Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a 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
• • 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
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

Definitions

• the present invention relates to active agent delivery systems and, more specifically, to compositions that comprise peptides covalently attached to active agents and methods for administering conjugated active agent compositions.
• Active agent delivery systems are often critical for the safe effective administration of a biologically active agent (active agent). Perhaps the importance of these systems is best realized when patient compliance and consistent dosing are taken under consideration. For instance, reducing the dosing requirement for a drug from four-times-a-day (QID) to a single dose per day would have significant value in terms of ensuring patient compliance. Increasing the stability of the active agent, will assure dosage reproducibility and perhaps may also reduce the number of dosages required. Furthermore, any benefit that modulated absorption can confer on an existing drug would certainly improve the safety of the drug. Finally, improving the absorption of drugs should have a significant impact on the safety and efficacy of the drug.
• QID biologically active agent
• ⁇ -blocker atenolol Another example of the benefit of more consistent dosing is provided by the ⁇ -blocker atenolol.
• the duration of effects for this commonly used drug is usually assumed to be 24 hours. However, at the normal dose range of 25-100 mg given once a day, the effect may wear off hours before the next dose begins acting. For patients being treated for angina, hypertension, or for the prevention of a heart attack, this may be particularly risky.
• One alternative is to give a larger dose than is necessary in order to get the desired level of action when the serum levels are the lowest. But this may cause side effects related to excessive concentrations in the initial hours of the dosing interval.
• statins are seemingly unrelated to the therapeutic effect of the drug.
• the toxic side effects of statins include, amongst other things, liver problems and rhabdomyolysis. Although the exact cause of statin-induced rhabdomyolysis and liver toxicity is not well understood, they have been linked to potent liver enzymes.
• the therapeutic effect of the statins is a result of the down-regulation of one of the key enzymes responsible for cholesterol production. Statin overdosing, however, can cause the reduced synthesis of non-sterol products that are important for cell growth, in addition to rhabdomyolysis.
• statins So with the statins, a case can be made that by modulating the absorption of the drug, the therapeutic effect can be obtained at lower doses thereby minimizing the risk of producing toxic side effects.
• increasing the absorption of an active agent can have a significant impact on its safety. Taking the example of statins, once more, statins are anywhere between 10 and 30% absorbed and dosing is based on the average of this range so for patients that absorb 30% of the statins administered, deleterious side effects can occur. The risk of manifesting these side effects would be greatly diminished if the bioavailability of the drug were more predictable. [013] In an attempt to address the need for improved bioavailability several drug release modulation technologies have been developed.
• Enteric coatings have been used as a protector of pharmaceuticals in the stomach and microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation.
• a wide range of pharmaceutical formulations purportedly provides sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids.
• Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.
• formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent.
• copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone.
• the microencapsulation of therapeutics and diagnostic agents is generally described for example in U.S. 5,238,714 to Wallace et al.
• microencapsulation and enteric coating technologies impart enhanced stability and time-release properties to active agent substances these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix or degradation of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water- soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.
• Active agents have been covalently attached to the amino acid side chains of polypeptides as pendant groups. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent.
• An example of a timed and targeted release pharmaceutical administered intravenously, intraperitoneally, or intra-arterially includes the linking of nitrogen mustard, via a stabilizing peptide spacer, to a macromolecular carrier, e.g. poly-[N 5 - (2-hydroxylethyl)-L-glutamine] (PHEG) which has an improved half-life when attached to the carrier and stabilizing unit.
• PHEG poly-[N 5 - (2-hydroxylethyl)-L-glutamine]
• Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid as a colon-specific drug delivery system, which is released by bacterial hydrolytic enzymes residing in the large intestines.
• the dexamethasone active agent was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream.
• Other examples include techniques for forming peptide-linked biotin, peptide-linked acridine, naphthylacetic acid bonds to LH-RH, and coumarinic acid cyclic bonds to opioid peptides.
• Several implantable drug delivery systems have utilized polypeptide attachment to drugs.
• An example includes the linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of a large polyglutamic acid polymeric carrier designed to biodegrade over long periods of time after implantation via injection or surgical procedures. Additionally, other large polymeric carriers incorporating drugs into their matrices are used as implants for the gradual release of drug. Examples of implant delivery systems are generally described in U.S. 4,356,166 to Peterson et al. and 4,976,962 to Bichon et al. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker.
• HARs highly ordered lipid films
• the polymeric carrier typically large, will not enter cells lacking receptors for the specific peptide attached.
• gastrin receptor directed tetragastrin and pentagastrin amides were attached to cytotoxic drug for testing in vitro. These systems are generally described in U.S. 5,948,750 to Garsky et al.; U.S. 5,087,616 Myers et al.; and Schmidt et al., Peptide Linked l,3-Dialkyl-3-acyltriazenes: Garstrin Receptor Directed Antineoplastic Alkylating Agents, Journal of Medical Chemistry, Nol. 37, No. 22, pp. 3812-17 (1994). [020] Several systems have been directed to the treatment of tumor cells.
• Daunorubicin bound poly-L-aspartic acid delivered intravenously or intraperitoneally, demonstrated lower cytotoxic effect.
• Daunorubicin was covalently attached via a methylketone side-chain of the drug to both poly-L-aspartic acid and poly-L-lysine
• the conjugates were prepared for intravenous and intraperitoneal administration.
• the poly-L-lysine was found to be ineffective, while the poly-L-aspartic acid conjugate showed preferential tumor cell uptake.
• a highly substituted polypetide conjugated to the active agent was designed for introduction into blood vessels that further penetrated the interstitium of tumors through long chain lengths.
• paclitaxel is conjugated to a high molecular weight polyglutamic acid, and was delivered via injection.
• Paclitaxel conjugates have also been used with implants, coatings and injectables. These systems are further described in U.S. Patent 6,306,993; Li et al. Complete Regression of Well-established Tumors Using a Novel Water-soluble Poly(L-Glutamic Acid)-Paclitaxel Conjugate, pp. 2404-2409; and U.S. 5,977,163 to Li et al.
• Delivery systems can be designed to utilize attachment to chemical moieties that are either specifically recognized by a specialized transporters or have enhanced adsorption into target cells through specific polypeptide sequence.
• a specialized transporters There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport.
• Evidence suggests that hydrophilic compounds are absorbed through the intestinal epithelia more efficiently through these specialized transporters than by passive diffusion. Active transport mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors.
• Facilitation of these transporters can also depend on some sort of specialized adjuvant, which can result in localized delivery of an active agent, increased absorption of the active agent or some combination of both.
• Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Increased bioavailability was found when peptides were bound to modified bile acids.
• the present invention also addresses the need for an active agent delivery system that is able to deliver active agents as an active agent peptide conjugate so that the molecular mass and physiochemical properties of the conjugate can be readily manipulated to achieve the desired release rate.
• the present invention accomplishes this by extending the period during which an active agent is absorbed, and providing a longer duration of action per dose than is currently expected. This leads to an overall improvement of dosing parameters such as, for example, taking an active agent twice a day where it has previously required four times a day dosing.
• the need still exists for a drug delivery system that enables the use of new molecular compositions, which can reduce the technical, regulatory, and financial risks associated with active agents while improving the performance of the active agent in terms of its absorption parameters, as described above, and stability. Further, the need exists for an active agent delivery system targeted to general systemic circulation wherein the release of the drug from the peptide can occur by enzymatic action on the peptide-drag conjugate in the bloodstream or by enzymatic action on the peptide-drag conjugate in the alimentary tract followed by absorption through the intestines or stomach.
• the present invention provides covalent attachment of active agents to a peptide.
• the invention may be distinguished from the above mentioned technologies by virtue of covalently attaching the active agent directly, which includes, for example, pharmaceutical drags and nutrients, to the N-terminus, the C- terminus or to the side chain of an amino acid, an oligopeptide or a polypeptide, also referred to herein as a carrier peptide.
• the active agent when the active agent is itself an amino acid active agent, then the active agent may be part of the chain at either the C-terminus or N-terminus through a peptide bond, or interspersed in the polypeptide via peptide bonds on both sides of the active agent.
• the peptide stabilizes the active agent, primarily in the stomach, through conformational protection.
• delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide.
• endigenous enzymes Upon entry into the upper intestinal tract, endigenous enzymes release the active ingredient for absorption by the body by hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces the second phase of the sustained release mechanism.
• the invention provides a composition comprising a peptide and an active agent covalently attached to the peptide.
• the peptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids (L or D isomers), or an isomer, analogue, or derivative thereof, (iii) a heteropolymer of two or more naturally occurring amino acids (L or D isomers), or an isomer, analogue, or derivative thereof, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.
• compositions comprising a carrier peptide and an active agent covalently attached to the carrier peptide.
• the carrier peptide is (i) an amino acid, (ii) a dipeptide, (iii) a tripeptide, (iv) an oligopeptide, or (v) polypeptide.
• the carrier peptide may also be (i) a homopolymer of a naturally occurring amino acids, (ii) a heteropolymer of two or more naturally occurring amino acids, (iii) a homopolymer of a synthetic amino acid, (iv) a heteropolymer of two or more synthetic amino acids, or (v) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.
• a further embodiment of the carrier and/or conjugate is that the unattached portion of the carrier/conjugate is in a free and unprotected state.
• the invention further provides a composition comprising an amino acid, a dipeptide or a tripeptide with an active agent covalently attached.
• the amino acid, dipeptide or tripeptide are (i) one of the twenty naturally occurring amino acids (L or D isomers), or an isomer, analogue, or derivative thereof, (ii) two or more naturally occurring amino acids (L or D isomers), or an isomer, analogue, or derivative thereof, (iii) a synthetic amino acid, (iv) two or more synthetic amino acids or (v) one or more naturally occurring amino acids and one or more synthetic amino acids.
• synthetic amino acids with alkyl side chains are selected from alkyls of -C ⁇ in length and more preferably from C ⁇ -C 6 in length.
• the active agent conjugate is attached to a single amino acid which is either naturally occurring or a synthetic amino acid.
• the active agent conjugate is attached to a dipeptide or tripeptide, which could be any combination of the naturally occurring amino acids and synthetic amino acids.
• the amino acids are selected from L-amino acids for digestion by proteases.
• the peptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.
• the peptide can be prepared through a fermentation process of recombinant microorganisms followed by harvesting and purification of the appropriate peptide. Alternatively, if a specific sequence of amino acids is desired, an automated peptide synthesizer can be used to produce a peptide with specific physicochemical properties for specific performance characteristics.
• the active agent is an inorganic acid or a carboxylic acid and the carboxylate or the acid group is covalently attached to the N- terminus of the peptide.
• the active agent is a sulfonamide or an amine and the amino group is covalently attached to the C- terminus of the peptide.
• the active agent is an alcohol and the alcohol group is covalently attached to the C-terminus of the peptide.
• the active agent is itself an amino acid and is preferably covalently interspersed in the peptide in a peptide-linked manner or covalently attached to a side chain, the N-terminus or the C-terminus of the peptide.
• the amino acid active agents when attached to the C- terminus or the N-terminus this results in the active agent being the end amino acid and is considered C-capped or N-capped, respectively.
• the active agent can be covalently attached to the side chains of the polypeptide using conventional techniques.
• a carboxylic acid containing active agent can be attached to the amine or alcohol group of the peptide side chain to form an amide or ester, respectively.
• an amine containing active agent can be attached to the carboxylate, carbamide or guanine group of the side chain to form an amide or a new guanine group.
• linkers can be selected from the group of all chemical classes of compounds such that virtually any side chain of the peptide can be attached.
• the amino acids used in either homopolymers or heteropolymers are selected from glutamic acid, aspartic acid, serine, lysine, cysteine, threonine, asparagine, arginine, tyrosine, and glutamine.
• Preferred examples of peptides include, Leu-Ser, Leu-Glu, homopolymers of Glu and Leu, and heteropolymers of (Glu)n-Leu-Ser.
• direct attachment of an active agent to the carrier peptide may not form a stable compound therefore the incorporation of a linker between the active agent and the peptide is required.
• the linker should have a functional pendant group, such as a carboxylate, an alcohol, thiol, oxime, hydraxone, hydrazide, or an amine group, to covalently attach to the carrier peptide.
• the active agent is an alcohol and the alcohol group is covalently attached to the N-terminus of the peptide via a linker.
• the active agent is a ketone or an aldehyde, which is attached to a linker through the formation of a ketal or acetal, respectively, and the linker has a pendant group that is attached to the carrier peptide.
• the active agent is an amide, an imide, an imidazole or a urea where the nitrogen is attached to the linker and the pendant group of the linker is attached to the carrier peptide.
• the invention also provides a method for preparing a composition comprising a peptide and an active agent covalently attached to the peptide.
• the method comprises the steps of:
• the active agent is a pharmaceutical agent or an adjuvant.
• steps (a) and (b) are repeated with a second active agent prior to step (c).
• the active agent and second active agent can be copolymerized in step (c).
• Step (b) can include an amino acid (e.g. Glycine, Alanine, etc.), without an active agent attached, such that the product in step (c) is a copolymer of the active agent/amino acid complex and an amino acid interspersed in a peptide-linked manner.
• the amino acid itself can be an active agent (e.g. Thyroxine or DOPA) such that combining the NCA of this bioactive amino acid NCA with other amino acid NCA's will produce a product in (c) of the bioactive amino acid interspersed in the peptide with the generic amino acid in a peptide-linked manner.
• the active agent/amino acid complex can serve as a synthetic module for solid-phase or solution-phase peptide synthesis.
• the drug can be attached to the selected amino acid by the ⁇ -amino group, the ⁇ -carboxylate or side chain functionality.
• the present invention provides for the synthesis whereby an active agent is conjugated to an amino acid, a dipeptide, a tripeptide, an oligopeptide or a polypeptide.
• Another embodiment of the present invention is dosage form reliability and batch-to-batch reproducibility.
• the active agent delivery is targeted into general systemic circulation.
• the release of the active agent from the peptide can occur by enzymatic action on the peptide-active agent conjugate in the bloodstream or by enzymatic action on the peptide-active agent conjugate in the alimentary tract followed by absorption through the intestines or stomach by the regular route of entry.
• the invention also provides a method for delivering an active agent to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a peptide and an active agent covalently attached to the peptide.
• the active agent is released from the composition by enzyme catalysis.
• the active agent is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release.
• the generic amino acid is glutamic acid and the side chain attached active agent/glutamic acid complex is released from the peptide upon hydrolysis of the peptide and then the active agent is released from the glutamic acid by coincident intramolecular transamination.
• the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a thioether, a carbonate, ah anhydride, an orthoester, a hydroxamic acid, a hydrazone, sulfonamide, sulfonic esters, other derivatives of sulfur, or a carbamate.
• the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, an urea, or an acid functionality.
• the composition of the invention can also include one or more microencapsulating agents, adjuvants and pharmaceutically acceptable excipients.
• the active agent can be bound to the microencapsulating agent, the adjuvant or the pharmaceutically acceptable excipient through covalent, ionic, hydrophilic interactions or by some other non-covalent means.
• the microencapsulating agent can be selected from polyethylene glycol (PEG), amino acids, carbohydrates or salts. If it is desired to delay peptide digestion, the microencapsulating agents can be used to delay protein unfolding.
• the adjuvant when an adjuvant is included in the composition, the adjuvant preferably imparts better absorption either through enhancing permeability of the intestinal or stomach membrane or activating an intestinal transporter.
• the intestinal wall is coated with a mucosa lining made primarily of mucin. Many reagents have been identified that can bind to mucin.
• the present invention provides the unique capability of binding a mucin-binding adjuvant to the peptide/drug conjugate to bioadhere the entire complex to the intestinal wall.
• the intestinal wall is impregnated with receptors for various reagents including many of the vitamins such as vitamin K. Binding vitamin K, for example, to the peptide-active agent conjugate will retain the entire complex in the intestines for a much longer time.
• the adjuvant can bioadhere to the mucosal lining of the intestine thereby lengthening the transit time of the drug-peptide conjugate in the gut and maximizing peptide digestion and thus drug bioavailability.
• the composition further comprises a microencapsulating agent and the active agent conjugate is released from the composition by swelling or dissolution of the microencapsulating agent followed by diffusion of the active agent conjugate which must then be acted upon by enzymes to release the active agent.
• the composition further comprises an adjuvant covalently attached to the peptide and release of the adjuvant from the composition is controlled by the enzymatic action on the peptide.
• the adjuvant can be microencapsulated into a carrier peptide-active agent conjugate for biphasic release of active agent.
• the peptide- active agent conjugate can be microencapsulated wherein the peptide-active agent conjugate is released in a biphasic manner, first through physicochemical means, such as through solvation or swelling, and then the active agent is released from the peptide carrier by enzymatic action.
• the active agent can be covalently attached to the microencapsulating agent via a peptide bond where the active agent is released first by peptidase action followed by migration of the active agent out of the microencapsulating medium.
• the active agents may be combined with peptides of varying amino acid content to impart specific physicochemical properties to the conjugate including, molecular weight, size, functional groups, pH sensitivity, solubility, three dimensional structure and digestibility in order to provide desired performance characteristics.
• peptides of varying amino acid content including, molecular weight, size, functional groups, pH sensitivity, solubility, three dimensional structure and digestibility in order to provide desired performance characteristics.
• a variety of active agents may also be used with specific preferred peptides to impart specific performance characteristics.
• Significant advantages with respect to the stability, release and/or adsorption characteristics of the active agent that are imparted through the use of one or more of the 20 naturally occurring amino acids are manifest in the peptide physicochemical properties that impart specific stability, digestibility and release properties to the conjugates formed with active agents.
• the amino acids that make up the carrier peptide is a tool set such that the carrier peptide can conform to the pharmacological demand and the chemical structure of the active agent such that maximum stability and optimal performance of the composition are achieved.
• the amino acid chain length can be varied to suit different delivery criteria.
• the active agent may be attached to a single amino acid to eight amino acids, with the range of two to five amino acids being preferred.
• the preferred length of the oligopeptide is between two and 50 amino acids in length.
• preferred amino acid lengths may be between 8 and 400 amino acids.
• the conjugates of the present invention are also suited for both large and small molecule active agents.
• the carrier peptide controls the solubility of the active agent-peptide conjugate and is not dependant on the solubility of the active agent. Therefore, the mechanism of sustained or zero-order kinetics afforded by the conjugate-drug composition avoids irregularities of release and cumbersome formulations encountered with typical dissolution controlled sustained release methods.
• the active agent conjugates can incorporate adjuvants such that the compositions are designed to interact with specific receptors so that targeted delivery may be achieved. These compositions provide targeted delivery in all regions of the gut and at specific sites along the intestinal wall.
• the active agent is released as the reference active agent from the peptide conjugate prior to entry into a target cell.
• the specific amino acid sequences used are not targeted to specific cell receptors or designed for recognition by a specific genetic sequence.
• the peptide carrier is designed for recognition and/or is not recognized by tumor promoting cells.
• the active agent delivery system does not require that the active agent be released within a specific cell or intracellularly.
• the carrier and/or the conjugate do result is specific recognition in the body. (e.g. by a cancer cell, by primers, for improving chemotactic activity, by sequence for a specific binding cite for serum proteins(e.g. kinins or eicosanoids).
• the active agent may be attached to an adjuvant recognized and taken up by an active transporter.
• the active transporter is not the bile acid active transporter.
• the present invention does not require the attachment of the active agent to an adjuvant recognized and taken up by an active transporter for delivery.
• microsphere/capsules may be used in combination with the compositions of the invention, the compositions are preferably not incorporated with microspheres/capsules and do not require further additives to improve sustained release.
• the active agent is not a hormone, glutamine, methotrexate, daunorubicin, a trypsin-kallikrein inhibitor, insulin, calmodulin, calcitonin, naltrexone, L-dopa, interleukins, gonadoliberin, norethindrone, tolmetin, valacyclovir, taxol, a GABA analog, an L-aromatic amino acid decarboxylase inhibitor, a catechol O-methyl transferase inhibitor, a naturally occurring amino acid, a bis-(2-chloroethyl)amine containing nitrogen mustard, a polypeptide, a peptidomimetic derived from a linear oligopeptide with greater the three amino acids, an oligonucleotide, a cyclophane derivative, a diehtylenetriaminopentaacetate derivative, histamine, a steroid or silver sulfadiazine.
• the active agent is not
• the invention provides a carrier and active agent which are bound to each other but otherwise unmodified in structure.
• This embodiment may further be described as the carrier having a free carboxy and/or amine terminal and/or side chain groups other than the location of attachment for the active agent.
• the carrier whether a single amino acid, dipeptide, tripeptide, oligopeptide or polypeptide is comprises only naturally occurring amino acids.
• the carrier is not a protein transporter (e.g. histone, insulin, transferrin, IGF, albumin or prolactin), Ala, Gly, Phe-Gly, or Phe- Phe.
• the carrier is also preferably not a amino acid copolymerized with a non-amino acid substitute such as PNP, a poly(alkylene oxide) amino acid copolymer, or an alkyloxycarbonyl (polyaspartate/polyglutamate) or an aryloxycarbonylmethyl (polyaspartate/polyglutamate).
• a non-amino acid substitute such as PNP, a poly(alkylene oxide) amino acid copolymer, or an alkyloxycarbonyl (polyaspartate/polyglutamate) or an aryloxycarbonylmethyl (polyaspartate/polyglutamate).
• a non-amino acid substitute such as PNP, a poly(alkylene oxide) amino acid copolymer, or an alkyloxycarbonyl (polyaspartate/polyglutamate) or an aryloxycarbonylmethyl (polyaspartate/polyglutamate).
• the conjugates provide the added benefit of allowing multiple attachments not only of active agents, but of active agents in combination with other active agents, or other modified molecules which can further modify delivery, enhance release, targeted delivery, and/or enhance adsorption.
• the conjugates may also be combined with adjuvants or be microencapsulated.
• the conjugates provide for a wide range of pharmaceutical applications including drug delivery, cell targeting, and enhanced biological responsiveness.
• the invention can stabilize the active agent and prevent digestion in the stomach.
• the pharmacologic effect can be prolonged by delayed or sustained release of the active agent.
• the sustained release can occur by virtue of the active agent being covalently attached to the peptide and or through the additional covalent attachment of an adjuvant that bioadheres to the intestinal mucosa.
• active agents can be combined to produce synergistic effects.
• absorption of the active agent in the intestinal tract can be enhanced either by virtue of being covalently attached to a peptide or through the synergistic effect of an added adjuvant.
• the composition of the invention is in the form of an ingestible tablet or capsule, an intravenous preparation, an intramuscular preparation, a subcutaneous preparation, a depot implant, a transdermal preparation, an oral suspension, a sublingual preparation, an intranasal preparation, inhalers, or anal suppositories.
• the peptide is capable of releasing the active agent from the composition in a pH-dependent manner.
• the active agent is prepared and/or administered through means other than implantation and/or injectables.
• the active agent conjugate is not bound to an immobilized carrier, rather it is designed for transport and transition through the digestive system.
• Embodiments of the present invention preferably are not bound to an adjuvant recognized and/or taken up by active transporters.
• the active agent conjugates of the present invention are not attached to active transporters, or antigenic agents such as receptor recognizing sequences found on cells and tumors.
• the active agent conjugate of the present invention is not connected to or constitutes an implantable polymer, which would not biodegrade in less than 48 hours, preferably between 12 and 24 hours.
• the active agent conjugates of the present invention are preferably designed to release the active agent into the blood, after absorption from the gut, as the reference active agent.
• the active agent conjugate following administration of the active agent conjugate by a method other than oral, first pass metabolism is prevented, by avoiding recognition of liver oxidation enzymes due to its peptidic structure.
• the active agent is directly attached to the amino acid without the use of a linker.
• the invention also provides a method for protecting an active agent from degradation comprising covalently attaching the active agent to a peptide such that the peptide will impart conformational protection to the active agent.
• the invention also provides a method for controlling release of an active agent from a composition wherein the composition comprises a peptide, the method comprising covalently attaching the active agent to the peptide.
• enhancement of the performance of active agents from a variety of chemical and therapeutic classes is accomplished by extending periods of sustained blood levels within the therapeutic window.
• the seram levels may peak too fast and too quickly for optimal clinical effect as illustrated below.
• Designing and synthesizing a specific peptide conjugate that releases the active agent upon digestion by intestinal enzymes mediates the release and absorption profile thus maintaining a comparable area under the curve while smoothing out active agent absorption over time.
• Conjugate prodrugs may afford sustained or extended release to the parent compound. Sustained release typically refers to shifting absorption toward slow first-order kinetics. Extended release typically refers to providing zero-order kinetics to the absorption of the compound. Bioavailability may also be affected by factors other than the absorption rate, such as first pass metabolism by the enterocytes and liver, and clearance rate by the kidneys. Mechanisms involving these factors require that the drag-conjugate is intact following absorption. The mechanism for timed release may be due to any or all of a number of factors.
• attachment of an amino acid, oligopepetide, or polypeptide may enhance absorption/bioavailability of the parent drug by any number of mechanisms, including conversion of the parent drug to a polymer-drag conjugate such that the amino acid-prodrugs may be taken up by amino acid receptors and/or di- and tri-peptide receptors (PEPT transporters).
• PPT transporters amino acid receptors and/or di- and tri-peptide receptors
• Adding an additional mechanism(s) for drag absorption may improve its bioavailability, particularly if the additional mechanism is more efficient than the mechanism for absorption of the parent drug.
• Many drags are absorbed by passive diffusion. Therefore, attaching an amino acid to the compound may convert the mechanism of absorption from passive to active or in some cases a combination of active and passive uptake, since the prodrug may be gradually converted to the parent drug by enzymatic activity in the gut lumen.
• active agent efficiency is enhanced by lower active agent serum concentrations.
• conjugating a variety of active agents to a carrier peptide and, thereby sustaining the release and absorption of the active agent would help achieve true once a day pharmacokinetics.
• peaks and troughs can be ameliorated such as what could be achieved with more constant atenolol levels, for example, following administration of a peptide-atenolol conjugate.
• the amino acids used can make the conjugate more or less labile at certain pH's or temperatures depending on the delivery required.
• the selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will include glycine, alanine, valine, leucine, isoleucine, phenylalanine and tyrosine. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the peptide.
• the amino acids with reactive side chains e.g., glutamine, asparagines, glutamic acid, lysine, aspartic acid, serine, threonine and cysteine
• the peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush- border membrane and so active agent release and subsequent absorption can occur in the jejunum or the ileum.
• the molecular weight of the carrier molecule can be controlled to provide reliable, reproducible and/or increased active agent loading.
• the invention provides methods of testing the conjugates using Caco-2 cells.
• the present invention also addresses the need for non-protected active agents, which provide for ease of manufacture and delivery.
• the present invention also addresses the need for an active agent delivery system that is able to deliver active agents through the stomach as active agent peptide conjugates so that the molecular mass and physiochemical properties of the conjugates can be readily manipulated to achieve the desired release rate.
• the present invention also addresses the need for an active agent delivery system that allows for the active agent to be released over an extended period of time, which is convenient for patient dosing.
• the present invention also addresses the need for an active agent delivery system that will provide protection through the stomach, but not require that the active agent be released within a specific cell or intracellularly.
• Embodiments of the present invention preferably do not produce an antigenic response or otherwise stimulate the immune system in the host.
• the active agent conjugate attached to the carrier peptide is used to create an immune response when administered.
• Figure 2 illustrates the in situ digestion of Polythroid in intestinal epithelial cell cultures
• Figure 3 illustrates the improved adsorption of T4 from PolyT4 compared to T4 alone;
• Figure 4 illustrates a decrease in the amount of Polythroid on the apical side over time (4hours) without intact Polythroid crossing the Caco-2 monolayer.
• peptide is meant to include a single amino acid, a dipeptide, a tripeptide, an oligopeptide, a polypeptide, or the carrier peptide. Oligopeptide is meant to include from 2 amino acids to 70 amino acids. Further, at times the invention is described as being an active agent attached to an amino acid, a dipeptide, a tripeptide, an oligopeptide, or polypeptide to illustrate specific embodiments for the active agent conjugate. Preferred lengths of the conjugates and other preferred embodiments are described herein.
• Modulation is meant to include at least the affecting of change, or otherwise changing total absorption, rate of adsorption and/or target delivery.
• Sustained release is at least meant to include an increase in the amount of reference drug in the blood stream for a period up to 36 hours following delivery of the carrier peptide active agent composition as compared to the reference drag delivered alone.
• Sustained release may further be defined as release of the active agent into systemic blood circulation over a prolonged period of time relative to the release of the active agent in conventional formulations through similar delivery routes.
• the active agent is released from the composition by a pH-dependent unfolding of the carrier peptide or it is released from the composition by enzyme- catalysis. In a preferred embodiment, the active agent is released from the composition by a combination of a pH-dependent unfolding of the carrier peptide and enzyme-catalysis in a time-dependent manner. The active agent is released from the composition in a sustained release manner. In another preferred embodiment, the sustained release of the active agent from the composition has zero order, or nearly zero order, pharmacokinetics. [094]
• the present invention provides several benefits for active agent delivery. First, the invention can stabilize the active agent and prevent digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed or sustained release of the active agent.
• the sustained release can occur by virtue of the active agent being covalently attached to the peptide and/or through the additional covalent attachment of an adjuvant that bioadheres to the intestinal mucosa.
• active agents can be combined to produce synergistic effects.
• absorption of the active agent in the intestinal tract can be enhanced either by virtue of being covalently attached to a peptide or through the synergistic effect of an added adjuvant.
• the invention also allows targeted delivery of active agents to specific sites of action.
• Proteins, oligopeptides, and polypeptides are polymers of amino acids that have primary, secondary, and tertiary structures.
• the secondary structure of the peptide is the local conformation of the peptide chain and consists of helices, pleated sheets, and turns.
• the peptide' s amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule.
• the folding of the secondary structure and the spatial arrangement of the side chains constitute the tertiary stracture.
• Peptides fold because of the dynamics associated between neighboring atoms on the peptide and solvent molecules.
• the thermodynamics of peptide folding and unfolding are defined by the free energy of a particular condition of the peptide that relies on a particular model.
• the process of peptide folding involves, amongst other things, amino acid residues packing into a hydrophobic core.
• the amino acid side chains inside the peptide core occupy the same volume as they do in amino acid crystals.
• the folded peptide interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to peptide stability is the solid reference state.
• the major forces contributing to the thermodynamics of peptide folding are Nan der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy, and the hydrophobic effect.
• the hydrophobic effect refers to the energetic consequences of removing apolar groups from the peptide interior and exposing them to water. Comparing the energy of amino acid hydrolysis with peptide unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the peptide fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic peptide core.
• the folded state of a peptide is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, peptide unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a peptide is often observed prior to the onset of irreversible chemical or conformation processes. Moreover, peptide conformation generally controls the rate and extent of deleterious chemical reactions. [099] Conformational protection of active agents by peptides depends on the stability of the peptide' s folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for peptide unfolding. [0100] Selection of the amino acids will depend on the physical properties desired.
• the carrier peptide will be enriched in the amino acids that have bulky, lipophilic side chains.
• Polar amino acids can be selected to increase the hydrophilicity of the peptide.
• Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid, and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine, and arginine, ionize in the stomach and are neutral in an alkaline environment.
• Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.
• the enzymes encountered in the lumen of the intestines, on the intestinal cell surface, and within the cells lining the intestine may completely remove the drag from the carrier peptide before it reaches the bloodstream. Accordingly, concerns about the safety of the novel composition are eliminated.
• Dextran has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug.
• polysaccharides are digested mainly in the colon and drug absorption is mainly limited to the colon.
• this invention has at least two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absorption can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.
• the active agent is attached to a peptide that ranges between a single amino acid and 450 amino acids in length.
• two to 50 amino acids are preferred, with the range of one to 12 amino acids being more preferred, and one to 8 amino acids being most preferred.
• the number of amino acids is selected from 1, 2, 3, 4, 5, 6, or 7 amino acids.
• the molecular weight of the carrier portion of the conjugate is below about 2,500, more preferably below about 1,000 and most preferably below about 500.
• the active agent conjugate is a dimer, of an active agent and a single amino acid. In another embodiment the active agent conjugate is attached to a dipeptide or tripeptide.
• compositions of the invention comprise four essential types of attachment. These types of attachment are termed: C-capped, N-capped, side-chain attached, and interspersed.
• C-capped comprises the covalent attachment of an active agent to the C-terminus of a peptide either directly or through a linker.
• N-capped comprises the covalent attachment of an active agent to the N-terminus of a peptide either directly or through a linker.
• Side-chain attachment comprises the covalent attachment of an active agent to the functional sidechain of a peptide either directly or through a linker.
• Interspersed comprises the attachment of active agents which themselves are amino acids. In this case the active agent would constitute a portion of the amino acid chain.
• Interspersed is herein meant to include the amino acid active agent (drug) being at the C-terminus, N-terminus, or interspersed throughout the peptide.
• amino acid active agents are attached to the C-terminus or the N- terminus this results in the active agent being the end amino and is considered C- capped or N-capped respectively.
• amino acids with reactive side chains e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine
• the present invention also envisions the use of multiple active agents or multiple attachment sites of active agents along a peptide chain. Further embodiments of the invention will become clear from the following disclosure.
• the alcohol, amine or carboxylic acid group of the active agent is covalently attached to the N-terminus, the C-terminus or the side chain of the peptide.
• the location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N-terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, one monomeric unit forming a new peptide bond in essence, adds a molecule to the end of the peptide.
• the active agent is an amine
• an alternate method of attaching the amine to the C-terminus of the peptide is to allow the amine to initiate polymerization of the amino acid NCA's.
• the active agent is an alcohol
• either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition.
• the active agent is an alcohol
• the alcohol can be converted into an alkylchloroformate with phosgene or triphosgene. This intermediate is then reacted with the N-terminus of the peptide carrier to produce an active agent peptide composition linked via a carbamate.
• the carbamate active ingredient may then be released from the peptide carrier by intestinal peptidases, amidases, or esterases.
• an alcohol active agent can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier.
• the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-carboxyanhydride.
• This intermediate can then be polymerized, as described above, using any suitable initiator.
• the product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved.
• other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.
• the alcohol can be added to the chloroformate of the side chain of polyserine as shown in section III of the Examples.
• the product is a carbonate, which upon hydrolysis of the peptide bond an intramolecular rearrangement occurs releasing the drag much of the same was as described above.
• the active agent is a ketone or an aldehyde than a ketal is formed with a linker that has a pendant group suitable for attachment to the N-terminus, C- terminus or side chain of the peptide.
• a ketal can be formed by the reaction of methylribofuranoside or glucose with methylnaltrexone as shown in example of glucose reacting with methylnaltrexone. The remaining free hydroxyl from the sugar moiety can then be treated as an alcohol for attachment to the C- terminus or a suitable side chain of the carrier peptide.
• the invention also provides a method of imparting the same mechanism of action for other peptides containing functional side chains.
• Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine.
• the mechanism can translate to these peptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-active agent dimer.
• the side-chain attached carrier peptide-active agent conjugate is preferably releases the active agent moiety through peptidase and not necessarily esterase activity.
• the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.
• the active agent is an amide or an imide then the nitrogen of the active agent can add in a Michael fashion to the dihydopyran carboxylic acid alkyl ester as shown in section VII:D of the Examples.
• the R group can either be an electron-withdrawing group such that transesterification with the side chain of the peptide can occur or the R group can be part of the side chain of the peptide.
• the release of the active agent from the linker is imparted by hydrolysis of the peptide carboxylate bond followed by a concerted decarboxylation/elimination reaction.
• the active agent can be covalently attached to the N-terminus, the C- terminus or the side chain of the peptide using known techniques.
• the active agent is an amino acid (e.g. Thyroxine, Triiodothyronine, DOPA, etc.)
• the active agent can be interspersed within the peptide chain in a peptide linked manner in addition to be covalently attached to the N-terminus, C-terminus or the side chains as described above. It is the preferred embodiment of the invention that the interspersed copolymer of the amino acid active agent and neutral amino acid be produced by polymerizing a mixture of the respective amino acid NCA's.
• the composition of the invention comprises a peptide and an active agent covalently attached to the peptide.
• active agents examples include, but are not limited to, those active agents listed in Table 2, either alone or in combination with other agents contained within Table 2.
• the active agents listed within Table 2 may exist in modified form to facilitate bioavailability and/or activity (e.g., a Sodium salt, halide-containing derivatives or HC1 forms of an active agent listed in Table 2). Accordingly, the invention encompasses variants (i.e., salts, halide derivatives, HC1 forms) of the active agents listed in Table 2.
• Nitrofurantoin Nitrofurantoin, Nitrofurantoin, Macrocrystalline
• the present invention allows for the combination of different active agents with a variety of peptides to impart specific characteristics according to the desired solubility, pH or folding. Similarly, the variety of peptides may be used to impart specific physicochemical properties to produce specific performance characteristics.
• the present invention provides significant advantages with respect to the stability and release and/or adsorption characteristics of the active agent(s).
• the conjugates of the present invention are also suited for delivery of both large and small molecules.
• the use of one or more of the 20 naturally occurring amino acids as individual amino acids, in oligopeptides, or in polypeptides impart specific stability, digestibility and release characteristics to the conjugates formed with active agents.
• the active agent conjugates are designed to interact with specific indigenous enzymes so that targeted delivery may be achieved. These conjugates provide targeted delivery in all regions of the gut and at specific sites along the intestinal wall.
• the active agent conjugates can incorporate adjuvants such that the compositions are designed to interact with specific receptors so that targeted delivery may be achieved. These compositions provide targeted delivery in all regions of the gut and at specific sites along the intestinal wall.
• the active agent is released as the reference active agent from the peptide conjugate prior to entry into a target cell.
• the specific amino acid sequences used are not targeted to specific cell receptors or designed for recognition by a specific genetic sequence, hi a more preferred embodiment, the peptide carrier is designed for recognition and/or is not recognized by tumor promoting cells.
• the active agent delivery system does not require that the active agent be released within a specific cell or intracellularly.
• the active agent conjugate allows for multiple active agents to be attached. The conjugates provide the added benefit of allowing multiple attachment not only of active agents, but of active agents in combination with other active agents, or other modified molecules which can further modify delivery, enhance release, target delivery, and/or enhance adsorption.
• the conjugates may also be combined with adjuvants or can be microencapsulated.
• the composition includes one or more adjuvants to enhance the bioavailability of the active agent.
• Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent.
• Suitable adjuvants include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the brush border membrane (BBM); and bile acids, which have been attached to peptides to enhance absorption of the peptides.
• absorption may be improved by increasing the solubility of the parent drag through selective attachment of an amino acid, oligopeptide, or polypeptide.
• solubility results in an increase in the dissolution rate. Consequently, there is an increase in the total amount of drug that is available for absorption; since the drag must be in solution for absorption to occur, bioavailability is increased.
• compositions provide for a wide range of pharmaceutical applications including active agent delivery, cell targeting, and enhanced biological responsiveness.
• the present invention provides several benefits for active agent delivery.
• the invention can stabilize the active agent and prevent digestion in the stomach.
• the pharmacologic effect can be prolonged by delayed or sustained release of the active agent.
• the sustained release can occur by virtue of the active agent being covalently attached to the peptide and/or through the additional covalent attachment of an adjuvant that bioadheres to the intestinal mucosa.
• active agents can be combined to produce synergistic effects.
• Absorption of the active agent in the intestinal tract can be enhanced either by virtue of being covalently attached to a peptide or through the synergistic effect of an added adjuvant.
• the absorption of the active agent is increased due to its covalent attachment to a peptide, hereafter to be referred to as a transporter peptide, which is a specialized example of a carrier peptide.
• the transporter peptide activates a specific peptide transporter.
• the peptide transporter is either the PepTl or the PepT2 transporters.
• the transporter peptide contains two amino acids.
• the transporter dipeptide is selected from the list of AlaSer, CysSer, AspSer, GluSer, PheSer, GlySer, HisSer, IleSer, LysSer, LeuSer, MetSer, AsnSer, ProSer, GlnSer, ArgSer, SerSer, ThrSer, ValSer, TrpSer, TyrSer.
• the present invention does not require the attachment of the active agent to an adjuvant that recognizes or is taken up by an active transporter.
• the invention also allows targeted delivery of active agents to specifics sites of action.
• the chain length of amino acid can be varied to suit different delivery criteria.
• the present invention allows for the delivery of active agents with sustained release.
• the invention may further be characterized by the following embodiments wherein the active agent is released as the reference active agent from the amino acid conjugate prior to entry into the target cell for the active agent.
• the active agent is prepared and/or administered through means other than implantation and/or injectibles. Embodiments of the present invention preferably do not produce and antigenic response or otherwise stimulate the immune system in the host.
• the intact conjugate may be less susceptible to first pass metabolism by the enterocytes, including biotransformation by cytochrome P450 (CYP) 3A4 and efflux by transporter P- glycoprotein. Immunogenicity and metabolic effects are avoided through maintenance of the three dimensional structure of the composition, maintaining blood levels below the threshold value for expression of these metabolic factors or some other means.
• the active agent is directly attached to the amino acid without the use of a linker.
• Caco-2 human intestinal epithelial cells are increasingly being used.
• Caco-2 cells are grown on the surface of collagen-coated wells in a 24 well format to form confluent monolayers that represent small segments of the intestine.
• the wells are removable and contain a top chamber representing the apical side (facing the lumen of the intestine) and a bottom chamber representing the basolateral side (site of serosal drug absorption). Testing the electrical resistance across the monolayer monitors the integrity of the epithelial barrier. Absorption of drags can be studied by adding sample to the apical side and assaying the concentration of the drag in the basolateral chamber following incubation.
• the small intestine has an extremely large surface area covered with highly specialized epithelial cells that produce both extracellular and intracellular enzymes.
• the Caco-2 cells also release enzymes similar to the epithelial cells of the small intestine.
• drag release from peptides on the apical side of Caco-2 transwell monolayers can be measured.
• the copolymer of glutamic acid and thyroxine enhanced the absorption of thyroxine across the Caco-2 monolayers.
• the invention provides methods of testing the conjugates using Caco-2 cells.
• the Table 3 provides a list of active agents that have been covalently attached to a peptide.
• the table also provides a list of typical areas of use for the active agent conjugate.
• compositions of the invention can be formulated in pharmaceutical compositions by combining the compound with a pharmaceutically acceptable excipient known in the art.
• the conjugates may be employed in powder or crystalline form, in liquid solution, or in suspension.
• the conjugates of the present invention may be administered by a variety of means, including but not limited to: topically, orally, parenterally by injection (intravenously, intramuscularly or subcutaneously), as a depot implant, an intranasal preparation, an inhaler, or as an anal suppository.
• the injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents.
• the conjugate may be in powder form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water.
• a suitable vehicle such as sterile water.
• the carrier is typically comprised of sterile water, saline or another injectable liquid, e.g., peanut oil for intramuscular injections.
• various buffering agents, preservatives and the like can be included.
• Topical applications may be formulated in carriers such as hydrophobic or hydrophilic bases to form ointments, creams, lotions, in aqueous, oleaginous or in dry diluents to form powders.
• Oral compositions may take such forms as tablets, capsules, oral suspensions and oral solutions.
• the oral compositions may utilize carriers such as conventional formulating agents, and may include sustained release properties as well as rapid delivery forms.
• the dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration.
• One embodiment of the methods of administration of the conjugates includes oral and parenteral methods, e.g., i.v. infusion, i.v. bolus and i.m. injection.
• the composition incorporates a microencapsulating agent.
• the composition of the invention is in the form of an ingestible tablet or capsule, an implantable device, a skin patch, a sublingual preparation, a subcutaneous preparation, an intravenous preparation, an intraperitoneal preparation, an intramuscular preparation or an oral suspension.
• the compositions are formulated for oral delivery.
• the figures are meant to describe the general scheme of attaching active agents through different functional groups to a variety of peptide conjugates resulting in different embodiments of the present invention.
• One skilled in the art would recognize other reagents, conditions, and properties necessary to conjugate other active agents to other peptides from the schemes, which are meant to be non-limiting examples.
• the N-terminus attachment of active agent to a peptide can be formed through a plurality of active agent functional groups.
• active agent functional groups include an alcohol group, a carboxylic acid group, an amine group or other reactive substiuents.
• the preferred active agent attaching functionalities for N-terminus attachment to a peptide include carboxylic acids, ketones and aldehydes.
• N-terminus of the peptide/active agent conjugate Any amino acid may be used as the N-terminus of the peptide/active agent conjugate.
• Preferred amino acids for attachment include glutamic acid, aspartic acid, serine, and lysine.
• Specific examples of an active agent attached to the N-terminus below are meant for example purposes only and are not meant to limit the invention to either specific active agents, amino acids or combinations thereof.
• Preferred drags for N-terminus attachment typically provide a carboxylic acid or an inorganic functional group for conjugation.
• ibuprofen, furosemide, gemfibrozil, naproxen may be attached to the N-terminus.
• R' Radical moiety attached to acid functionality on drug
• R Side chain of amino acid or peptide
• HOBt HydroxybenzotriazoIe
• an acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0 C.
• the solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier.
• the reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.
• GPC gel permeation chromatography
• R' Radical moiety attached to alcohol functionality on drug
• R Side chain of amino acid or peptide
• solvents examples include dimethylsulfoxide (DMSO), ethers such as tetrahydrofuran(THF) or chlorinated solvents such as chloroform (CHC1 3 ).
• activating agents include dicyclohexylcarbodiimide or thionyl chloride.
• An example of another co-catalyst is N-hydroxysuccinimide (NHS).
• bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine (Et 3 N) or tributylamine.
• the C-terminus attachment of an active agent to a peptide can be formed through a plurality of active agent functional groups.
• the functional groups include amines and their equivalents and alcohols and their equivalents. While any amino acid may be used to connect the active agent to the C-terminus, glutamic acid, aspartic acid, serine and lysine are preferred amino acids.
• Preferred active agents for C-terminus attachment are active agents with alcohol and amino functional groups. More preferred active agents include atenolol, metropolol, propanolol, methylphenidate and sertraline.
• R' Radical moiety attached to acid functionality on drug
• R Side chain of amino acid or peptide
• HOBt Hydroxybenzotriazole
• This example can be used to generically describe the process of an amine active agent initiating polymerization of an amino acid NCA. The following procedure was successfully used to synthesize the polyglutamic acid conjugate of atenolol through its amine functionality. It should also be noted that atenolol is also an alcohol active agent and can also initiate polymerization of amino acid NCA's. This procedure can readily be applied to other amine drags described herein.
• Table 4 describes the relative proportions that would be used in a typical atenolol preparation. Other amine drags that would be used to initiate polymerization of an amino acid NCA would be expected to utilize similar proportions. [158] The procedure is further described below, although those skilled in the art would recognize other solvents, proportions and reaction conditions that could be utilzed to achieve the desired results.
• DMF is dimethylformamide, anhydrous, and was purchased from Aldrich. The glassware was oven-dried prior to use.
• Glu-NCA 500 mg, 2.89 mmoles was dissolved in 4 mL of DMF and stirred under argon in a 15 mL round bottom flask equipped with a gas inlet tube.
• Atenolol dissolved in 1 mL of DMF, was added to this solution of Glu-NCA and allowed to stir at room temperature for 72 h. hi general, the reactions can be run until there is no free amine initiator by Thin Layer Chromatography (TLC).
• TLC Thin Layer Chromatography
• aqueous layers were brought to a pH of 6 with 6N hydrochloric acid (HC1) and reduced to a volume of about 20 mL by rotary evaporation.
• This solution was then cooled in the refrigerator for > 3 hours.
• the aqueous solution was then acidified to a pH of about 2 using 6N HC1 and placed back in the refrigerator for 1-2 hours.
• the suspension was poured by portions into a 10 mL test tube and centrifuged for 15 minutes until the precipitate formed a solid pack at the bottom of the tube from which the water could be decanted. (At this point in the general procedure, it is preferable that the solid be filtered through a filter funnel and washed with acidic water.
• the centrifuge was used for atenolol because the solid was too thin to filter.)
• the solid was then resuspended in acidic water (pH about 2) and vortexed before being centrifuged again and the water decanted. This procedure was repeated once more for a total of three washes.
• the solid was then dried by high vacuum overnight yielding 262 mg (59%) of polymer. NMR analysis indicated that the Glu/ Atenolol ratio was about 30/1.
• the below example describes the attachment of an amino acid active agent to the C-terminus of a peptide.
• the example uses glutamic acid NCA to produce a polyGlu attached to Cephalexin.
• Cephalexin attached to a single amino acid may be produced via the below method if an excess amount of Cephalexin is added to the procedure.
• Glu(OtBu)NCA 1.000 g, 4.4 mmol
• Cephalexin»HCl (0.106 g, 0.3 mmol) were dissolved in anhydrous DMF (5 mL). The reaction was then allowed to stir at room temperature under argon. After 3 days, the solvent was removed by rotary-evaporation under vacuum.
• the resulting solid was then placed under argon and then dissolved in 4N HC1 in Dioxane (2mL) and then allowed to stir at room temperature under a blanket of argon. After 1 hour, the dioxane and HC1 were removed by rotary-evaporation under vacuum. The solid was then suspended in methanol (2 mL) and once more brought to dryness by rotary- evaporation in order to remove residual HC1 and dioxane. This material was then resuspended in methanol (2 mL) and precipitated by the addition of water (20 mL). The aqueous suspension was then stored at 4°C for 4 hours, and the solid isolated by centrifugation. The pelleted material was then allowed to dry under vacuum over night.
• DMF is dimethylformamide, anhydrous, and was purchased from Aldrich. Glassware was oven-dried prior to use.
• Glu-NCA (2 g, 11.56 mmoles) was dissolved in 8 mL of DMF and stirred under Ar in a 25 mL roundbottom flask equipped with a gas inlet tube. Atenolol, dissolved in 2 mL of DMF, was added to this solution of Glu-NCA and allowed to stir at room temperature for 93 h. Bubbles were observed at the beginning of the reaction.
• the DMF was reduced by rotary evaporation and the oil was transferred into a 125 mL Erlenmeyer, rinsing the round bottom well with water.
• the pH of the solution was adjusted to 3 with 1 N HCL This solution (60 mL total volume) was then cooled in the refrigerator for > 3 hours. The suspension was filtered through a sintered glass funnel and washed with 3 X 30 mL of 1%
• Boc-Glu(OtBu)-OH (0.44 g, 1.46 mmol) and PyBOP (0.84 g, 1.60 mmol) were dissolved in dry DMF (15 mL) with stirring.
• DIEA (0.31 mL, 1.75 mmol) was added and the amino acid derivative was allowed to activate for 15 minutes.
• Sertraline hydrochloride (0.50 g, 1.46 mmol) was added to the stirring mixture followed by an additional 0.31 mL DIEA. The mixture was allowed to stir for 16 hours. The solution was stripped yielding brown oil.
• the oil was dissolved in EtOAc (100 mL) and the resulting solution was washed with 10 % HC1 (3 x 30 mL), saturated NaHCO 3 , 4M NaHSO 4 , and brine (2 x 30 mL, respectively).
• the solution was dried over MgSO , filtered and the solvent was removed by rotary evaporation under reduced pressure, yielding light brown oil.
• the oil was dried on the vacuum manifold and the product was purified by column chromatography on silica gel using EtOAc/Hexanes 1:5 to 1:4 solvent system. The product fractions were pooled and solvent was again removed by rotary evaporation yielding 0.85 g (99%) of the final product, Sertraline-NH-C(O)-Glu-NH3+.
• the preparation was dried on the vacuum manifold.
• Boc-Glu(OtBu)-OH (0.44 g, 1.46 mmol) and PyBOP (0.84 g, 1.60 mmol) were dissolved in dry DMF (15 mL) with stirring.
• DIEA (0.31 mL, 1.75 mmol) was added and the amino acid derivative was allowed to activate for 15 minutes.
• Propranolol hydrochloride (0.43 g, 1.46 mmol) was added to the stirring mixture followed by an additional 0.31 mL DIEA. The mixture was allowed to stir for 16 h. The solution was stripped yielding a brown oil.
• the oil was dissolved in EtOAc (100 mL) and the resulting solution was washed with 10 % HC1 (3 x 30 mL), saturated NaHCO 3 , 4M NaHSO 4 , and brine (2 x 30 mL, respectively).
• the solution was dried over MgSO 4 , filtered and the solvent was removed by rotary evaporation under reduced pressure, yielding light brown oil.
• the oil was dried on the vacuum manifold and the product was purified by column chromatography on silica gel using EtOAc/Hexanes 1:5 to 1:4 solvent system. The product fractions were pooled and solvent was again removed by rotary evaporation the final product, Propranolol- NH-C(O)-Glu-NH3+.
• the preparation was dried on the vacuum manifold.
• the attachment of active agents to the side-chain of a peptide can be formed through a plurality of a combination of functional groups that can be selected from the active agent or amino acid used for conjugation. Unlike, when the active agent is conjugated to the N-terminus or C-terminus, where the functional group of the amino acid is restricted to either an amine or carboxylate group respectively, side-chain attachment allows for variability in the selection of specific amino acid side-chain functionalities. Additionally, where applicable the active agent functional group can be selected to conform to the amino acid side-chain utilized for attachment.
• the functional groups depend on the functionality on the side chain of a peptide utilized for conjugation. The diversity of side-chain attachment allows any active agent to be directly attached to the side chain of amino acids with appropriate functional groups.
• Active agents containing alcohols, amines and/or carboxylic acids are directly amenable to attachment through and may dictate the side-chain of the amino acid selected.
• active agents that lack these functional groups it is preferred that the incorporation of a linker contain an alcohol, amine, or carboxylic group.
• More preferred amino acids used to create the attachment and/or the peptide are glutamic acid, aspartic acid, serine, lysine, cysteine, threonine, and glutamine. While homopolymers are often used, heteropolymers may be utilized to impart specific performance parameters. These heteropolymers may be of various chain length and degree of heterogeneity. Preferred examples include, but are not limited to, dipetides including Leu-Ser, Leu-Glu, homopolyers of Glu and Leu and heteropolymers of (Glu)n-Leu-Ser. [185] An example of side-chain attachment conjugation to an acid drag is depicted in the scheme below:
• R' Drug with carboxylic acid functionality
• HOBt Hydroxybenzotriazole
• DIPC Diisopropylcarbodiimide
• R Side chain of amino acid or peptide
• R' Radical moiety attached to acid functionality
• HOBt Hydroxybenzotriazole
• DIPC Diisopropylcarbodiimide
• R Side chain of amino acid
• R ' Drug with amine functionality
• R ' Drug with alcohol functionality
• Examples III: A - 1H:G describe the attachment of active agents to a peptide through the alcohol group.
• One of the examples describes the attachment of Naltrexone to aspartic acid, while the others show different active agents attached to glutamic acid.
• Examples III:H - III:I are illustrative of the conversion of one of the naturally occurring amino acids, in this case Glu, to a glutamic acid derivative for subsequent incorporation into a peptide either through the NCA method or through the use of a peptide synthesizer.
• Example IH:J is further illustrative of the conversion of one of the namrally occurring amino acids, in this case Glu, to a glutamic acid derivative which can be further incorporated into a linear or dendritic peptide either through the NCA method or through the use of a peptide synthesizer.
• Examples III:K - D- N show a carboxylic acid attached to the side chain of an amino acid.
• the active agent is attached to Polylysine through the amino group.
• Example 111:0 is describes a sulfonamide attached to the side-chain of a Polyglutamic acid.
• active agent attached to the side-chain attachment are meant for example purposes only and are not meant to limit the invention to either specific active agents, amino acids or combinations thereof. Those skilled in the art would recognize from the present disclosure other active agents, which can be attached to the side-chain of a peptide.
• R Drug attached via Alcohol Group as an Ester
• Boc-Asp(Nal)-OtBu was obtained in 41% isolate yield using a similar protocol as the one used to prepare Boc-Glu(Nal)-OtBu. [196] 1H-NMR (360 MHz, CDC1 3 ): ⁇ 6.84 (d, IH, naltrexone aromatic),
• naltrexone While naltrexone has a complex NMR spectrum, there are several key protons that have distinct chemical shifts and are unique to naltrexone.
• Glu- ⁇ H and AZT 2' CH 2 2.58 (m, 2H, Glu- ⁇ H), 3.70 (t, IH, Glu- ⁇ H), 4.05-4.41 (m, 4H, AZT 4' CH, 3' CH and 5' CH 2 ), 6.18 (t, IH, AZT 1' CH), 7.51 (s, IH, AZT 6 CH).
• Glu- ⁇ H 2.39 (br m, 2H, Glu- ⁇ H), 2.72 (br m, 2H, urea), 3.32 (br m, 6H, acyclovir CH 2 and urea), 3.83 (br m, 3H, urea), 4.38 (br d, 3H, Glu- ⁇ H), 5.47 (br s, 2H, acyclovir 1' CH2), 7.94 (br s, IH, acyclovir 8 CH).
• the crude product was purified using ultrafiltration. Product was then collected from ultrafiltration using acid precipitation (0.965 g, 35%).
• Glu- ⁇ H 2.12 (s, 3H, acetaminophen CH 3 ), 2.25 (m, IH, Glu- ⁇ H), 2.60 (m, 2H, Glu- ⁇ H), 4.25 (m, IH, Glu- ⁇ H), 7.04 (d, 2H, acetaminophen aromatic), 7.48 (d, 2H, acetaminophen aromatic).
• R Boc-Glu-OtBu or H
• N-methyl morpholine (1.26 mL, 11.5 mmol) and b ⁇ omo-tris- pyrrolidino-phosphonium hexafluorophosphate (PyBrOP) (2.04 g, 4.38 mmol).
• PyBrOP b ⁇ omo-tris- pyrrolidino-phosphonium hexafluorophosphate
• the resulting mixture was allowed to stir at room temperature for 30 minutes. After this time, N-methyl morpholine (0.78 mL, 7.11 mmol) and 4-dimethylaminopyridine (0.133 g, 1.09 mmol) (DMAP) followed by Furosemide (1.63 g, 4.92 mmol) were added. The resulting solution was stirred at room temperature for 24 hours.
• Poly-lysine-HBr (Sigma, 100 mg, 34.5 nmol) was dissolved in 1 mL of water that had been brought to a pH of 8 with sodium bicarbonate, and stirred at room temperature. To this solution was added a solution of ibuprofen-O- succinimide (116 mg, 380 nmol) in 2 mL of dioxane. After stirring overnight, the dioxane was removed by rotary evaporation and diluted with 10 mL of pH 8 sodium bicarbonate in water. The precipitated product was filtered through a sintered glass funnel and washed with 3 x 10 mL of water and 4 x 10 mL of diethyl ether.
• Boc-Leu-Glu-OtBu [266] To a solution of Boc-Leu-OSu and Glu-OtBu in DMF was added
• Boc-Leu-Glu-OtBu [267] To a solution of Boc-Leu-Glu-OtBu in DMF was added NMM and
• Amino acid active agents allow for a distinct embodiment of the present invention. This embodiment occurs when the active agent itself is an amino acid and thus capable of both amine and carboxylate bonding allowing for the interspersement of the active agent into the peptide chain. Amino acid drags can be
• the active agent may be interspersed within the peptide chain through a peptide bond at both ends of the active agent.
• Most preferred drag conjugates for the interspersed attachment of amino acid active agents include amoxicillin; amoxicillin and clavulanate; baclofen; benazepril hydrochloride; candoxatril; captopril; carbidopa/Levodapa; cefaclor; cefadroxil; cephalexin; cilastatin sodium and imipenem; ciprofloxacin; diclofenac sodium; diclofenac sodium and misoprostol; enalapril; enapril maleate; gabapentin; levothyroxine; lisinopril; lisinopril and hydroichlorothiazide; loracarbef; mesalamine; pregabalin; quinapril hydrochloride; sitafloxacin; tirofiban hydrochloride; trandolapril; and trovafloxacin mesylate.
• An example An example
• R Side chain of amino acid or peptide
• R' Radical moiety attached to amine functionality on drug
• amino acid active agent is attached to the N-terminus of different amino acids. These examples do not only describe an N- terminus attachment, but also represent the peptide linkage of the amino acid active agent as an amino acid to form either a dipeptide or a peptide conjugate.
• T4 conjugated to amino acid polymers were either prepared by coupling (protected) T4 to commercially available amino acid homopolymers or incorporated by addition of T4-NCA to the corresponding polypeptide in situ.
• N-TeocT4 (0.017 g, 17 ⁇ mol) in 1 mL dry DMF was added dicyclohexylcarbodiimide (0.004 g, 18 ⁇ mol). After stirring for 30 minutes N- dimethyl-4-aminopyridine (0.004 g, 36 ⁇ mol) and Gly 18 (0.017 g, 17 ⁇ mol) were added and the reaction stirred overnight. The cloudy solution was poured into 20 mL H 2 O and extracted twice with 10 mL CH 2 C1 2 . The aqueous component was acidified to pH 3 with 1 N HCl and chilled to 4°C. The material was isolated by centrifugation and the pellet washed 3 times with 8 mL H 2 O.
• N-carboxyanhydrides of the amino acids listed below were prepared using a protocol similar to that reported for glutamic acid. Minor variations in their final workups are noted below.
• Thr-OtBu a mixture of Thr-OtBu (0.500 g, 2.85 mmol) in THF (25mL) was added triphosgene (0.677 g, 2.28 mmol). The resulting solution was stirred at reflux for 3 hours. The solution was evaporated to dryness to obtain Thr-NCA (0.500 g, 2.48 mmol, 87%) as a white solid. Thr-NCA was used without further characterization.
• Thr-NCA was used without further characterization.
• Leu-NCA 1H NMR (500 MHz, CDC1 3 ): ⁇ 6.65 (s, IH, NH), 4.33 (dd, IH, ⁇ ), 1.82 (m, 2H, ⁇ ), 1.68 (m, IH, ⁇ ), 0.98 (dd, 6H, ⁇ ).
• Trp(Boc)-NCA 1H NMR (500 MHz, CDC1 3 ): ⁇ 8.14 (d, IH), 7.49 (d, 2H),
• Lys(Boc)-NCA IH NMR (500 MHz, CDC13): D 6.65 (bs, IH, NtH), 4.64 (s, IH, carbamate NH), 4.31 (t, IH, ⁇ ), 3.13 (s, 2H, ⁇ ), 2.04 (m, 2H, ⁇ ), 1.84 (m, 2H, ⁇ ), 1.48 (m, llH, ⁇ , t-Bu).
• NCA 200 mg, 0.93 mmol
• anhydrous DMF 5 mL
• T4-NCA 27 mg, 0.03 mmol
• Polymer was then precipitated by the addition of water (50 mL) to each flask. The resulting solids were collected by filtration and dried over night under vacuum.
• T4-Asp n (Flask A) consisted of a mixture of polymers of varying lengths: T4-Asp 3 - ⁇ 2 .
• Table 7 Amino Acid Conjugates of T4 -
• T4 Conjugation to Preformed Homopolymer in situ are listed below:
• Trp ⁇ s-T4 [290] To T4 (0.078 g, 100 ⁇ mol) in 10 mL dry DMF was added Trp(Boc)-
• Lys ⁇ 5 -T4 was prepared using a similar protocol than the one used for
• Trp 5 -T4.
• Random T4/Lys ⁇ 5 was prepared using a protocol similar to the one used to prepare Random T4/Trp.
• TeocT3C8 material (0.187 g, 0.21 mmol) was dissolved in 10 mL of CH 2 C1 2 and 5 mL of trifluoroacetic acid, TFA. After stirring for lh the solvent was removed by rotary evaporation target as a white solid (0.177 g, 100%): R f (1:1 hexane:EtOAc) 0.78; 1H NMR (DMSO 500 MHz) 7.83 (s, 2H), 6.95-6.66 (m, 3H),

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