexanoate ester contains 6-aminohexanoic acid as its spacer group.
• This spacer group is linked to the pharmaceutical agent via an ester linkage.
• the half-life for 1-(4-nitrophenyl)-2-propanol 6-(O-acetyl-L- carnitinamido) hexanoate ester is 21.4 minutes. If the spacer group is changed to 6-aminohexanol and the spacer group is linked to the pharmaceutical agent via a carbonate linkage, then the serum half-life becomes 3.5 minutes.
• a functionality modifier is a chemical entity which possesses at least one chemical functionality which can be covalently bound to the pharmaceutical agent-chemical modifier complex (optionally via a spacer group), which does not necessarily carry a charge, and which serves to affect or modify a chemical, physical, or biological property of the complex, including providing a means for detection, for increasing the excretion half-life of the pharmaceutical agent, for targeting the pharmaceutical agent, for increasing avidity, for decreasing aggregation, for decreasing the inflammation and/or irritation accompanying the delivery of the pharmaceutical agent across membranes, and for facilitating receptor crosslinking.
• chemical modifiers constitute a species within the broader class of functionality modifiers.
• the functionality modifier can be bound to either the pharmaceutical agent, a chemical modifier, or a spacer group.
• a chemical modifier and a functionality modifier are used, because the chemical modifier is cleaved from the pharmaceutical agent-chemical modifier complex after membrane transport, the functionality modifier will be covalently coupled to the pharmaceutical agent or the spacer group.
• An example of a functionality modifier which serves to provide a means for detection is a radiolabeling site, including radiolabeled chelates for cancer imaging or radiotherapy and for assessing dose regiments in different tissues, such as kemptide.
• modifiers are capable of extending the excretion half-life of a pharmaceutical agent.
• these modifiers will find use with peptide and protein drugs or other pharmaceutical agents with short excretion half-lives.
• this modifier will comprise a moiety capable of binding to a serum protein, such as human serum albumin.
• those moieties will be bound to plasma more than 60%, preferably more than 70%, more preferably more than 80%, and most preferably more than 90%, as measured by the procedures known in the art.
• effector groups include naproxen, fluoxetine, oxazepam, nitrazepam, phenylbutazone, nortriptyline, methadone hydrochloride, lorazepam, imipramine, haloperidol, flurazepam, doxycycline, ditonin, diflunisal, diazoxide, diazepam, nordazepam, desipramine, dapsone, clofibrate, amantadine, chlorthalidone, clonazepam, chlorpropamide, chlorpromazine, chlorpheiramine, chloroquine, carbamazepine, auranofin, amitriptyline, amphotericin B, piroxicam, warfarin, pimozide, doxorubicin, pyrimethamine, amidoarone, protriptylene, desipramine, nortriptyline, oxazepam, nitraze
• the excretion half-life functionality modifier will comprise a circulating carrier protein, for example, an antibody, or antigen-binding fragment, or genetically-engineered binding protein derived therefrom. See, e .g. , Huston et al . (1988) Biochem. 85:5879-5883.
• the antibody, fragment, or derivative thereof should have a relatively long half-life in the circulation, generally on the order of about 1-24 hours, and preferably about 12-24 hours. Because the half-lives of antibody fragments are generally shorter than that of intact antibodies, it may be necessary to increase the half-life of the fragments by attaching polyethylene glycol or polyamino acid chains to the fragment.
• the functionality modifier will serve as a "targeting" modifier and serve to increase tissue or organ specificity, increase the local concentration of drug in the target tissue, or intracellular or transcellular targeting of the active drug.
• this type of “targeting” modifier will comprise a peptide that binds to a cell surface receptor and preferably, peptides that bind to an internalized receptor, such as transferrin.
• the simultaneous use of two different targeting modifiers could result in greater specificity as well as high avidity.
• the targeting modifier can also serve to "target” or direct the pharmaceutical agent-chemical modifier complex inside a cell, i.e., to achieve internalization of the complex.
• endocytotic vesicles generally fuse with acidic vesicles wherein ligand-receptor dissociation occurs at low pH.
• acid vesicles contain diverse hydrolytic enzymes including esterases and proteases. These factors can be exploited for the dissociation or cleavage of the pharmaceutical agent-chemical modifier complex.
• fusion of endocytotic vesicles with intracellular acid vesicles does not always occur, especially in cells such as epithelial cells (and some endothelial cells) that engage in transcellularly directed molecular trafficking.
• the directed movement of peptides across gut epithelial cells involves several pathways, including the directed movement of vesicles from the gut lumen to the basal cell surface (transcytosis). At this surface, fusion of the vesicles with the basal cell plasma membrane releases the vesicle contents into the extracellular space allowing their subsequent transfer into the gut microcirculation or lymph.
• the transcytotic movement of endocytotic vesicles across the endothelial cells constituting the blood-brain barrier is a similar process, yielding a net flux of endocytosed molecules from the blood to the brain compartment.
• direction of a pharmaceutical agent to an intracellular vesicle compartment via receptor-mediated endocytosis can represent a route for drug effects on the epithelial/endothelial cell, as well as a route for drug transport across otherwise drug impermeable cell monolayers.
• a cell internalization modifier typically will comprise a receptor specific targeting modifier, for example, folic acid or folate, which is implicated in receptor mediated endocytosis (clathrin pits).
• the modifier could comprise a membrane active molecule, such as the peptide melittin, for the transport of small, hydrophobic drugs across the cell membrane.
• Membrane active peptides such as derivatives of domain II of Pseudomonas exotoxin, can be used to transport the pharmaceutical agent-chemical modifier complex from vesicles into the cytoplasm.
• Functionality modifiers can also serve to increase the avidity of receptor binding.
• a dimerization peptide such as the peptide linker SKVILF will be used. Formation of the high-avidity dimer will occur preferentially on tissues with high receptor concentration and thus, will also provide additional specificity for tissues with high receptor densities.
• a receptor crosslinking functionality modifier is essentially a targeting modifier.
• Crosslinking of cell surface receptors is a useful ability for a pharmaceutical agent in that crosslinking is often a required step before receptor internalization.
• the crosslinking modifier can be used as a means to incorporate a pharmaceutical agent into a cell.
• the presence of two receptor binding sites i.e., targeting modifiers gives the pharmaceutical agent increased avidity.
• each pharmaceutical agent will have a targeting modifier and an avidity modifier (i.e., a dimerization peptide).
• the dimerization of two peptides will effectively form one molecule with two targeting modifiers, thus allowing receptor crosslinking.
• a functionality modifier may serve to prevent aggregation.
• many peptide and protein pharmaceutical agents form dimers or larger aggregates which may limit their permeability or otherwise affect properties related to dosage form or bioavailability.
• the hexameric form of insulin can be inhibited through the use of an appropriate functionality modifier and thus, result in greater diffusability of the monomeric form of insulin.
• Bi- and multi-functional chemical modifiers and multi-functional spacer groups are particularly preferred in that they provide an additional means of altering the physicochemical properties, and hence, the pharmacokinetics and transmembrane delivery of the pharmaceutical agent-chemical modifier complex through derivatization of the extra functionality.
• the derivatization may occur either prior to or after coupling of the chemical modifier or spacer group to the pharmaceutical agent.
• Carnitine is a particularly preferred bifunctional chemical modifier because of the ease with which its derivatization may be accomplished.
• the hydroxyl group of carnitine may be utilized in a reaction with fatty acids of varying chain lengths. The carboxy group may then be exploited to form a bond with a pharmaceutical agent.
• An example of a pharmaceutical agent-chemical modifier complex containing a derivatized carnitine is digitoxigenin-3-(O-palmitoyl-L-carnitine) ester, chloride salt which contains the palmitoyl derivative of carnitine.
• protecting groups may be used to block or protect a chemical functionality.
• the choice of protecting group will depend on many factors including sensitivity of the molecule to reaction conditions and other functionality in the molecule.
• the protecting group may be chosen for its effect on serum half-life of the resulting pharmaceutical agent-chemical modifier complex. Examples of protecting groups and techniques for protection and deprotection can be found in Greene et al . Protective Groups in Organic Svnthesis. 2nd Ed., John Wiley & Sons: New York (1991), which is incorporated herein by reference.
• the pharmaceutical agent-chemical modifier complex may be produced using either chemical or biochemical techniques.
• Biochemical techniques are used primarily in the case of protein drugs and include recombinant expression methods.
• the pharmaceutical agent is linked to a chemical modifier using standard chemical techniques through their respective chemical functionalities.
• the chemical modifier or pharmaceutical agent may be coupled to a spacer group prior to formation of the pharmaceutical agent-chemical modifier complex.
• the complex may be represented as pharmaceutical agent-[(spacer) x -(chemical modifier) y ] z or A-(S ⁇ -M y ) z wherein x is 0-10, y is y-10 and z is 1-10 and A is the pharmaceutical agent, S is the spacer group and M is the chemical modifier.
• the spacer groups may be equivalent or different when used in combination.
• the chemical modifiers may be equivalent or different.
• At least one functionality modifier will also be covalently linked, optionally via a spacer group(s), to the pharmaceutical agent, chemical modifier, and/or spacer group.
• the ratio of pharmaceutical agent to chemical modifier can be about 1:10 (i.e., y is 10). to about 1:5 (i.e., y is 5), and most preferably, about 1:1 (i.e., y is 1).
• the pharmaceutical agent-chemical modifier complex will have a molecular weight of less than about 50,000, more often less than about 25,000 daltons, and most preferably, less than about 15,000 daltons.
• the pharmaceutical agent has more than one chemical functionality, it can be coupled to more than one chemical modifier.
• a bis-adduct will result if the pharmaceutical agent binds to two molecules of a single chemical modifier.
• a pharmaceutical agent may bind with several different chemical modifiers.
• one of the chemical functionalities of the pharmaceutical agent is first selectively protected.
• a chemical modifier is then joined to the remaining chemical functionalities of the pharmaceutical agent.
• the protecting group on the pharmaceutical agent is removed to release the chemical functionality which may then be coupled to a second chemical modifier.
• Preferred examples of bis adducts include piroxicam-N,O-bis (choline chloride) carbonate ester; pregn-5-ene-3,20-dione 3, 20-bis (glycerol-(O-acetyl-L-carnitinate))ketal; digoxin 3',3'',12-tris-(6-trimethylaminohexanoyloxymethyl carbonate)-3''',4'''-cycliccarbonate, tribromide salt; 5-fluorouracil-1,3-bis-(2- trimethylaminoethoxycarbonyloxymethyl), diiodide salt; and 6-mercaptopurine-S,9-bis-[6-(N,N,N-trimethylamino)hexanoyloxymethyl], diiodide salt.
• Many protein drugs including INF- ⁇ 2 or chemical modifiers are manufactured in recombinant expression systems. These recombinant systems allow for the introduction or deletion of charged (positively or negatively) residues from the protein thus, allowing for enhanced transport and delivery of the modified protein through membranes without requiring the attachment of a separate chemical modifier.
• one mode of addition of charged residues is as N- or C- terminal tails. These could be designed to be labile to proteases in the skin or serum, effecting the removal of the charged tail after delivery.
• a further aspect of this embodiment would relate to the use of propeptide sequences as target sites for mutagenesis to enhance the electrotransport of proteins. For example, mature insulin results from two discreet processing steps, from preproinsulin to proinsulin to mature insulin.
• proinsulin The conversion of proinsulin to the mature form is accompanied by the proteolytic removal of the 35 amino acid C-chain.
• the aminp acid residues within this removable C-chain that are nonessential for processing and folding can be modified, e.g., by increasing or decreasing the net charge or by altering the charge localization or distribution, to enhance the delivery and transport of proinsulin and hence, insulin, through membranes.
• the prodrug i.e., proinsulin
• the active drug i.e., insulin
• the removable C-chain the active drug
• This approach will also find use with other proteins and peptides having a pro-form, such as thymosin alpha 1 which results from the processing of its precursor form, prothymosin.
• fusion protein consisting of the pharmaceutical agent covalently bound to various highly charged peptides can be produced. This can be achieved by fusing the cloned gene encoding the pharmaceutical agent to a segment that encodes a charged peptide residue containing several charged (positively or negatively) amino acids. These charged peptide residues would be rich in lysine, arginine and/or histidine. The net charge on the residues, as well as the charge distribution and localization, can be varied.
• the actual genetic constructs engineered for expression may incorporate oligonucleotides encoding the recognition sequence from maltose binding protein joined to the desired fusion protein by a bond that can be cleaved after passage through a column of immobilized maltose. See U.S. application No. 07/876,288, filed April 29, 1992, which is incorporated herein by reference.
• protein drugs or chemical modifiers which contain a plurality of charged residues may be engineered to delete some of these residues.
• the charge distribution and localization of the protein drugs or chemical modifiers can be altered.
• Enzymes also can be employed as a means for modifying protein and peptide drugs to enhance their delivery and transport through membranes.
• the enzyme will comprise an esterase, i.e., an hydrolase that can convert an ester into an acid residue and an alcohol residue.
• the acid may be a carboxylic, a phosphoric, or a sulfuric acid; and the ester may be an alcoholic or a thiol ester.
• a protein or peptide drug i.e., the acid residue
• an esterase such as cholinesterase
• a large excess of the alcohol residue is utilized to drive the equilibrium towards ester formation thus, incorporating the alcohol residue.
• ester formation will result in the introduction of charge to the protein or peptide drug and hence, the enhancement of the drug's transport and delivery through membranes.
• this method will be applicable with any enzyme capable of posttranslational modification of a protein and can result in either the introduction of positive charge or the deletion of negative charge.
• these enzymes include, but are not limited to, those enzymes responsible for the following amino acid modifications: hydroxylation of proline and lysine residues to form the hydroxyproline and hydroxylysine residues in Collagen; phosphorylation of serine to phosphoserine, carboxylation of glutamate to ⁇ -carboxyglutamate; the introduction of amide groups to C-terminal residues, e.g., glycinamide; the methylation, acetylation or phosphorylation of the ⁇ -amino group of lysine; glycosylation; and the attachment of prosthetic groups, e.g., the attachment of carbohydrates toglycoproteins.
• DNA construction principles can be exploited using known construction techniques involving the use of various restriction enzymes which make sequence specific cuts in DNA to produce blunt ends or cohesive ends, DNA ligases, techniques enabling enzymatic addition of sticky ends to blunt-ended DNA, construction of synthetic DNAs by assembly of short oligonucleotides, cDNA synthesis techniques, and synthetic probes for isolating genes having a particular function.
• Various promoter sequences and other regulatory DNA sequences used in achieving expression, and various types of host cells are also known and available.
• Conventional transfection techniques, and equally conventional techniques for cloning and subcloning DNA may also be used and are known to those of skill in the art.
• vectors may be used such as plasmids and viruses including animal viruses and bacteriophages.
• the vectors may exploit various marker genes which impart to successfully transfected cells a detectable phenotypic characteristic that can be used to identify which of a family of cells has successfully incorporated the recombinant DNA of the vector.
• the production of various proteins of interest may be achieved by expressing fused protein which is collected, purified, and then cleaved to remove the extraneous portion of the molecule.
• the invention provides pharmaceutical agent-chemical modifier complexes with a charge-to-mass ratio that allows the complex to be delivered in therapeutically effective amounts.
• the charge-to-mass ratio of such a complex will be equal to or exceed one charge per 5000 daltons.
• the charge-to-mass ratio will be equal to or exceed one charge per 2500 daltons.
• the charge-to-mass ratio will be equal to or exceed one charge per 1000 daltons.
• the pharmaceutical agent-modifier complex can be admixed with an acceptable physiological carrier, such as water, aqueous alcohols, propylene glycol, dimethylsulfoxide, to make a composition suitable for contact with the various membranes and transport and delivery through these membranes.
• an acceptable physiological carrier such as water, aqueous alcohols, propylene glycol, dimethylsulfoxide.
• Well known techniques for choosing appropriate carriers and formulating the proper mixtures are exemplified in Banga et al . supra ; Lattin et al . (1991) Ann. N.Y. Acad. Sci. 618:450; and Remington's Pharmaceutical Science, 15th Ed. , Mack Publishing Company, Easton, PA (1980) and Goodman and Gillman supra , which are incorporated herein by reference.
• composition may contain other materials such as dyes, pigments, inert fillers, or other permeation enhancers, excipients, and conventional components of pharmaceutical products or transdermal therapeutic systems as known in the art.
• the activity of the pharmaceutical agent-modifier complex may be ascertained through studies of the hydrolytic or enzymatic conversion of the complex to the unbound pharmaceutical agent. Generally, good correlation between in vitro and in vivo activity is found using this method. See, e.g., Phipps et al . (1989) J. Pharm. Sciences 78:365. The rates of conversion may be readily determined, for example by spectrophotometric methods or by gas-liquid or high pressure liquid chromatography. Half-lives and other kinetic parameters may then be calculated using standard techniques. See, e.g., Lowry et al . Mechanism and Theory in Organic Chemistry. 2nd Ed., Harper & Row, Publishers, New York (1981).
• the in vitro skin permeation rate of a pharmaceutical agent-chemical modifier complex can be measured using flow-through diffusion cells. Typically these cells will have an active area of 1 cm 2 and a receiving volume of 3 ml.
• the receptor fluid generally isotonic saline, is pumped into and through the cells, by a peristaltic pump. Samples are collected in glass vials arranged in an automatic fraction collector. Human, mouse, or porcine skin is placed on the lower half of the diffusion cell with the stratum corneum facing the donor compartment. The transdermal device is placed on the stratum corneum and the amount of drug permeating across the skin ( ⁇ g/cm 2 ⁇ hr) is calculated from the cumulative release.
• a solution of the complex can be placed into the donor compartment and the amount of transported drug can be calculated.
• the electrotransport behavior of the charged complex in comparison with the pharmaceutical agent may also be assessed using the above analytical techniques and gel or capillary electrophoresis.
• the complex merits further study for improved iontophoretic transdermal deliverability. Preparation measurements may also be performed on excised skin in conventional diffusion cell tests. See Lattin et al . supra .
• compositions described herein will find use in the treatment of a variety of diseases, including but not limited to those described below.
• pharmaceutical agents can be designed to prevent expression of diverse potential target genes, including oncogenes, fungal genes, and any other gene known to be activated specifically in the skin.
• compositions of pharmaceutical agent- chemical modifier complexes directly to block mediators of inflammation, including cytokines, growth factors, cell adhesion molecules or their ligands and receptors thereof, as well as key enzymes in pathways leading to inflammation.
• these blocking actions include preventing the expression of cytokines (such as IL-1), growth factors (such as TGF- ⁇ and EGF), or cell adhesion molecules (such as ELAM and ICAM); or the receptors for cytokines (such as IL-1), growth factors, or cell adhesion molecules.
• cytokines such as IL-1
• growth factors such as TGF- ⁇ and EGF
• cell adhesion molecules such as ELAM and ICAM
• cytokines such as IL-1
• Key enzymes whose expression may be blocked include protein kinase C and phospholipase A or C.
• the complexes described herein can be used to treat conditions in which improper immune or inflammatory responses have been implicated such as psoriasis (e.g., by blocking the expression of IL-1, TGF ⁇ , amphiregulin, or IL-6); atopic dermatitis and eczema (e.g., by blocking the overexpression of IgE); rheumatoid arthritis; allergic rhinitis (e.g., by blocking the expression of IL-4); and the like.
• psoriasis e.g., by blocking the expression of IL-1, TGF ⁇ , amphiregulin, or IL-6
• atopic dermatitis and eczema e.g., by blocking the overexpression of IgE
• rheumatoid arthritis e.g., by blocking the expression of IL-4
• allergic rhinitis e.g., by blocking the expression of IL-4
• the complexes described herein can be used to treat certain cancers of the skin and mucous membranes, such as melanoma, mycosis fungoides, and squamous cell carcinoma (including of the cervix), for example, by blocking the expression of certain factors which promote cell growth and/or adhesion and which are believed to be involved in metastasis.
• the methods described herein can find use in the amelioration of side effects associated with a given pharmaceutical agent. Specifically, introduction of a charged chemical modifier to the 12-position of digoxin has been found to substantially decrease the cytotoxicity of digoxin.
• Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient.
• the skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.
• Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time.
• Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body.
• Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material.
• Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.
• transdermal patch can be prepared from a backing material and an acrylate adhesive.
• the pharmaceutical agent-chemical modifier complex and any enhancer are formulated into the adhesive casting solution and allowed to mix thoroughly.
• the solution is cast directly onto the backing material and the casting solvent is evaporated in an oven, leaving an adhesive film.
• the release liner can be attached to complete the system.
• a polyurethane matrix patch can be employed to deliver the pharmaceutical agent-chemical modifier complex.
• the layers of this patch comprise a backing, a polyurethane drug/enhancer matrix, a membrane, an adhesive, and a release liner.
• the polyurethane matrix is prepared using a room temperature curing polyurethane prepolymer. Addition of water, alcohol, and complex to the prepolymer results in the formation of a tacky firm elastomer that can be directly cast only the backing material.
• a further embodiment of this invention will utilize a hydrogel matrix patch.
• the hydrogel matrix will comprise alcohol, water, drug, and several hydrophilic polymers.
• This hydrogel matrix can be incorporated into a transdermal patch between the backing and the adhesive layer.
• the liquid reservoir patch will also find use in the methods described herein.
• This patch comprises an impermeable or semi-permeable, heat sealable backing material, a heat sealable membrane, an acrylate based pressure sensitive skin adhesive, and a siliconized release liner. The backing is heat sealed to the membrane to form a reservoir which can then be filled with a solution of the complex, enhancers, gelling agent, and other excipients.
• Foam matrix patches are similar in design and components to the liquid reservoir system, except that the gelled pharmaceutical agent-chemical modifier solution is constrained in a thin foam layer, typically a polyurethane. This foam layer is situated between the backing and the membrane which have been heat sealed at the periphery of the patch.
• the rate of release is typically controlled by a membrane placed between the reservoir and the skin, by diffusion from a monolithic device, or by the skin itself serving as a rate-controlling barrier in the delivery system. See U.S. Patents 4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062; and the like.
• the rate of drug delivery will be dependent, in part, upon the nature of the membrane. For example, the rate of drug delivery across membranes within the body is generally higher than across dermal barriers.
• the rate at which the complex is delivered from the device to the membrane is most advantageously controlled by the use of rate-limiting membranes which are placed between the reservoir and the skin. Assuming that the skin is sufficiently permeable to the complex (i.e., absorption through the skin is greater than the rate of passage through the membrane), the membrane will serve to control the dosage rate experienced by the patient.
• Suitable permeable membrane materials may be selected based on the desired degree of permeability, the nature of the complex, and the mechanical considerations related to constructing the device.
• Exemplary permeable membrane materials include a wide variety of natural and synthetic polymers, such as polydimethylsiloxanes (silicone rubbers), ethylenevinylacetate copolymer (EVA), polyurethanes, polyurethane-polyether copolymers, polyethylenes, polyamides, polyvinylchlorides (PVC), polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulose triacetate and cellulose nitrate/acetate, and hydrogels, e.g., 2-hydroxyethylmethacrylate (HEMA).
• siloxanes silicone rubbers
• EVA ethylenevinylacetate copolymer
• PVC polyurethanes
• polyurethane-polyether copolymers
• compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like.
• preservatives or bacteriostatic agents e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like.
• bacteriostatic agents e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like.
• active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.
• the therapeutic composition will be delivered by a standard iontophoretic device.
• iontophoresis is an introduction, by means of electric current, of ions of soluble salts into the tissues of the body. More specifically, iontophoresis is a process and technique which involves the transfer of ionic (charged) species into a tissue (for example through the skin of a patient) by the passage of a electric current through an electrolyte solution containing ionic molecules to be delivered (or precursors for those ions), upon application of an appropriate electrode polarity. That is, ions are transferred into the tissue, from an electrolyte reservoir, by application of electromotive force to the electrolyte reservoir. In iontophoretic systems, the rate of release is primarily controlled by the voltage or current.
• the active electrode includes the therapeutic species as a charged ion, or a precursor for the charged ion, and the transport occurs through application of the electromotive force to the charged therapeutic species.
• the therapeutic species will be delivered in an uncharged form, transfer being motivated, however, by electromotive force.
• the applied current may induce movement of a non-therapeutic species, which carries with it water into the subject. The water may have dissolved therein the therapeutic species.
• electrotransport of the non-therapeutic charged species induces movement of the therapeutic but non-charged species.
• either positively charged pharmaceutical agent-chemical modifier complexes or negatively charged complexes can be readily transported through the skin and into the patient. This is done by setting up an appropriate potential between two electrode systems (anode and cathode) in electrical contact with the skin. If a positively charged drug is to be delivered through the skin, then an appropriate electromotive force can be generated by orienting the positively charged drug species at a reservoir associated with the anode. Similarly, if the ion to be transferred across the skin is negatively charged, then an appropriate electromotive force can be generated by positioning the drug in a reservoir at the cathode.
• a single system can be utilized to transfer both positively charged and negatively charged drugs into a patient at a given time; and, more than one cathodic drug and/or more than one anodic drug may be delivered from a single system during a selected operation.
• iontophoresis see, e.g., Tyle (1989) J. Pharm. Sci. 75:318; Burnette, Iontophoresis (Chapter 11) in Transdermal Drug Delivery Hadgraft and Guy (eds.) Marcel Dekker, Inc.: New York, NY; Phipps et al . (1988) Solid State Ionics 28-30:1778-1783; Phipps et al . (1989) J. Pharm.
• One electrode is the electrode from which the pharmaceutical agent-chemical modifier complex is delivered or driven into the body by application of the electromotive force.
• the other electrode typically referred to as an “indifferent” or “ground” electrode, serves to close the electrical circuit through the body.
• both electrodes may be “active", i.e. drugs may be delivered from both.
• electrode, or variants thereof, when used in this context refers to an electrically conductive member, through which a current passes during operation.
• Electrodes ranging from platinum to silver-silver chloride, are available for these devices. The primary difference in these materials is not in their ability to generate an electric potential across the skin, but rather in certain nuances associated with their performance of this function.
• platinum electrodes hydrolyze water, thus liberating hydrogen ions and subsequently, changes in pH. Obviously, changes in pH can influence the ionization state of therapeutic agents and their resulting rate of iontophoretic transport.
• Silver-silver chloride electrodes do not hydrolyze water. However, these electrodes require the presence of chloride ion which may compete for current-induced transport.
• Electrotransport devices generally require a reservoir as a source of the species (or a precursor of such species) which is to be moved or introduced into the body.
• the reservoir typically will comprise a pool of electrolyte solution, for example an aqueous electrolyte solution or a hydrophilic, electrolyte-containing, gel or gel matrix, semi-solid, foam, or absorbent material.
• Such pharmaceutical agent-chemical modifier complex reservoirs when electrically connected to the anode or the cathode of an iontophoresis device, provide a source of one or more ionic species for electrotransport.
• Many iontophoresis devices employ a selectively permeable membrane.
• composition of this membrane will vary with the particular needs of the system and will depend upon the composition of the electrolyte reservoir, i.e., the nature of the pharmaceutical agent or pharmaceutical agent-chemical modifier complex, the transference of current out of the reservoir, and the desired selectivity to transport of particular types of charged and uncharged species.
• a microporous polymer or hydrogel such as is known in the art can be utilized. See, e.g., U.S. Patent No. 4,,927,408.
• Suitable permeable membrane materials can be selected based on the desired degree of permeability, the nature of the complex, and the mechanical considerations related to constructing the device.
• Exemplary permeable membrane materials include a wide variety of natural and synthetic polymers, such as polydimethylsiloxanes (silicone rubbers), ethylenevinylacetate copolymer (EVA), polyurethanes, polyurethane-polyether copolymers, polyethylenes, polyamides, polyvinylchlorides (PVC), polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulose triacetate and cellulose nitrate/acetate, and hydrogels, e.g., 2-hydroxyethylmethacrylate (HEMA)'.
• polydimethylsiloxanes silicone rubbers
• EVA ethylenevinylacetate copolymer
• PVC polyurethanes
• polyurethane-polyether copolymers polyethylenes
• polyamides polyamides
• PVC polyvinylchlorides
• PTFE polypropylenes
• buffers will also be incorporated into the reservoir to maintain the reservoir environment at the same charge as the electrode.
• a buffer having the opposite charge to the drug will be employed.
• the drug may act as its own buffer.
• the circuit is completed by connection of the two electrodes to a source of electrical energy as a direct current; for example, a battery or a source of appropriately modified alternating current.
• a source of electrical energy for example, a battery or a source of appropriately modified alternating current.
• Chemical enhancers and electroporation can also be utilized to alter the iontophoretic transport rate.
• the coapplication of oleic acid to the skin causes a large decrease in the skin impedance or resistance which is inversely related to permeability or transport.
• Solid State Ionics 53-56: 165-169 instead of the current passing primarily through the shunt pathways (e.g., the follicles and sweat ducts), the ions constituting the current can more uniformly permeate the lipid milieu of the stratum corneum at a lower current density.
• the epidermis, as well as the peripheral neurons surrounding the hair follicles and sweat ducts will be able to experience the electrical stimulation.
• the backing or enclosure of the drug delivery system is intended primarily as a mechanical support for the reservoir or matrix.
• the matrix is exposed directly to the skin or membrane of the host, and the backing is a strip or patch capable of being secured to the skin, typically with the matrix acting as an adhesive.
• the backing will usually be impermeable to the complex. This impermeability inhibits the loss of the complex.
• Suitable backing materials will generally be thin, flexible films or fabrics such as woven and non-woven fabrics and polymeric films, such as polyethylene, polypropylene, and silicone rubber; metal films and foils; and the like.
• the delivery device can be held in place with the adhesive of the matrix, with an adhesive along the perimeter of the matrix, with tape or elastic, or any other means, so long as the device allows the pharmaceutical agent-chemical modifier complex to be transported through the skin.
• the device can be placed on any portion of the skin or dermal surface, such as the arm, abdomen, thigh, and the like.
• the device can be in various shapes and can consist of one or more complexes and/or transport areas. Other items can be contained in the device, such as other conventional components of therapeutic products, depending upon the desired device characteristics.
• the procedure for use can vary.
• the manufacturer's instructions should be followed for appropriate pharmaceutical agent delivery.
• Body fluid or blood levels of the uncomplexed pharmaceutical agent will be determined to measure the effectiveness of the transport and bioconversion.
• the direct current is applied through moist pad-type electrodes with size corresponding to that of the skin region to be treated.
• the interposition of a moist pad between the electrode plate and the skin is necessary for making a perfect contact, preventing any skin burns, overcoming skin resistance, and protecting the skin from absorbing any caustic metal compounds formed on the metal plate surface.
• the drug is administered through an electrode having the same charge as the drug, and a return electrode opposite in charge to the drug is placed at a neutral site on the body surface.
• the operator selects a current intensity below the pain threshold level of the patient and allows the current to flow for an appropriate length of time. Ions transferred through the skin are taken up by the micro-circulation at the dermal-epidermal junction, while the current proceeds through the skin tissues to the return electrode.
• the current intensity should be increased slowly, maintained for the length of time of the treatment, and then decreased slowly at the end of the treatment.
• the current must be within comfortable toleration of the patient, with a current density which is generally less than 0.5 mA/cm 2 of the electrode surface.
• One aspect of this invention provides for the topical delivery of therapeutic compositions of pharmaceutical agent-chemical modifier complexes of pharmaceutical agents.
• This treatment regimen is suitable either for the systemic administration of the pharmaceutical agent or for localized therapy, i.e., directly to pathological or diseased tissue.
• the topical formulations will comprise a preparation for delivering the pharmaceutical agent-chemical modifier complex directly to the affected skin comprising the complex, typically in concentrations in the range' of from about 0.001% to 10%; preferably, from about 0.01 to about 10%; more preferably, from about 0.1 to about 5%; and most preferably, from about 1 to about 5%, together with a nontoxic, pharmaceutically acceptable topical carrier.
• a preparation for delivering the pharmaceutical agent-chemical modifier complex directly to the affected skin comprising the complex, typically in concentrations in the range' of from about 0.001% to 10%; preferably, from about 0.01 to about 10%; more preferably, from about 0.1 to about 5%; and most preferably, from about 1 to about 5%, together with a nontoxic, pharmaceutically acceptable topical carrier.
• Topical preparations can be prepared by combining the pharmaceutical agent-chemical modifier complex with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, cream and aerosol formulations.
• Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
• bases may include water and/or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil.
• Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and the like.
• Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like.
• Powders may be formed with the aid of any suitable powder base, e.g., talc, lactose, starch, and the like.
• Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, solubilizing agents, and the like.
• Dosage forms for the topical administration of a complex of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the active compound may be mixed under sterile conditions with a pharmaceutically- acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
• the ointments, pastes, creams and gels also may contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• Powders and sprays also can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
• Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
• the methods of the present invention are also applicable to the enhanced transport and delivery of pharmaceutical agents through mucosal membranes, such as gastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal, corneal, and ocular membranes.
• mucosal membranes such as gastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal, corneal, and ocular membranes.
• mucosal membranes such as gastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal, corneal, and ocular membranes.
• mucosal membranes such as gastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal, corneal, and ocular membranes.
• Transmucosal (i.e., sublingual, buccal and vaginal) drug delivery provides for an efficient entry of active substances to systemic circulation and reduce immediate metabolism by the liver and intestinal wall flora.
• Transmucosal drug dosage forms e.g., tablet, suppository, ointment, gel, pessary, membrane, and powder
• buccal Administration For delivery to the buccal or sublingual membranes, typically an oral formulation, such as a lozenge, tablet, or capsule will be used.
• the method of manufacture of these formulations are known in the art, including but not limited to, the addition of the pharmaceutical agent-chemical modifier complex to a pre-manufactured tablet; cold compression of an inert filler, a binder, and either a pharmaceutical agent-chemical modifier complex or a substance containing the complex (as described in U.S. Patent No. 4,806,356); and encapsulation.
• Another oral formulation is one that can be applied with an adhesive, such as the cellulose derivative, hydroxypropyl cellulose, to the oral mucosa, for example as described in U.S. Pat. No. 4,940,587.
• This buccal adhesive formulation when applied to the buccal mucosa, allows for controlled release of the pharmaceutical agent-chemical modifier complex into the mouth and through the buccal mucosa.
• aerosol For delivery to the nasal and/or pulmonary membranes, typically an aerosol formulation will be employed.
• aerosol includes any gas-borne suspended phase of the pharmaceutical agent-chemical modifier complex which is capable of being inhaled into the bronchioles or nasal passages.
• aerosol includes a gas-borne suspension of droplets of the compounds of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition of the pharmaceutical agent-chemical modifier complex suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example.
• the preferred range of concentration of the pharmaceutical agent-chemical modifier complex is 0.1-100 milligrams (mg)/ milliliter (ml), more preferably 0.1-30 mg/ml, and most preferably, 1-10 mg/ml.
• the solutions are buffered with a physiologically compatible buffer such as phosphate or bicarbonate.
• the usual pH range is 5 to 9, preferably 6.5 to 7.8, and more preferably 7.0 to 7.6.
• sodium chloride is added to adjust the osmolarity to the physiological range, preferably within 10% of isotonic.
• Solutions of the pharmaceutical agent-chemical modifier complex may be converted into aerosols by any of the known means routinely used for making aerosol inhalant pharmaceuticals.
• such methods comprise pressurizing or providing a means of pressurizing a container of the solution, usually with an inert carrier gas, and passing the pressurized gas through a small orifice, thereby pulling droplets of the solution into the mouth and trachea of the animal to which the drug is to be administered.
• a mouthpiece is fitted to the outlet of the orifice to facilitate delivery into the mouth and trachea.
• the pharmaceutical agent is coupled to the chemical modifier via a covalent bond.
• This covalent bond may be non-reversible, partially reversible, or preferably, reversible.
• the degree of reversibility corresponds to the ability of the pharmaceutical agent-chemical modifier complex to hydrolyze in vivo.
• the bond will be reversible (i.e., easily hydrolyzed) or partially reversible (i.e., partially or slowly hydrolyzed).
• Cleavage of the bond can occur through biological or physiological processes.
• the physiological processes will cleave bonds at other locations within the complex (e.g., removing an ester group or other protecting group that is coupled to an otherwise sensitive chemical functionality) before cleaving the bond between the pharmaceutical agent and chemical modifier, resulting in partially degraded complexes; or multiple cleavages will . occur, for example, between the spacer and agent and then the spacer and modifier.
• circulating enzymes in the plasma can be used to cleave the chemical modifier from the pharmaceutical agent.
• These enzymes can include non-specific aminopeptidases and esterases, dipeptidyl carboxy peptidases, proteases of the blood clotting cascade, and the like.
• cleavage may be brought about by nonenzymatic processes.
• chemical hydrolysis may be initiated by differences in pH experienced by the complex following delivery.
• the pharmaceutical agent-chemical modifier complex may be characterized by a high degree of chemical lability at physiological pH of 7.4, while exhibiting higher stability at an acidic or basic pH in the reservoir of the delivery device.
• Examples of a pharmaceutical agent-chemical modifier complex which may be cleaved in such a process are those with N-Mannich base linkages. Conversion of the complex to the pharmaceutical agent may also involve a combination of both enzymatic and nonenzymatic processes.
• cleavage of the complex will occur during or shortly after transport through the skin or mucosa. However, in certain instances, cleavage is not desired until the complex reaches the pharmaceutical agent's site of action. Furthermore, in some cases, particularly with peptide and protein drugs produced via recombinant expression techniques, one may not desire cleavage of the complex. Of course, alternatively, these peptide and protein drugs can be engineered to have specific protease cleavage sites.
• Norcarnitine was prepared according to the procedure of Keller et al . (1963) J. Medicinal Chem., 6:202.
• the solid was further purified by twice boiling with acetone (100 ml), cooling and filtering, and finally rinsed with acetone/ether and dried in vacuo to give 15.37 g (35.3 mmol, 87% yield, mp 164-174°C, dec. 185°C).
• D,L-4-dimethylamino-3-hydroxybutyric acid (D,L-norcarnitine) was prepared by treatment of a methanol solution of this acid with ethereal diazomethane. The structure was verified by NMR.
• O-Palmityl-L-carnitine chloride (436 mg, 1 mmol) was suspended in dichloromethane (7 ml) containing one drop of dimethylformamide and oxalyl chloride (250 microliters ( ⁇ l) 2.87 mmol) was added with stirring at room temperature. The cloudy suspension became clear after 5-10 minutes and stirring was continued for one hour. The solution was concentrated under reduced pressure and the residue was dried under vacuum. The product was used immediately for further reactions.
• Oxalyl chloride (220 ⁇ l, 2.5 mmol) was added to a solution of indomethacin (430 mg, 1.2 mmol) in dichloromethane (7 ml) containing one drop of dimethylformamide and the reaction mixture was stirred at room temperature for one hour. The solvent was evaporated and the solid residue was used without further purification.
• Digitoxigenin 300 mg, 0.8 mmol in pyridine (2.5 ml) was treated with (choline chloride) chloroformate (720 mg, 3.5 mmol) and a catalytic amount (5 mg) of 4-dimethylaminopyridine.
• the reaction mixture was vigorously stirred and sonicated to form a viscous paste and then stirred at 40 °C for 72 hours.
• the reaction mixture was diluted with dichloromethane (50 ml), filtered and the solid, mostly unreacted choline chloride, was washed with more dichloromethane (50 ml). The filtrate was evaporated todryness and the residue heated with acetone (50 ml).
• a solution of digitoxigenin (300 mg, 0.8 mmol) in dichloromethane (7 ml) was treated with a dichloromethane solution (7 ml) of freshly prepared O-acetyl-carnitine chloride acid chloride (1 mmol) at room temperature.
• the reaction mixture was kept under an argon atmosphere and was evacuated every five minutes by means of an aspirator in order to remove the evolved hydrogen chloride gas. This process was required to prevent dehydration of digitoxigenin to the 14-anhydro compound.
• the reaction mixture was stirred at room temperature for 4.5 hours. while maintaining a total volume of 3-5 ml by replacement of dichloromethane lost to evaporation during the evacuation procedure.
• O-Acetyl-L-carnitine chloride acid chloride prepared from 2.4 g (10 mmol) of O-acetyl-L-carnitine hydrochloride, was dissolved in dry dichloromethane (50 ml) and estradiol (2.72 g, 10 mmol) was added as a solid. The solid appeared to dissolve slowly at first while at the same time being replaced by an oily, milky upper layer in the stirred mixture. Stirring was continued at room temperature for 66 hours during which time the oily layer was replaced by a fine white solid. The solid was removed by filtration and washed with dichloromethane (50 ml).
• Indomethacin acid chloride (430 mg, 1.2 mmol) was dissolved in chloroform (3 ml) and added to a solution of D,L-norcarnitine methyl ester (363 mg, 2.5 mmol) in chloroform (3 ml) at 0°C with stirring. The ice bath was then removed and stirring continued at room temperature for 2 hours. The reaction mixture was diluted with chloroform and extracted with saturated sodium bicarbonate. The chloroform layer was dried over sodium sulfate, filtered and rotary evaporated to give an oil which was purified by column chromatography on alumina (grade III, elution with 2.5% methanol in chloroform).
• the hydroxyl diester (74 mg, 0.14 mmol) was dissolved in dichloromethane (2 ml) containing diisopropylethylamine (35 mg, 0.28 mmol) and stirred at 0°C while a solution of 3-O-acetyl-L-carnitine acid chloride (prepared from 33 mg of 3-O-acetyl-L-carnitine and oxalyl chloride) in dichloromethane (2 ml) was added. The mixture was stirred at room temperature for 12 hours, decanted from salts and rotary evaporated to dryness. The oily residue was dissolved in toluene (2 ml) and added dropwise to ether/hexane (30 ml, 1/1).
• the resulting precipitate was separated by centrifugation, washed with hexane and redissolved in toluene, centrifuged to remove a small amount of insoluble salts, and the supernatant was evaporated to give 60 mg of the desired carnitine derivative.
• estradiol-3-acetate 280 mg, 0.89 mmol
• tetrahydrofuran 10 ml
• phosgene 5.7 ml, 1 M solution
• the solvent was evaporated and the resulting solid dried under vacuum.
• the residue was redissolved in dichloromethane (10 ml) and added dropwise at 0°C to a solution of benzyl alcohol (95 mg, 0.89 mmol) in pyridine (10 ml). After stirring for one hour at room temperature the mixture was poured into 1 N hydrochloric acid and extracted with dichloromethane.
• the final step to give the desired compound was accomplished by methylation with methyl iodide.
• the dimethylaminoethyl ester 110 mg, 0.25 mmol
• tetrahydrofuran 2 ml
• ether 20 ml
• Methyl iodide 50 mg was then added and the solution stirred at room temperature overnight.
• the precipitated product was removed by centrifugation, washed twice with ether and dried in vacuo to give the final desired compound (83 mg, 57% yield).
• the protecting group was removed by dissolving the ester 712 mg, 1.9 mmol) in trifluoroacetic acid(TFA)/dichloromethane (1 ml, 1/2) and stirring at room temperature for 3 hours. Addition of another ml of TFA and an additional 1 hour of stirring was required to complete the reaction after which the solvents were removed to give 1.01 g of oily residue. The residue was redissolved in dichloromethane, washed with saturated sodium bicarbonate, dried over sodium sulfate, filtered and evaporated to give 508 mg (98% yield) of the free amine.
• the protecting group was removed by dissolving the ester (664 mg, 1.73 mmol) in 1/1 trifluoroacetic acid/dichloromethane (3 ml) and stirring at room temperature for one hour. The solvent was evaporated and the residue redissolved in dichloromethane (50 ml), washed with saturated sodium bicarbonate, filtered, dried over sodium sulfate and evaporated to give the free amine.
• the crude amine (500 mg, 1.7 mmol) was dissolved in dichloromethane along with triethylamine (1.6 mmol) and a solution of O-acetyl-L-carnitine acid chloride (from 1.75 mmol of O-acetyl-L-carnitine) in dichloromethane (7 ml) was added dropwise at 0°C. After stirring for 30 minutes the mixture was warmed to room temperature and stirring continued for an additional 30 minutes. The solvent was evaporated and the residue triturated with ether (2 ⁇ 20 ml) and redissolved in acetone (20 ml).
• the NHS active ester (291 mg, 1 mmol) was dissolved in dichloromethane (5 ml) and the mixture was added to a solution of 2-aminoethanol (143 mg, 2 mmol) in the same solvent (15 ml). After stirring for 30 minutes a white precipitate had formed. The mixture was diluted with additional solvent, washed with water and saturated sodium bicarbonate, and the organic layer dried over sodium sulfate, filtered and evaporated to give 210 mg (89% yield), of desired amide alcohol.
• acetonitrile solution was evaporated to give a viscous oil which was further purified by trituration with toluene, redissolving in acetonitrile and precipitation in ether to give 290 mg (35% yield) of the desired thioester.
• 6-Bromohexanoyl chloride (2 g, 9.37 mmol) was added to t-butanol (6.98 g, 93.7 mmol) under an inert atmosphere. The reaction mixture was stirred overnight and then was concentrated in vacuo , dissolved in ethyl acetate (150 ml), washed with saturated aqueous sodium bicarbonate and 10% aqueous citric acid, and dried over magnesium sulfate to yield t-butyl 6-bromohexanoate (2.02 g, 80% yield).
• reaction mixture was diluted with dichloromethane (100 ml), washed with saturated aqueous sodium bicarbonate, 10% aqueous citric acid, and water, dried over magnesium sulfate, and concentrated in vacuo to yield the desired t-butyl 6-hydroxyhexanoate (640 mg) in 52% yield.
• dichloromethane 100 ml
• saturated aqueous sodium bicarbonate 10% aqueous citric acid
• water dried over magnesium sulfate, and concentrated in vacuo to yield the desired t-butyl 6-hydroxyhexanoate (640 mg) in 52% yield.
• the structure was confirmed by NMR.
• the diester prepared above (650 mg, 1.928 mmol) was treated with trifluoroacetic acid (1.5 ml) and dichloromethane (3 ml). The reaction mixture was stirred at room temperature for 3 hours and then concentrated in vacuo to yield the carboxylic acid (about 660 mg) as an oil. The oil was treated with oxalyl chloride (350 ⁇ l, 3.94 mmol) in dichloromethane (5 ml) and DMF (1 drop) to form the acyl chloride. The reaction mixture was concentrated in vacuo and the acyl chloride was used immediately.
• the dichloromethane layer was separated, washed with water, concentrated in vacuo and filtered through a 5 cm alumina column (eluting with dichloromethane:methanol 20:1), and again concentrated in vacuo to yield the desired chloromethyl ester (3.09 g, 95% yield).
• the structure was confirmed by NMR.
• the reaction mixture was filtered and concentrated in vacuo .
• the residue was triturated with acetone:ether (1:3, 40 ml).
• the oily precipitate was dried in vacuo, dissolved in acetonitrile (3 ml), and centrifuged.
• the filtrate was diluted with isopropyl ether and the precipitate was centrifuged out.
• the precipitate was dissolved in acetone (6 ml) and the precipitate was centrifuged out.
• the supernate was concentrated in vacuo and triturated with ether to yield the desired trimethylammonium salt (160 mg) with a molecular mass of 686 by FAB mass spectrometry.
• the reaction mixture was diluted with dichloromethane (70 ml), washed with water (2 ⁇ 20 ml), dried over sodium sulfate, and concentrated in vacuo to yield a solid (2.0 g) which was purified by column chromatography (flash silica, eluting first with dichloromethane: ethyl acetate, 2:1, 500 ml, then dichloromethane: ethyl acetate, 1:1, 450 ml), and finally dichloromethane:methanol, 95:5, 450 ml).
• the desired carbonate (1.06 g) was produced in 66.9% yield.
• the precipitate was heated with acetonitrile (10 ml) and then cooled to room temperature. The precipitate was centrifuged and washed with acetonitrile (5 ml). The supernate was concentrated in vacuo to yield precipitate II which was further purified by precipitation from dichloromethane:methanol (10:1, 3 ml), followed by precipitation from ether.
• Glycine t-butyl ester hydrochloride (2.51 g, 15 mmol) was treated with 10 N aqueous sodium hydroxide (1.6 ml) and extracted with dichloromethane (50 ml). The dichloromethane solution was back washed with saturated aqueous sodium chloride (2 ⁇ 5 ml), dried over sodium sulfate, and concentrated in vacuo to yield the glycine t-butyl ester (1.38 g, 70% yield).
• Trimethylamine was bubbled into a solution of the bromo carbamate prepared above (165 mg, 0.137 mmol) in acetonitrile (10 ml) for four minutes. The reaction flask was then closed and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and then triturated with ether. The precipitate was centrifuged out and redissolved in methanol:dichloromethane (1:8, 1.5 ml) and then precipitated out with ether to yield the desired trimethylammonium salt (103 mg).
• the reaction mixture was concentrated in vacuo at a temperature ⁇ 30°C and diluted with saturated aqueous sodium chloride.
• the white precipitate was extracted with dichloromethane (75 ml).
• the dichloromethane solution was washed saturated aqueous sodium bicarbonate (3 ⁇ 30 ml), saturated aqueous sodium chloride (2 ⁇ 30 ml), saturated aqueous sodium carbonate (2 ⁇ 30 ml), and saturated aqueous sodium chloride (2 ⁇ 30 ml).
• the dichloromethane solution was concentrated in vacuo to yield a yellowish solid (1.60 g).
• the reaction mixture was concentrated in vacuo and dissolved in dichloromethane.
• the dichloromethane solution was washed with saturated aqueous sodium chloride, saturated aqueous sodium bicarbonate, saturated aqueous sodium chloride buffered to pH 4, and saturated aqueous sodium chloride, dried, and concentrated in vacuo to yield the desired carbonate (167 mg, 78% yield) whose structure was confirmed by NMR.
• the supernatant was concentrated on the rotary evaporator to about 2 ml and then diluted with toluene (30 ml) to give a second precipitate.
• the first precipitate was triturated with dichloromethane (3 ⁇ 10 ml) and acetone (3 ⁇ 10 ml) and the residue was partially dissolved in warm acetonitrile (5 ml). After filtration, the solvent was evaporated and the residue further triturated with acetone and ether.
• the remaining solid (30 mg) was shown by NMR to be a bis adduct of choline chloride to the piroxicam which may be the carbonate ester of the phenolic hydroxyl along with an acyl derivative of either the nitrogen or the oxygen of the amide group.
• the bis-ketal diacetate (1.56 g, 2.86 mmol) was hydrolyzed by stirring its methanol solution (50 ml) containing two drops of 10 N sodium hydroxide at room temperature for one hour. The mixture was then diluted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate and filtered. Evaporation of the solvent gave 674 mg of crude product which was further purified by precipitation from an acetone with hexane.
• This material (140 mg, 0.3 mmol) was then esterified by reaction with 3-O-lauryl-D,L-carnitine (230 mg, 0.6 mmol) in dichloromethane solution using oxalyl chloride (0.6 mmol) and triethylamine (0.6 mmol) to give 53 mg of the desired product as a mixture of isomers.
• methotrexate-bis-choline ester, dibromide salt A solution of methotrexate (233 mg, 0.512 mmol) and cerium carbonate (171 mg, 0.523 mmol) in anhydrous DMSO (8 ml) was sonicated and stirred at room temperature for 1.5 hours. To this reaction mixture was then added a solution of 1,2-dibromoethane (188 mg, 1 mmol) in DMSO (2 ml). The reaction mixture was stirred at room temperature for 39 hours and concentrated in vacuo . The residue was triturated with pH 4 acetate buffer and the resulting yellow solid was filtered and dried in an vacuum oven.
• the desired diester can be prepared in higher yield by the following procedure.
• methotrexate 100 mg, 0.22 mmol
• 2-bromoethanol 5 ml
• concentrated hydrochloric acid 25 ⁇ l
• the reaction was stirred in the dark for two days and then concentrated in vacuo .
• the residue was triturated with aqueous sodium bicarbonate and extracted with chloroform 93 ⁇ 25 ml).
• the organic layer was washed with 50 mM pH 7.3 phosphate buffer, dried over sodium sulfate, and concentrated in vacuo to yield crude diester (160 mg).
• reaction mixture was stirred at 0°C for one hour, diluted with dichloromethane (80 ml), washed with saturated aqueous sodium chloride, 10% aqueous citric acid, and saturated aqueous sodium chloride, dried over sodium sulfate, and concentrated in vacuo to yield the desired carbonate (3.42 g, 73.6% yield) which was used without further purification.
• the dichloromethane solution was washed with 5% aqueous sodium thiosulfate and saturated aqueous sodium chloride, dried over sodium sulfate, and concentrated in vacuo to yield the desired diiodo carbonate (2.56 g, 83% yield) which was used without further purification.
• the structure of the carbonate was confirmed by NMR.
• the carbonate prepared above (710 mg, 1.212 mmol) was treated with a solution of trimethylamine in acetonitrile (1.43 M, 16 ml). The reaction mixture was stirred at room temperature for 4 hours and at 40°C overnight and was then concentrated in vacuo . The residue was triturated with acetonitrile and ether. The precipitate was dried in vacuo to yield the desired trimethylammonium salt (860 mg, quantitative yield, mp 142°C, dec. 184-192°C). The structure of the salt was confirmed by NMR and mass spectroscopy.
• the bis ester prepared above (148 mg, 0.224 mmol) was treated with a solution of trimethylamine in acetonitrile (1.15 M, 6 ml) at room temperature for 22 hours.
• the reaction mixture was concentrated in vacuo .
• the residue was dissolved in dichloromethane:methanol (8:1) and precipitated out with ether.
• the precipitate was separated by centrifuge and dried in vacuo to yield the desired bis-trimethylammonium salt (185 mg, quantitative yield, mp foam at 86°C, clear at 150°C).
• the monoester (240 mg, 0.58 mmol) was treated with a solution of trimethylamine in acetonitrile (1.15 M, 7 ml) at room temperature.
• Estradiol dimethylaminoethyl carbonate ester (500 mg, 1.29 mmol) was dissolved in tetrahydrofuran (10 ml) and iodomethane (200 mg) was added with stirring at room temperature. After 5 hours the precipitated product was separated by centrifugation and vacuum dried at 40°C for 2 hours to give 390 mg (68% yield) of desired product whose structure was confirmed by NMR. 10.2 Preparation of 3-O-(indomethacinyl)-D,L-carnitine methyl ester
• oligonucleotides (less than 15-20 residues in length) can be prepared on a commercially-available oligonucleotide synthesizer (e.g., Applied Biosystems Model 394 Oligonucleotide Synthesizer), using radioactive end-labeling with 32 P and T4 polynucleotide kinase.
• oligonucleotide synthesizer e.g., Applied Biosystems Model 394 Oligonucleotide Synthesizer
• any of a variety of other methodologies can be used, including Bal 31 nuclease digestion of DNA followed by radioactive labeling, "nick translation” or “random primer synthesis”, which uses Dnase 1 or random oligonucleotide primers, respectively, to create primer-template junctions for the incorporation of radioactively-labeled deoxynucleosides by DNA polymerases, etc.
• the labeled DNA's should be in sufficient molar excess over their templates, as well as devoid of detectable secondary structures (unless engineered into the template sequence), to ensure that no higher order, macromolecular structures are formed.
• the size distribution of a sample of a mixture of labeled fragments can be assessed by electrophoresis using a standard DNA sequencing gel and autoradiography. See, e . g . , Sambrook et al . Molecular Cloning. Typically, a distribution of uniformly labeled fragments extending from approximately 5-200 nucleotides is created.
• Stock solutions (6 mM) of the pharmaceutical agent-modifier complexes were prepared in ethanol (by vortexing, sonication or warming at 37°C if necessary). If required for solubility, a drop of dimethylsulfoxide, dimethylformamide or methanol may be added. (Acetonitrile cannot be used because it deactivates the serum enzymes.) HPLC separation methods were established for each study by dilution of the above stock solution (6 ⁇ l) in acetonitrile (1 ml) to give a 36 ⁇ M solution.
• Samples (10 ⁇ l) were injected onto a Nucleosil 5C8 (250 ⁇ 4 mmID) and guard column (11 ⁇ 4 mm ID) and eluted with a mobile phase of approximately 65% HPLC grade water and 35% of a solution of 0.1% trifluoroacetic acid in acetonitrile, the exact proportions being adjusted in each case to give optimum separation and run times.
• Synthetic membrane, hairless guinea pig, hairless mouse, or human (either living or cadaverous) skin can be prepared by techniques known in the art.
• the skin or membrane is inserted into a standard electrotransport cell between two chambers.
• the compound to be tested is placed in an appropriate buffer in a "donor” chamber on the exterior side of the skin or membrane, along with a negative electrode.
• a "counter" chamber containing suitable buffer and the positive electrode is placed on the interior side of the skin or membrane.
• a sample is withdrawn from the counter chamber and analyzed.
• the transdermal delivery of a labeled-test compound can be assessed by electrophoresis analysis of the sample using standard DNA sequencing followed by autoradiography. Transport can also be assessed using an antibody-mediated reaction, an activity assay, or by radioactively prelabeling the test compound, either enzymatically or metabolically, and monitoring the radioactivity.
• the in vivo iontophoretic delivery of pharmaceutical agent from the pharmaceutical agent-chemical modifier complexes may be accomplished with a battery-powered control module and two hydrogel-electrode patches.
• the power source is a one-channel constant-current device compliant to within 5% of the set point value and can contain a current of up to 2 mA into a resistive load of 10 kiloohms.
• the hydrogel-electrode patches consist of a conductive polyvinyl acetate (PVA) hydrogel matrix that is in contact with a metallic electrode mesh and is housed in a circular section of polyethylene foam tape.
• the hydrogel contact area with the skin is 25 cm 2 .
• the "active" patch has a silver electrode and a hydrogel composed of 26% pharmaceutical agent-chemical modifier complex, 15% PVA and 59% water by weight.
• the other patch has a silver chloride electrode and a hydrogel composed of 2% sodium chloride, 15% PVA and 83% water by weight.
• the hydrogel formulations are prepared by dissolving PVA in water at 90°C, adding the appropriate substrate, pouring the solution in electrode housings, and. freezing at -20°C for a minimum of 4 hr.
• Iontophoretic delivery of the pharmaceutical agent-chemical modifier complex is performed at currents of up to 1 mA for a duration of 24 hr. After initiating iontophoresis, blood samples are collected every 2 hr by means of an indwelling jugular catheter. Each pig is utilized on consecutive days for two iontophoretic studies at two different currents. New skin sites and new patches are used for each experiment.
• Plasma concentrations of the pharmaceutical agent, chemical modifier, spacer group, and pharmaceutical agent-chemical modifier complex are determined using an HPLC method. The area under the plasma concentration versus time curve is used to estimate the value of the systemic clearance for each pig.

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  • 1993-06-11 AU AU45345/93A patent/AU4534593A/en not_active Abandoned
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