EP1229793A1 - Bifunctional molecules having modulated pharmacokinetic properties and therapies based thereon - Google Patents

Abstract

Bifunctional molecules and methods for their use are provided. The subject bifunctional molecules are conjugates of a drug moiety and a pharmacokinetic modulating moiety, where these two moieties are optionally joined by a linking group. The bifunctional molecules are further characterized in that they exhibit at least one modulated pharmacokinetic property upon administration to a host as compared to a free drug control. The subject bifunctional molecules find use in a variety of therapeutic applications.

Description

BIFUNCTIONAL MOLECULES HAVING MODULATED PHARMACOKINETIC PROPERTIES AND THERAPIES BASED THEREON

ACKNOWLEDGMENT This invention was made with United States Government support under Grant No CA39612 awarded by National Institutes of Health The United States Government has certain rights in this invention

CROSS REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U S C § 119 (e), this application claims pnoπty to the filing date of the United States Provisional Patent Application Senal No 60/166,633 filed November 19, 1999, the disclosure of which is herein incorporated by reference

INTRODUCTION Technical Field The field of this invention is pharmacology

Background of the Invention

Any chemical agent that affects any process of living is a drug Drugs are a critical tool for health care practitioners, as they are used m the prevention, diagnosis and treatment of disease Because of their cnticahty to the health care profession, annual world investment to the research and development of new chemical agents with therapeutic potential reaches mto the billions of dollars As a result, a large number of drugs have been developed to date and new chemical agents havmg potential therapeutic utility are frequently discovered Chemical agents that find, or have found, use as drugs mclude naturally occurring and synthetic small molecules, as well as larger molecules, such as protemaceous compounds

A major challenge in the development of drugs is the predictable modulation of pharmacokinetic properties Major pharmacokmetic parameters that effect the ability of a particular drug to treat a given condition mclude the drug half-life, the hepatic first-pass metabolism of the drag, the volume of distnbution of the drug, the degree of albumin binding of the drug, etc Each of the above parameters can have a profound effect on the efficacy of a given drag agent

As such, of great mterest to the pharmaceutical industry and related fields would be the development of a methods for predictably modulating one or more of these pharmacokmetic properties so as to improve a given drug, e g the efficacy of a given drug Of particular mterest would be the development of a method which retained the small molecule nature of the drag, so as to retain the advantages of small molecule compounds with respect to drag delivery. Relevant Literature

Patent publications of interest include: WO 91/01743; WO 94/18317; WO 95/02684; WO 95/10302; WO 96/06111; W096/12796; W096/13613; WO 97/25074; W097/29372; W098/11437; WO 98/47916; U.S. Patent No. 5, 714,142; U.S. Patent No. 5,830,462; U.S. Patent No. 5,843,440; and U.S. Patent No. 5,871,753. References of interest include: Briesewitz et al., Proc. Nat'l Acad. Sci. USA (March 1999) 96: 1953-1958; Clardy, Proc. Nat'l Acad. Sci. USA (March 1999) 1826-1827; Crabtree & Schreiber, Elsevier Trends Journal (November 1996) 418-422; Spencer et al., Curr. Biol. (July 1996) 6:839-847; Spencer et al., Science (1993) 262: 1019; Chakraborty et al., Chem. & Biol. (March 1995) 2:157-161; Ho et al., Nature (1996) 382: 822; Riviera et al., Nature Medicine (1996) 2: 1028; Klemm et al., Current Biology (1997) 7: 638; Belshaw et al., Proc. Nat'l. Acad. Sci. USA (1996) 93: 4604; Livnah et al., Science (1996) 273: 464; Johnson et al., Chemistry and Biology, (1997) 4: 939; Garboczi et al., Nature (1996) 384:134; Kissenger et al., Natiire (1995) 378:641; Griffith et al., Cell (1995) 82: 507; Choi et al., Science (1996) 273:239. Also of interest are Kramer et al., J. Biol. Chem. (1992) 267:18598-18604; and Varshavsky, Proc. Nat'l Acad. Sci. USA (March 1998) 95: 2094-2099; Varshavsky, Proc. Nat'l Acad. Sci. USA (April 1995) 92:3663-3667; and Mu et al., Biochem. Biophys. Res. Comm. (1999)255:75-79.

SUMMARY OF THE INVENTION Bifunctional molecules and methods for their use are provided. The subject bifunctional molecules are conjugates of a drug moiety and a pharmacokinetic modulating moiety, where these two moieties are optionally joined by a linking group. The bifunctional molecules are ftirther characterized in that they exhibit at least one modulated pharmacokinetic property upon administration to a host as compared to a free drug control. The subject bifunctional molecules find use in a variety of therapeutic applications.

DEFINITIONS The term "bifunctional molecule" refers to a non-naturally occurring molecule that includes a pharmacokinetic modulating moiety and a drug moiety, where these two components may be covalently bonded to each other either directly or through a linking group. The term "drug" refers to any active agent that affects any biological process. Active agents which are considered drugs for purposes of this application are agents that exhibit a pharmacological activity. Examples of drugs include active agents that are used in the prevention, diagnosis, alleviation, treatment or cure of a disease condition.

By "pharmacologic activity" is meant an activity that modulates or alters a biological process so as to result in a phenotypic change, e.g. cell death, cell proliferation etc. By "pharmacokinetic property" is meant a parameter the describes the disposition of an active agent in an organism or host. Representative pharmacokinetic properties include: drug half-life, hepatic first-pass metabolism, volume of distribution, degree of blood serum protein, e.g. albumin, binding, etc.

By "half-life" is meant the time for one-half of an administered drug to be eliminated through biological processes, e.g. metabolism, excretion, etc.

By "hepatic first-pass metabolism" is meant the propensity of a drag to be metabolized upon first contact with the liver, i.e. during its first pass through the liver.

By "volume of distribution" is meant the distribution and degree of retention of a drag throughout the various compartments of an organisms, e.g. intracellular and extracellular spaces, tissues and organs, etc. By "degree of blood serum binding" is meant the propensity of a drug to be bound by by a blood serum protein, such as albumin, in manner such that the activity of the drag is substantially dissipated if not abolished. This property is also referred to herein as the blood serum binding effect. In those embodiments where the blood serum protein is albumin, this property is also referred to as the albumin binding effect.

The term "efficacy" refers to the effectiveness of a particular active agent for its intended purpose, i.e. the ability of a given active agent to cause its desired pharmacologic effect.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Bifunctional molecules and methods for their use are provided. The subject bifunctional molecules are conjugates of a drug moiety and a pharmacokinetic modulating moiety, where these two moieties are optionally joined by a linking group. The bifunctional molecules are further characterized in that they exhibit at least one modulated pharmacokinetic property upon administration to a host as compared to a free drug control. The subject bifunctional molecules find use in a variety of therapeutic applications.

Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. BIFUNCTIONAL MOLECULE

The bifunctional molecule of the subject invention is a non-naturally occurring or synthetic compound that is a conjugate of a drag or derivative thereof and a pharmacokinetic modulating moiety, where these two moieties are optionally joined by a linking group. The targeted bifunctional molecule is further characterized in that the pharmacokinetic modulating and drag moieties are different, such that the bifunctional molecule may be viewed as a heterodimeric compound produced by the joining of two different moieties. In many embodiments, the pharmacokinetic modulating moiety and the drag moiety are chosen such that the corresponding drag target and binding partner of the pharmacokinetic modulating moiety, e.g. corresponding pharmacokinetic modulating protein to which the pharmacokinetic modulating moiety binds, do not naturally associate with each other to produce a biological effect. As indicated above, the subject bifunctional molecules are small. As such, the molecular weight of the bifunctional molecule is generally at least about 100 D, usually at least about 400 D and more usually at least about 500 D, and maybe as great as 2000 D or greater, but usually does not exceed about 5000 D.

The bifunctional molecule is further characterized in that it exhibits at least one modulated pharmacokinetic property, e.g. half-life, hepatic first-pass metabolism, volume of distribution, degree of albumin binding, etc., upon administration to a host as compared to free drag control. By modulated pharmacokinetic property is meant that the bifunctional molecule exhibits a change with respect to at least one pharmacokinetic property as compared to a free drug control. For example, a bifunctional molecule of the subject invention may exhibit a modulated, e.g. longer, half-life than its corresponding free drug control. Similarly, a bifunctional molecule may exhibit a reduced propensity to be eliminated or metabolized upon its first pass through the liver as compared to a free drug control. Likewise, a given bifunctional molecule may exhibit a different volume of distribution that its corresponding free drag control, e.g. a higher amount of the bifunctional molecule may be found in the intracellular space as compared to a corresponding free drug control. Analogously, a given bifunctional molecule may exhibit a modulated degree of albumin binding such that the drug moiety's activity is not as reduced, if at all, upon binding to albumin as compared to its corresponding free drug control. In evaluating whether a given bifunctional molecule has at least one modulated pharmacokinetic property, as described above, the pharmacokinetic parameter of interest is typically assessed at a time at least 1 week , usually at least 3 days and more usually at least 1 day following administration, but preferably within about 6 hours and more preferably within about 1 hour following administration.

Bifunctional molecules of the subject invention are generally described by the formula:

Z-L-X wherein:

X is a drag moiety; L is bond or linking group; and

Z is pharmacokinetic modulating moiety; with the proviso that X and Z are different. Drug Moiety X

The drug moiety X may be any molecule, as well as a bmdmg portion or fragment, e g denvative, thereof, that is capable of modulating a biological process m a living host, either by itself or in the context of the pharmacokmetic modulating protein/bifunctional molecule binary complex Generally, X is a small organic molecule that is capable of bmdmg to the target of mterest As the drag moiety of the bifunctional molecule is a small molecule, it generally has a molecular weight of at least about 50 D, usually at least about 100 D, where the molecular weight may be as high as 500 D or higher, but will usually not exceed about 2000 D

The drug moiety is capable of interacting with a target m the host mto which the bifunctional molecule is administered during practice of the subject methods The target may be a number of different types of naturally occurring structures, where targets of mterest include both intracellular and extracellular targets, where such targets may be proteins, phosphohpids, nucleic acids and the like, where protems are of particular mterest Specific protemaceous targets of mterest include, without limitation, enzymes, e g kinases, phosphatases, reductases, cyclooxygenases, proteases and the like, targets compnsmg domains mvolved in protem-protem interactions, such as the SH2, SH3, PTB and PDZ domains, structural protems, e g actin, tubulin, etc , membrane receptors, mrmunoglobuhns, e g IgE, cell adhesion receptors, such as integπns, etc, ion channels, transmembrane pumps, transcription factors, signaling protems, and the like

The drag moiety of the bifunctional compound will mclude one or more functional groups necessary for structural interaction with the target, e g groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions, dependmg on the particular drug and its intended target Where the target is a protein, the drug moiety will mclude functional groups necessary for structural interaction with protems, such as hydrogen bond g, hydrophobic-hydrophobic interactions, electrostatic interactions, etc , and will typically mclude at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups As descnbed m greater detail below, the drug moiety will also comprise a region that may be modified and/or participate in covalent linkage to the other components of the bifunctional molecule, such as the targeting moiety or linker, without substantially adversely affecting the moiety's ability to b d to its target

The drag moieties often compnse cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups Also of mterest as drug moieties are structures found among biomolecules, including peptides, sacchandes, fatty acids, steroids, puπnes, pyrrmidines, denvatives, structural analogs or combinations thereof Such compounds may be screened to identify those of mterest, where a vanety of different screemng protocols are known in the art

The drug moiety of the bifunctional molecule may be denved from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, mcludmg hbranes of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, mcludmg the preparation of randomized oligonucleotides and ohgopeptides Alternatively, hbranes of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

As such, the drug moiety may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e. a compound diversity combinatorial library. When obtained from such libraries, the drug moiety employed will have demonstrated some desirable activity in an appropriate screening assay for the activity. Combinatorial libraries, as well as methods for the production and screening, are known in the art and described in:

5,741,713; 5,734,018; 5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711; 5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698; 5,565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564; 5,440,016; 5,438,119; 5,223,409, the disclosures of which are herein incorporated by reference. Specific drugs of interest from which the drag moiety may be derived include, but are not limited to: psychopharmacological agents, such as (1) central nervous system depressants, e.g. general anesthetics (barbiturates, benzodiazepines, steroids, cyclohexanone derivatives, and miscellaneous agents), sedative- hypnotics (benzodiazepines, barbiturates, piperidinediones and triones, quinazoline derivatives, carbamates, aldehydes and derivatives, amides, acyclic ureides, benzazepines and related drugs, phenothiazines, etc.), central voluntary muscle tone modifying drags (anticonvulsants, such as hydantoins, barbiturates, oxazolidinediones, succinimides, acylureides, glutarimides, benzodiazepines, secondary and tertiary alcohols, dibenzazepine derivatives, valproic acid and derivatives, GABA analogs, etc.) , analgesics (morphine and derivatives, oripavine derivatives, morphinan derivatives, phenylpiperidines, 2,6-methane-3- benzazocaine derivatives, diphenylpropylamines and isosteres, salicylates, p-aminophenol derivatives, 5- pyrazolone derivatives, arylacetic acid derivatives, fenamates and isosteres, etc.) and antiemetics

(anticholinergics, antihistamines, antidopaminergics, etc.), (2) central nervous system stimulants, e.g. analeptics (respiratory stimulants, convulsant stimulants, psychomotor stimulants), narcotic antagonists (morphine derivatives, oripavine derivatives, 2,6-methane-3-benzoxacine derivatives, morphinan derivatives) nootropics, (3) psychopharmacologicals, e.g. anxiolytic sedatives (benzodiazepines, propanediol carbamates) antipsychotics (phenothiazine derivatives, tiuoxanfhine derivatives, other tricyclic compounds, butyrophenone derivatives and isosteres, diphenylbutylamine derivatives, substituted benzamides, arylpiperazine derivatives, indole derivatives, etc.), antidepressants (tricyclic compounds, MAO inhibitors, etc.), (4) respiratory tract drags, e.g. central antitussives (opium alkaloids and their derivatives); pharmacodynamic agents, such as (1) peripheral nervous system drags, e.g. local anesthetics (ester derivatives, amide derivatives), (2) drags acting at synaptic or neuroeffector junctional sites, e.g. cholinergic agents, cholinergic blocking agents, neuromuscular blocking agents, adrenergic agents, antiadrenergic agents, (3) smooth muscle active drugs, e.g. spasmolytics (anticholinergics, musculotropic spasmolytics), vasodilators, smooth muscle stimulants, (4) histamines and antihistamines, e.g. histamine and derivative thereof (betazole), antihistamines (Hi -antagonists, H2-antagonists), histamine metabolism drugs, (5) cardiovascular drags, e.g. cardiotonics (plant extracts, butenolides, pentadienolids, alkaloids from erythrophleum species, ionophores, -adrenoceptor stimulants, etc), antiarrhythmic drags, antihypertensive agents, antilipidemic agents (clofibric acid derivatives, nicotinic acid derivatives, hormones and analogs, antibiotics, salicylic acid and derivatives), antivaricose drags, hemostyptics, (6) blood and hemopoietic system drags, e.g. antianemia drugs, blood coagulation drags (hemostatics, anticoagulants, antithrombotics, thrombolytics, blood proteins and their fractions), (7) gastrointestinal tract drags, e.g. digestants (stomachics, choleretics), antiulcer drugs, antidiarrheal agents, (8) locally acting drags; chemotherapeutic agents, such as (1) anti-infective agents, e.g. ectoparasiticides (chlorinated hydrocarbons, pyrethins, sulfurated compounds), anthelmintics, antiprotozoal agents, antimalarial agents, antiamebic agents, antileiscmanial drags, antitrichomonal agents, antitrypanosomal agents, sulfonamides, antimycobacterial drugs, antiviral chemotherapeutics, etc., and (2) cytostatics, i.e. antineoplastic agents or cytotoxic drugs, such as alkylating agents, e.g. Mechlorethamine hydrochloride (Nitrogen Mustard, Mustargen, HN2), Cyclophosphamide (Cytovan, Endoxana), Ifosfamide (IFEX), Chlorarnbucil (Leukeran), Melphalan (Phenylalanine Mustard, L-sarcolysin, Alkeran, L-PAM), Busulfan (Myleran), Thiotepa (Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine (CeeNU, CCNU), Streptozocin (Zanosar) and the like; plant alkaloids, e.g. Vincristine (Oncovin), Vinblastine (Velban, Velbe), Paclitaxel (Taxoi), and the like; antimetabolites, e.g. Methotrexate (MTX), Mercaptopurine (Purinethol, 6-MP), Thioguanine (6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U, Ara-C), Azacitidine (Mylosar, 5-AZA) and the like; antibiotics, e.g. Dactinomycin (Actinomycin D, Cosmegen), Doxorabicin (Adriamycin), Daunorubicin (duanomycin, Cerabidine), Idarubicin (Idamycin), Bleomycin (Blenoxane), Picamycin (Mithramycin, Mithracin), Mitomycin (Mutamycin) and the like, and other anticellular proliferative agents, e.g. Hydroxyurea (Hydrea), Procarbazine (Mutalane), Dacarbazine (DTIC-Dome), Cisplatin (Platinol) Carboplatin (Paraplatin), Asparaginase (Elspar) Etoposide (VePesid, VP-16-213), Amsarcrine (AMSA, m- AMSA), Mitotane (Lysodren), Mitoxantrone (Novatrone), and the like;

Antibiotics, such as: aminoglycosides, e.g. amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin, gentamicin, isepamicin, kanamycin, micronomcin, neomycin, neti nicin, paromycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, trospectomycin; amphenicols, e.g. azidamfenicol, chloramphenicol, florfenicol, and theimaphenicol; ansamycins, e.g. rifamide, rifampin, rifamycin, rifapentine, rifaximin; β-lactams, e.g. carbacephems, carbapenems, cephalosporins, cehpamycins, monobactams, oxaphems, penicillins; lincosamides, e.g. clinamycin, lincomycin; macrolides, e.g. clarithromycin, dirthromycin, erythromycin, etc.; polypeptides, e.g. amphomycin, bacitracin, capreomycin, etc.; tetracyclines, e.g. apicycline, chlortetracycline, clomocycline, etc.; synthetic antibacterial agents, such as 2,4-diaminopyrimidines, nitrofurans, quinolones and analogs thereof, sulfonamides, sulfones; Antifungal agents, such as: polyenes, e.g. amphotericin B, candicidin, dermostatin, filipin, fungichromin, hachirnycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin; synthetic antifungals, such as allylamines, e.g. butenafine, naftifϊne, terbinafine; imidazoles, e.g. bifonazole, butoconazole, chlordantoin, chlormidazole, etc., thiocarbamates, e.g. tolciclate, triazoles, e.g. fluconazole, itraconazole, terconazole;

Anmelmintics, such as: arecoline, aspidin, aspidinol, dichlorophene, embelin, kosin, napthalene, niclosamide, pelletierine, quinacrine, alantolactone, amocarzine, amoscanate, ascaridole, bephenium, bitoscanate, carbon tetrachloride, carvacrol, cyclobendazole, diethylcarbamazine, etc.;

Antimalarials, such as: acedapsone, amodiaquin, arteether, artemether, artemisinin, artesunate, atovaquone, bebeerine, berberine, chirata, chlorguanide, chloroquine, chlorprogaunil, cinchona, cinchonidine, cinchonine, cycloguanil, gentiopicrin, halofantrine, hydroxychloroquine, mefloquine hydrochloride, 3-methylarsacetin, pamaquine, plasmocid, primaquine, pyrimethamine, quinacrine, quinidine, quinine, quinocide, quinoline, dibasic sodium arsenate;

Antiprotozoan agents, such as: acranil, tinidazole, ipronidazole, ethylstibamine, pentamidine, acetarsone, aminitrozole, anisomycin, nifuratel, tinidazole, benzidazole, suramin, and the like.

Name brand drugs of interest include, but are not limited to: Rezulin™, Lovastatin™, Enalapril™, Prozac™, Prilosec™, Lipotor™, Claritin™, Zocor™, Ciprofloxacin™, Viagra™, Crixivan™, Ritalin™, and the like.

Drug compounds of interest from which drag moieties may be derived are also listed in: Goodman & Gilman's, The Pharmacological Basis of Therapeutics (9th Ed) (Goodman et al. eds) (McGraw-Hill) (1996); and 1999 Physician's Desk Reference (1998).

Specific compounds of interest also include, but are not limited to: antineoplastic agents, as disclosed in U.S. Patent no.'s 5880161, 5877206, 5786344, 5760041, 5753668, 5698529, 5684004, 5665715, 5654484, 5624924, 5618813, 5610292, 5597831, 5530026, 5525633, 5525606, 5512678, 5508277, 5463181, 5409893, 5358952, 5318965, 5223503, 5214068, 5196424, 5109024, 5106996, 5101072, 5077404, 5071848, 5066493, 5019390, 4996229, 4996206, 4970318, 4968800, 4962114, 4927828, 4892887, 4889859, 4886790, 4882334, 4882333, 4871746, 4863955, 4849563, 4845216, 4833145, 4824955, 4785085, 476925, 4684747, 4618685, 4611066, 4550187, 4550186, 4544501, 4541956, 4532327, 4490540, 4399283, 4391982, 4383994, 4294763, 4283394, 4246411, 4214089, 4150231, 4147798, 4056673, 4029661, 4012448; psycopharmacological/psychotropic agents, as disclosed in U.S. Patent no.'s 5192799, 5036070, 4778800, 4753951, 4590180, 4690930, 4645773, 4427694, 4424202, 4440781, 5686482, 5478828, 5461062, 5387593, 5387586, 5256664, 5192799, 5120733, 5036070, 4977167, 4904663, 4788188, 4778800, 4753951, 4690930, 4645773, 4631285, 4617314, 4613600, 4590180, 4560684, 4548938, 4529727, 4459306, 4443451, 4440781, 4427694, 4424202, 4397853, 4358451, 4324787, 4314081, 4313896, 4294828, 4277476, 4267328, 4264499, 4231930, 4194009, 4188388, 4148796, 4128717, 4062858, 4031226, 4020072, 4018895, 4018779, 4013672, 3994898, 3968125, 3939152, 3928356, 3880834,3668210; cardiovascular agents, as disclosed in U.S. Patent no.'s 4966967, 5661129, 5552411, 5332737, 5389675, 5198449, 5079247, 4966967, 4874760, 4954526, 5051423, 4888335, 4853391, 4906634, 4775757, 4727072, 4542160, 4522949 4524151, 4525479, 4474804, 4520026, 4520026 5869478, 5859239, 5837702, 5807889, 5731322 5726171, 5723457, 5705523, 5696111, 5691332 5679672, 5661129, 5654294, 5646276, 5637586 5631251, 5612370, 5612323, 5574037, 5563170 5552411, 5552397, 5547966, 5482925, 5457118 5414017, 5414013, 5401758, 5393771, 5362902 5332737, 5310731, 5260444, 5223516, 5217958 5208245, 5202330, 5198449, 5189036, 5185362 5140031, 5128349, 5116861, 5079247, 5070099 5061813, 5055466, 5051423, 5036065, 5026712 5011931, 5006542, 4981843, 4977144, 49719844966967, 4959383, 4954526, 4952692, 49391374906634, 4889866, 4888335, 4883872, 4883811 4847379, 4835157, 4824831, 4780538, 47757574774239, 4771047, 4769371, 4767756, 47628374753946, 4752616, 4749715, 4738978, 4735962 4734426, 4734425, 4734424, 4730052, 4727072 4721796, 4707550, 4704382, 4703120, 46819704681882, 4670560, 4670453, 4668787, 46633374663336, 4661506, 4656267, 4656185, 46543574654356, 4654355, 4654335, 4652578, 4652576 4650874, 4650797, 4649139, 4647585, 4647573 4647565, 4647561, 4645836, 4639461, 4638012 4638011, 4632931, 4631283, 4628095, 46265484614825, 4611007,4611006,4611005,4609671 4608386, 4607049, 4607048, 4595692, 4593042 4593029, 4591603, 4588743, 4588742, 4588741 4582854, 4575512, 4568762, 4560698, 45567394556675, 4555571, 4555570, 4555523, 45501204542160, 4542157, 4542156, 4542155, 4542151 4537981, 4537904, 4536514, 4536513, 4533673 4526901, 4526900, 4525479, 4524151, 45229494521539, 4520026, 4517188, 4482562, 4474804_; 4474803, 4472411, 4466979, 4463015, 44566174456616, 4456615, 4418076, 4416896, 4252815 4220594, 4190587, 4177280, 4164586, 41512974145443, 4143054, 4123550, 4083968, 40768344064259, 4064258, 4064257, 4058620, 4001421 3993639, 3991057, 3982010, 3980652, 39681173959296, 3951950, 3933834, 3925369, 3923818_: 3898210,

3897442, 3897441, 3886157, 3883540 3873715, 3867383, 3873715, 3867383, 36912163624126; antimicrobial agents as disclosed in U.S. Patentno.^'s 5902594, 5874476, 5874436, 5859027,

5856320, 5854242, 5811091, 5786350 5783177, 5773469, 5762919, 5753715, 5741526 5709870,

5707990, 5696117, 5684042, 5683709 5656591, 5643971, 5643950, 5610196, 5608056 5604262, 5595742, 5576341, 5554373, 5541233 5534546, 5534508, 5514715, 5508417, 5464832 5428073, 5428016, 5424396, 5399553, 5391544 5385902, 5359066, 5356803, 5354862, 5346913 5302592, 5288693, 5266567, 5254685, 5252745 5209930, 5196441, 5190961, 5175160, 5157051 5096700, 5093342, 5089251, 5073570, 5061702_; 5037809, 5036077, 5010109, 4970226, 49161564888434, 4870093, 4855318, 4784991, 47465044686221, 4599228, 4552882, 4492700, 44890984489085, 4487776, 4479953, 4477448, 44748074470994, 4370484, 4337199, 4311709, 4308283 4304910, 4260634, 4233311, 4215131, 41661224141981, 4130664, 4089977, 4089900, 4069341 4055655, 4049665, 4044139, 4002775, 3991201, 3966968, 3954868, 3936393, 3917476, 3915889, 3867548, 3865748, 3867548, 3865748, 3783160, 3764676, 3764677; anti-inflammatory agents as disclosed in U.S. Patent no.'s 5872109, 5837735, 5827837, 5821250, 5814648, 5780026, 5776946, 5760002, 5750543, 5741798, 5739279, 5733939, 5723481, 5716967, 5688949, 5686488, 5686471, 5686434, 5684204, 5684041, 5684031, 5684002, 5677318, 5674891, 5672620 5665752, 5656661, 5635516, 5631283, 5622948, 5618835, 5607959, 5593980, 5593960, 5580888, 5552424, 55524225516764, 5510361, 5508026, 5500417, 5498405, 5494927 : 547687654729735470885, 5470842, 5464856, 54648495462952, 5459151, 5451686, 54440435436265, 5432181, RE034918, 5393756, 5380738, 5376670, 5360811, 5354768, 5348957, 5347029, 5340815, 5338753, 5324648, 5319099, 5318971, 5312821, 5302597, 5298633, 5298522, 5298498, 5290800, 5290788, 5284949, 5280045, 5270319, 5266562, 5256680, 5250700, 5250552, 5248682, 5244917, 5240929, 5234939, 5234937, 5232939, 5225571, 5225418, 5220025, 5212189, 5212172, 5208250, 5204365, 5202350, 5196431, 5191084, 5187175, 5185326, 5183906, 5177079, 5171864, 5169963, 5155122, 5143929, 5143928, 5143927, 5124455, 5124347, 5114958, 5112846, 5104656, 5098613, 5095037, 5095019, 5086064, 5081261, 5081147, 5081126, 5075330, 5066668, 5059602, 5043457, 5037835, 5037811,

5036088,5013850,5013751,5013736,500654,4992448,4992447,4988733,4988728,4981865,4962119, 4959378, 4954519, 4945099, 4942236, 4931457, 4927835, 4912248, 4910192, 4904786, 4904685, 4904674, 4904671, 4897397, 4895953, 4891370, 4870210, 4859686, 4857644, 4853392, 4851412, 4847303, 4847290, 4845242, 4835166, 4826990, 4803216, 4801598, 4791129, 4788205, 4778818, 4775679, 4772703, 4767776, 4764525, 4760051, 4748153, 4725616, 4721712, 4713393, 4708966, 4695571, 4686235, 4686224, 4680298, 4678802, 4652564, 4644005, 4632923, 4629793, 4614741, 4599360, 4596828, 4595694, 4595686, 4594357, 4585755, 4579866, 4578390, 4569942, 4567201, 4563476, 4559348, 4558067, 4556672, 4556669, 4539326, 4537903, 4536503, 4518608, 4514415, 4512990, 4501755, 4495197, 4493839, 4465687, 4440779, 4440763, 4435420, 4412995, 4400534, 4355034, 4335141, 4322420, 4275064, 4244963, 4235908, 4234593, 4226887, 4201778, 4181720, 4173650, 4173634, 4145444, 4128664, 4125612, 4124726, 4124707, 4117135, 4027031, 4024284, 4021553, 4021550, 4018923, 4012527, 4011326, 3998970, 3998954, 3993763, 3991212, 3984405, 3978227, 3978219, 3978202, 3975543, 3968224, 3959368, 3949082, 3949081, 3947475, 3936450, 3934018, 3930005, 3857955, 3856962, 3821377, 3821401, 3789121, 3789123, 3726978, 3694471, 3691214,3678169,3624216; immunosuppressive agents, as disclosed inU.S. Patentno.'s 4450159, 4450159, 5905085,5883119, 5880280, 5877184, 5874594, 5843452, 5817672, 5817661, 5817660, 5801193, 5776974, 5763478, 5739169, 5723466, 5719176, 5696156, 5695753, 5693648, 5693645, 5691346, 5686469, 5686424, 5679705, 5679640, 5670504, 5665774, 5665772, 5648376, 5639455, 5633277, 5624930, 5622970, 5605903, 5604229, 5574041, 5565560, 5550233, 5545734, 5540931, 5532248, 5527820, 5516797, 5514688, 5512687, 5506233, 5506228, 5494895, 5484788, 5470857, 5464615, 5432183, 5431896, 5385918, 5349061, 5344925, 5330993, 5308837, 5290783, 5290772, 5284877, 5284840, 5273979, 5262533, 5260300, 5252732, 5250678, 5247076, 5244896, 5238689, 5219884, 5208241, 5208228, 5202332, 5192773, 5189042, 5169851, 5162334, 5151413, 5149701, 5147877, 5143918, 5138051, 5093338, 5091389, 5068323, 5068247, 5064835, 5061728, 5055290, 4981792, 4810692, 4410696, 4346096, 4342769, 4317825, 4256766, 4180588, 4000275, 3759921; analgesic agents, as disclosed in U.S. Patentno.'s 5292736, 5688825, 5554789, 5455230, 5292736, 5298522, 5216165, 5438064, 5204365, 5017578, 4906655, 4906655, 4994450, 4749792, 4980365, 4794110, 4670541, 4737493, 4622326, 4536512, 4719231, 4533671, 4552866, 4539312, 4569942, 4681879, 4511724, 4556672, 4721712, 4474806, 4595686, 4440779, 4434175, 4608374, 4395402, 4400534, 4374139, 4361583, 4252816, 4251530, 5874459, 5688825, 5554789, 5455230, 5438064, 5298522, 5216165, 5204365, 5030639, 5017578, 5008264, 4994450, 4980365, 4906655, 4847290, 4844907, 4794110, 4791129, 4774256, 4749792, 4737493, 4721712, 4719231, 4681879, 4670541, 4667039, 4658037, 4634708, 4623648, 4622326, 4608374, 4595686, 4594188, 4569942, 4556672, 4552866, 4539312, 4536512, 4533671, 4511724, 4440779, 4434175, 4400534, 4395402, 4391827, 4374139,4361583,4322420,4306097,4252816,4251530,4244955,4232018,4209520,41645144147872, 4133819, 4124713, 4117012, 4064272, 4022836, 3966944; cholinergic agents, as disclosed in U.S. Patentno.'s 5219872, 5219873, 5073560, 5073560, 5346911, 5424301, 5073560, 5219872, 4900748, 4786648, 4798841, 4782071, 4710508, 5482938, 5464842, 5378723, 5346911, 5318978, 5219873, 5219872, 5084281, 5073560, 5002955, 4988710, 4900748, 4798841, 4786648, 4782071, 4745123, 4710508; adrenergicagents, as disclosedinU.S. Patentno.'s 5091528, 5091528, 4835157, 5708015, 5594027, 5580892, 5576332, 5510376, 5482961, 5334601, 5202347, 5135926, 5116867, 5091528, 5017618, 4835157, 4829086, 4579867, 4568679, 4469690, 4395559, 4381309, 4363808, 4343800, 4329289, 4314943, 4311708, 4304721, 4296117, 4285873, 4281189, 4278608, 4247710, 4145550, 4145425, 4139535, 4082843, 4011321, 4001421, 3982010, 3940407, 3852468, 3832470; antihistamine agents, as disclosed in U.S. Patentno.'s 5874479, 5863938, 5856364, 5770612, 5702688, 5674912, 5663208, 5658957, 5652274, 5648380, 5646190, 5641814, 5633285, 5614561, 5602183, 4923892, 4782058, 4393210, 4180583, 3965257, 3946022, 3931197; steroidal agents, as disclosed in U.S. Patentno.'s 5863538, 5855907, 5855866, 5780592, 5776427, 5651987, 5346887, 5256408, 5252319, 5209926, 4996335, 4927807, 4910192, 4710495, 4049805, 4004005, 3670079, 3608076, 5892028, 5888995, 5883087, 5880115, 5869475, 5866558, 5861390, 5861388, 5854235, 5837698, 5834452, 5830886, 5792758, 5792757, 5763361, 5744462, 5741787, 5741786, 5733899, 5731345, 5723638, 5721226, 5712264, 5712263, 5710144, 5707984, 5705494, 5700793, 5698720, 5698545, 5696106, 5677293, 5674861, 5661141, 5656621, 5646136, 5637691, 5616574, 5614514, 5604215, 5604213, 5599807, 5585482, 5565588, 5563259, 5563131, 5561124, 5556845, 5547949, 5536714, 5527806, 5506354, 5506221, 5494907, 5491136, 5478956, 5426179, 5422262, 5391776, 5382661, 5380841, 5380840, 5380839, 5373095, 5371078, 5352809, 5344827, 5344826, 5338837, 5336686, 5292906, 5292878, 5281587, 5272140, 5244886, 5236912, 5232915, 5219879, 5218109, 5215972, 5212166, 5206415, 5194602, 5166201, 5166055, 5126488, 5116829, 5108996, 5099037, 5096892, 5093502, 5086047, 5084450, 5082835, 5081114, 5053404, 5041433, 5041432, 5034548, 5032586, 5026882, 4996335, 4975537, 4970205, 4954446, 4950428, 4946834, 4937237, 4921846, 4920099, 4910226, 4900725, 4892867, 4888336, 4885280, 4882322, 4882319, 4882315, 4874855, 4868167, 4865767, 4861875, 4861765, 4861763, 4847014, 4774236, 4753932, 4711856, 4710495, 4701450, 4701449, 4689410, 4680290, 4670551, 4664850, 4659516, 4647410, 4634695, 4634693, 4588530, 4567000, 4560557, 4558041, 4552871, 4552868, 4541956, 4519946, 4515787, 4512986, 4502989, 4495102; the disclosures of which are herein incorporated by reference. The drug moiety of the bifunctional molecule may be the whole compound or a derivative thereof, e.g. a binding fragment or portion thereof, that retains its affinity and specificity for the target of interest, and therefor its desired activity, while having a linkage site for covalent bonding to the targeting moiety or linker.

Pharmacokinetic Modulating Moiety: Z

Z is a pharmacokinetic modulating moiety that is a ligand for a biological entity endogenous to the host to which the bifunctional molecule is administered, where binding of the modulating moiety to this biological entity results in modulation of at least one pharmacokinetic property of the drag moiety of the bifunctional molecule, as compared to the drug moiety's corresponding free drug control. In many embodiments, this biological entity to which the modulating moiety of the bifunctional molecule binds is a protein, e.g. an intracellular or extracellular protein. As such, in many embodiments this biological entity is properly referred to the endogenous pharmacokinetic modulating protein.

The binding interaction between the modulating moiety of the bifunctional molecule and the endogenous biological entity, e.g. endogenous pharmacokinetic modulating protein, is non-covalent, such that no covalent bonds are produced between the bifunctional molecule and the pharmacokinetic modulating protein upon binding of the two entities. As the pharmacokinetic modulating moiety of the bifunctional molecule is a small molecule, it generally has a molecular weight of at least about 50 D, usually at least about 100 D, where the molecular weight may be as high as 500 D or higher, but will usually not exceed about 2000 D. In certain embodiments, the pharmacokinetic modulating moiety, in the context of the bifunctional molecule, has substantially no pharmacological activity at its effective concentration beyond binding to its corresponding endogenous pharmacokinetic modulating protein, i.e. it does not directly cause a pharmacokinetic modulating protein-mediated pharmacological event to occur upon binding at its effective concentration to the pharmacokinetic modulating protein, where a pharmacokinetic modulating protein mediated pharmacological event is a pharmacologically relevant event which is directly modulated by the pharmacokinetic modulating protein in the absence of the subject bifunctional molecules. In other certain embodiments, the modulating moiety may have some pharmacological activity, where this pharmacological activity does not adversely effect the host to the extent that the therapy in which the bifunctional molecule is employed places the host in a worst condition than prior to the therapy. In other words, pharmacological activity in the modulating moiety may be tolerated in these embodiments to the extent that any consequences of such activity, if any, are outweighed by the benefits provided by the bifunctional molecule. As used herein, pharmacological event is an event that is distinct from a biochemical event (e.g. inhibition a prolyl isomerase activity) or a biological event (e.g. inducement of a cell to express new genes).

The pharmacokinetic modulating protein to which the modulating moiety of the bifunctional molecule binds may be any protein that is present in the host at the time the bifunctional molecule is introduced to the host, i.e. the pharmacokinetic modulating protein is one that is endogenous to the host. The pharmacokinetic modulating protein may or may not have one or more modified residues, e.g. residues that are glycosylated, such that it may or may not be a glycoprotein. Furthermore, the pharmacokinetic modulating protein to which the bifimctional molecule binds via the pharmacokinetic modulating moiety may or may not be part of a complex or structure of a plurality of biological molecules, e.g. lipids, where such complexes or structures may include lipoproteins, lipid bilayers, and the like. However, in many embodiments, the pharmacokinetic modulating protein to which the bifunctional molecule binds will be by itself, i.e. will not be part of a larger structure of a plurality of biological molecules. Though the pharmacokinetic modulating protein may be a protein that is not native to the host but has been introduced at some time prior to introduction of the bifunctional molecule, e.g. through prior administration of the protein or a nucleic acid composition encoding the same, such as through gene therapy, the pharmacokinetic modulating protein will, in many embodiments, be a protein that is native to and naturally expressed by at least some of the host's cells, i.e. a naturally occurring protein in the host. The pharmacokinetic modulating protein is a protein that is present in the region of host occupied by the drag target. As such, where the drug target is an intracellular drag target, the pharmacokinetic protein will be an intracellular protein present in the cell comprising the target, typically expressed in the cell comprising the target, i.e. the pharmacokinetic modulating protein and target are co-expressed in the same cell. Likewise, where the drug target is an extracellular drug target, the pharmacokinetic modulating protein will be an extracellular protein that is found in the vicinity of the target.

Although not a requirement in certain embodiments, in many preferred embodiments the pharmacokinetic modulating protein is one that is present in the host in sufficient quantities such that, upon binding of at least a portion of pharmacokinetic modulating protein present in the host to the bifunctional molecule, adverse pharmacological effects do not occur. In other words, the pharmacokinetic modulating protein in these preferred embodiments is one in which its native and desirable biological activity, if any, is not dimimshed by an unacceptable amount following binding of the portion of the pharmacokinetic modulating protein population to the bifunctional molecule. The amount of diminished activity of the pharmacokinetic modulating protein that is acceptable in a given situation is determined with respect to the condition being treated in view of the benefits of treatment versus the reduction of overall pharmacokinetic modulating protein activity, if any. In certain situations, a large decrease in overall pharmacokinetic modulating protein activity may be acceptable, e g where the pharmacokmetic modulating protem activity aggravates the condition being treated

The specific pharmacokmetic modulating protein to which the modulating moiety of the subject bifunctional molecule b ds may vary greatly dependmg on the desired modulation of the one or more pharmacokmetic properties or parameters of mterest For example, where one wishes to modulated the half- life, hepatic first-pass metabolism, , or volume of distribution, intracellular protems are often of mterest, where representative intracellular proteins of mterest include peptidyl-prolyl isomerases, e g FKBPs and cyclophilins, ubiquitously expressed molecular chaperones, e g Heat Shock Protein 90 (Hsp90), steroid hormone receptors, e g estrogen receptors, glucocorticoid receptors, androgen receptors, retinoic acid bmdmg protem, cytoskeletal proteins, such as tubulrn and actin, etc Of particular mterest as intracellular pharmacokmetic modulating protems are cis-trans peptidyl-prolyl isomerases which interact with many proteins because of their chaperonin/isomerase activity, e g FKBPs and cyclophilins Peptidyl-prolyl isomerases of mterest mclude FKBPs A number of different FKBPs are known in the art, and mclude those described m Sabatini et al , Mol Neurobiol (Oct 1997) 15 223-239, Marks, Physiol Rev (July 1996) 76 631-649, Kay, Biochem J (March, 1996) 314 361-385, Braun et al , FASEB J (January 1995) 9 63-72, Fruman et al, FASEB J (Apnl 1994) 8 391-400, and Hacker et al , Mol Microbiol (November 1993) 10 445-456 FKBPs of mterest mclude FKBP 12, FKBP 52, FKBP 14 6 (descnbed m U S Patent No 5,525,523, the disclosure of which is herem mcorporated by reference), FKBP 12 6 (described in U S Patent No 5,457,182 the disclosure of which is herem mcorporated by reference), FKBP 13 (described m U S Patent No 5,498,597, the disclosure of which is herem mcorporated by reference), and HCB (descnbed in U S Patent No 5,196,352 the disclosure of which is herein incorporated by reference), where FKBP 12 and FKBP 52 are of particular mterest as mtracellular pharmacokmetic modulating protems Also of specific mterest as intracellular pharmacokinetic modulating protems are cyclophilins A number of cyclophilins are known m the art and are descnbed m Trandmh et al , FASEB J (December 1992) 6 3410-3420, Harding et al , Transplantation (August 1988) 46 29S-35S Specific cyclophilins of interest as mtracellular pharmacokmetic modulating protems include cyclophilin A, B, C, D, E, and the like, where cyclophilin A is of particular mterest

Instead of bemg an intracellular protem, in certain embodiments the endogenous pharmacokmetic modulating protem is an extracellular or serum protem, e g where extracellular pharmacokmetic modulating proteins find use in the modulating of half-life, volume of distnbution, and degree of albumin bindmg or albumin bmdmg effect Serum pharmacokmetic modulating protems of particular mterest are those that are relatively abundant m the serum of the host and meet the above cntena for suitable endogenous pharmacokmetic modulating protems By relatively abundant is meant that the concentration of the serum pharmacokmetic modulating protem is at least about 1 ng/ml, usually at least about 10 μg/ml and more usually at least about 15 μg/ml Specific serum protems of mterest as pharmacokinetic modulating protems mclude albumin, Vitamin A bmdmg protems and Vitamin D bmdmg protems, β-2 macroglobultn, α-1 acid glycoprotein, with albumin being a particularly preferred pharmacokinetic modulating protein in many embodiments.

The Z moiety of the subject bifunctional molecules will therefore be chosen in view of the endogenous pharmacokinetic modulating protein that is to be used to bind to the bifunctional molecule and thereby achieve the desired pharmacokinetic property modulation. As such, the Z moiety may be a number of different ligands, depending on the particular endogenous pharmacokinetic modulating protein to which it is intended to bind. In many preferred embodiments, the Z moiety has an affinity for its pharmacokinetic modulating protein of at least about 10^"4 M, usually at least about 10^"6 molar and more usually at least about

10^"8 M, where in many embodiments the Z moiety has an affinity for its pharmacokinetic modulating protein

-9 -12 of between about 10 and 10 M. The Z moiety portion of the bifunctional molecule should also be specific for the pharmacokinetic modulating protein in the context of its binding activity when present in the bifunctional molecule, in that it does not significantly bind or substantially affect non- pharmacokinetic modulating proteins when it is present in the bifunctional molecule.

Representative ligands capable of serving as the Z moiety of the bifunctional molecule include ligands for intracellular proteins, such as: peptidyl-prolyl isomerase ligands, e.g. FK506, rapamycin, cyclosporin A and the like; Hsp90 ligands, e.g. geldanamycin; steroid hormone receptor ligands, e.g. naturally occurring steroid hormones, such as estrogen, progestin, testosterone, and the like, as well as synthetic derivatives and mirnetics thereof, particularly those which bind with high specificity and affinity but do not activate their respective receptors; small molecules that bind to cytoskeletal proteins, e.g. antimitotic agents, such as taxanes, colchicine, colcemid, nocadozole, vinblastine, and vincristine, actin binding agents, such as cytochalasin, latrunculin, phalloidin, and the like.

As mentioned above, in certain preferred embodiments the intracellular pharmacokinetic modulating proteins are members of the peptidyl-prolyl isomerase family, particularly the FKBP and cyclophilin subsets of this family. Where peptidyl-prolyl isomerase pharmacokinetic modulating proteins are employed, the bifunctional molecule/peptidyl-prolyl isomerase complex will preferably not substantially bind to the natural peptidyl-prolyl isomerase/ligand target calcineurin so as to result in significant immunosuppression. A variety of ligands are known that bind to FKBPs and may be used in the subject invention. The ligands should specifically bind to an FKBP and have an affinity for the FKBP that is between about 10^" and 10^"

M. Of interest are both naturally occurring FKBP ligands, including FK506 and rapamycin. Also of interest are synthetic FKBP ligands, including those described in U.S. Patent Nos.: 5,665,774; 5,622,970; 5,516,797;

5,614,547; and 5,403,833, the disclosures of which are herein incorporated by reference.

Also of interest in this particular set of preferred embodiments are cyclophilin ligands, where such

-6 -9 ligands should specifically bind to cyclophilin with an affinity that is between about 10 and 10 M. A variety of ligands that bind to cyclophilins are also known, where such ligands include the naturally occurring cyclosporins, such as cyclosporin A, as well as synthetic derivatives and mimetics thereof, including those described in U.S. Patent Nos.: 5,401,649; 5,318,901; 5,236,899; 5,227,467; 5,214,130; 5,122,511, 5,116,816, 5,089,390, 5,079,341, 5,017,597, 4,940,719, 4,914,188, 4,885,276, 4,798,823, 4,771,122, 4,703,033, 4,554,351 , 4,396,542, 4,289,851, 4,288,431, 4,220,61 and 4,210,581, the disclosures of which are herem incorporated by reference

Representative ligands for use as the Z moiety m the bifunctional molecule also include ligands that bmd to extracellular pharmacokmetic modulating protems Such ligands should specifically bmd to their

_4 respective extracellular pharmacokmetic modulating protem with an affinity of at least about 10 M

Ligands of interest for use m bmdmg to extracellular pharmacokinetic modulating protems mclude albumin ligands, such as arachidonate, bilirubin, hemin, aspirin, lbuprofen, para-ammo salicylic acid, myristylate, plamitate, lmoleate, warfarin, sulfisoxazole, chlorpromazine, etc , α-1 acid glycoprotem ligands, e g small neutral or basic molecules, e g propanolol, chlorpromazine, dipyπmadole, metoclopramide, apπndme, verapamil, and the like, Vitamin A and denvatives thereof, Vitamin D and derivatives thereof, and the like

Linking Moiety L

The Z and X moieties of the bifunctional molecule are jomed together through linking moiety L, where L may be either a bond or a linking group Where linking groups are employed, such groups are chosen to provide for covalent attachment of the drag and ligand moieties through the linking group, as well as the desired structural relationship of the bifunctional molecule with respect to its intended pharmacokmetic modulating protein Linking groups of mterest may vary widely dependmg on the nature of the drug and ligand moieties The linking group, when present, should preferably be biologically inert Appropriate linkers can readily be identified usmg the affinity, specificity or selectivity assays descnbed supra A variety of linking groups are known to those of skill m the art and find use in the subject bifunctional molecules The linker groups should be sufficiently small so as to provide a bifunctional molecule havmg the overall size charactenstics as descnbed above, the size of the linker group, when present, is generally at least about 50 daltons, usually at least about 100 daltons and may be as large as 1000 daltons or larger, but generally will not exceed about 500 daltons and usually will not exceed about 300 daltons Generally, such linkers will compnse a spacer group terminated at either end with a reactive functionality capable of covalently bondmg to the drag or ligand moieties Spacer groups of interest mclude aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may possibly contam heteroatoms Spacer groups may also be compnsed of ligands that bmd to metals such that the presence of a metal ion coordmates two or more ligands to form a complex Specific spacer elements mclude 1,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenedιanune-N,N-dιacetιc acid, l,r-ethylenebis(5-oxo-3-pyrrolidmecarboxylic acid), 4,4'- efhylenedipipendine Potential reactive functionalities include nucleophihc functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, lsocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or bmdmg to metals Specific examples mclude primary and secondary amines, hydroxamic acids, N- hydroxysuccinimidyl esters, N-hydroxysuccmimidyl carbonates, oxycarbonylumdazoles, mtrophenylesters, tπfluoroethyl esters, glycidyl ethers, vmylsulfones, and maleimides Specific linker groups that may find use m the subject bifunctional molecules mclude heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p-azιdosalιcylammo)butyl]-3 '-[2'-pyπdyldιthιo]propιonamιd), bis-sulfosuccmimidyl suberate, dnnethyladipimidate, disuccmimidyltartrate, N- -maleimidobutyryloxysuccmimide ester, N-hydroxy sulfosuccmrmιdyl-4-azιdobenzoate, N-succmmiidyl [4-azιdophenyl]-l,3'-dιthιopropιonate, N-succmimidyl [4-ιodoacetyl]ammobenzoate, glutaraldehyde, and succimmidyl 4- [N-maleimidomethyl] cyclohexane- 1- carboxylate, 3-(2-pyndyldιthιo)propιomc acid N-hydroxysuccimmide ester (SPDP), 4-(N- maleιmιdomethyl)-cyclohexane-l-carboxylιc acid N-hydroxysuccimmide ester (SMCC), and the like

METHODS OF MAKING BIFUNCTIONAL MOLECULES

The bifunctional molecules of the subject invention may be prepared usmg any convenient methodology In many embodiments of the subject invention, the invention is used to modulate the pharmacokmetic properties of an identified and at least partially characterized small molecule drug Generally, a small molecule drag of interest is first identified The drug may be a previously identified biologically active agent or compound having the desired target bmdmg activity, or one that has been newly discovered usmg one or more drag discovery techniques The bifunctional molecule is then generally produced from the drag usmg a rational or combmatonal approach

In a rational approach, the bifunctional molecules are constructed from their individual components, e g pharmacokmetic modulating moiety, linker and drag The components can be covalentiy bonded to one another through functional groups, as is known in the art, where such functional groups may be present on the components or introduced onto the components usmg one or more steps, e g oxidation reactions, reduction reactions, cleavage reactions and the like Functional groups that may be used m covalentiy bonding the components together to produce the bifunctional molecule mclude hydroxy, sulfhydryl, ammo, and the like The particular portion of the different components that are modified to provide for covalent linkage will be chosen so as not to substantially adversely interfere with that components desired bmdmg activity, e g for the drug moiety, a region that does not affect the target bmdmg activity will be modified, such that a sufficient amount of the desired drag activity is preserved Where necessary and/or desired, certain moieties on the components may be protected usmg blockmg groups, as is known m the art, see, e g Green & Wuts, Protective Groups m Organic Synthesis (John Wiley & Sons) (1991)

The above component approach to production of the bifunctional molecule is best suited for situations where the crystal structures of the pharmacokmetic modulating protem, the pharmacokmetic modulating moiety, the drag and the target are known, such that molecular modelmg can be used to determine the optimal linker size, if any, to be employed to jom the different components Alternatively, the bifunctional molecule can be produced usmg combmatonal methods to produce large hbranes of potential bifunctional molecules which may then be screened for identification of a bifunctional molecule with the pharmacokmetic profile Methods for producmg and screening combmatonal libraries of molecules include: 5,741,713; 5,734,018; 5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711; 5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698; 5,565,324; 5,549,974; 5,545,568; 5,541,061 ; 5,525,735; 5,463,564; 5,440,016; 5,438,119; 5,223,409, the disclosures of which are herein incorporated by reference. Alternatively, the bifunctional molecule may be produced using medicinal chemistry and known structure-activity relationships for the targeting moiety and the drug. In particular, this approach will provide insight as to where to join the two moieties to the linker.

METHODS FOR MAKING BIFUNCTIONAL MOLECULES FOR PEPTIDYL-PROLYL ISOMERASE PHARMACOKINEΗC MODULATING PROTEINS

As mentioned above, one class of preferred embodiments of the subject invention are those embodiments in which the bifunctional molecules comprise a pharmacokinetic modulating moiety that binds to an intracellular protein. In these embodiments, of particular interest are those bifunctional molecules in which the pharmacokinetic modulating moiety specifically binds to endogenous peptidyl-prolyl isomerase pharmacokinetic modulating proteins present in the host into which the bifunctional molecule is introduced.

Thus, bifunctional molecules of interest include those in which the endogenous pharmacokinetic modulating protein is either an FKBP or a cyclophilin.

In preparing bifunctional molecules from FK506, a suitable attachment site on the FK506 structure is identified, modified as necessary, and then covalentiy attached to the linker or drug moiety. The structure of FK506 (also known as tacrolimus) is:

The site to which the linker/drag moiety is covalentiy attached is one that, upon covalent attachmen does not ablate the affinity and/or specificity of FK506 for its FKBP pharmacokinetic modulating protein, e.g. FKBP 12 or FKBP 52. As such, positions suitable for use as covalent linkage sites include atoms located between carbon 15 and carbon 25 and the substituents attached to these atoms. For example, oxidation of the allyl group or oxidation of the carbon 18 methylene group; modification of the carbon 22 ketone or the carbon 24 hydroxyl group or alkylation at carbon 21 or carbon 23; as well as the secondary hydroxyl group located on the cyclohexyl ring (carbon 32); are potential specific covalent linkage sites. With FK506, depending on the drug moiety and/or linker to be attached, it may be desirable to introduce one or more functional moieties onto the FK506 stracture. Functional moieties of interest that may be introduced include: hydroxyl groups, amino groups, carboxyl groups, aldehydes, carbonates, carbamates, azides, thiols, and esters, etc. Such groups may be introduced using known protocols, such as oxidation reactions, reduction reactions, cleavage reactions and the like, with or without the use of one or more blocking groups to prevent unwanted side reactions.

In some instances, it is desirable to covalentiy attach the drug moiety directly to FK506, often activated FK506. In such instances, the reactive functional group(s) introduced onto the FK506 stracture will depend primarily on the nature of the drag moiety to be attached. Thus, for peptidic drag moieties, specific pairings of interest include: FK506 carbonates for reacting with amino groups of peptides; FK506 carboxylic acids for reacting with amino groups of peptides; FK506 amines for reacting with carboxylic acid groups of peptides; FK506 maleimide for reacting with thiol groups of peptides; and the like. Alternatively, where the drug moiety is a steroid, potential pairings of interest include: FK506 N-hydroxysuccinimidyl carbonate and partner amine; FK506 aldehyde and partner amine; FK506 aldehyde and partner hydrazide; FK506 hydroxy group and partner carboxylic acid OR alkyl halide; FK506 thiol and partner maleimide and the like.

Following introduction of the reactive functional group(s) onto the FK506 structure, the activated FK506 is then combined with the drug moiety/linker under conditions sufficient for covalent bonding to occur.

Another embodiment of particular interest are bifunctional molecules of cyclosporin A or analogs thereof. The structure of cyclosporin A is:

As with the FK506 bifunctional molecules, the cyclosporin A will be conjugated to the drug moiety in a manner such that cyclosporin A does not substantially lose its affinity for cyclophilin. Preferred positions on the cyclosporin A structure that may serve as covalent linkage sites include: residues 4, 5, 6, 7, 8; while less preferred but still possible residues include: 1,2, 3, 9, 10 and 11. Where necessary, reactive functionalities may be introduced onto the cyclosporin structure, where such functionalities include: hydroxyl groups, amino groups, carboxyl groups, aldehydes, carbonates, carbamates, azides, thiols, and esters, etc., with the particular functionality of interest being chosen with respect to the specific linker or drug moiety to be attached.

SCREENING BIFUNCTIONAL COMPOUNDS

The resultant bifunctional molecules are then screened for those molecules that exhibit the desired pharmacokinetic profile, e.g. desired half-life, desired hepatic first-pass metabolism, desired volume of distribution and/or desired degree of albumin binding. Any convenient screening assay may be employed, where the particular screening assay may be one known to those of skill in the art or one developed in view of the specific molecule and property being studied. Suitable screening assays for identifying those bifunctional molecules that exhibit at least a modulated pharmacokinetic property as compared to a free drag control, e.g. for evaluating drag half-life, volume of distribution, first-pass metabolism and degree of albumin binding, are known to those of skill in the art and need not be described further here.

SPECIFIC IMPROVEMENTS AS COMPARED TO FREE DRUG

As mentioned above, the bifunctional molecules of the subject invention exhibit at least one modulated pharmacokinetic property upon aiiministration of to a host as compared to their corresponding free drug, i.e. a free drug control. In other words, at least one of the pharmacokinetic properties of the subject bifunctional molecules differ from that of the corresponding free drag. Specific pharmacokinetic properties that may differ in the subject bifunctional molecules are drug half-life, drug first-pass metabolism, drug volume of distribution and degree of drag binding to a serum protein, e.g. albumin. In certain embodiments, the above improvements are achieved through the formation of binary or tripartite complexes, as described in application serial no. 09/316,932 entitled Bifunctional Molecules and Therapies Based Thereon, the disclosure of which is herein incorporated by reference.

In those embodiments where the half-life of a drug is modulated, e.g. prolonged, by incorporating it into a bifunctional molecule, the modulating moiety of the bifunctional molecule may be a ligand for an intracellular or extracellular protein, as described above. In these embodiments, the modulating moiety is a ligand for an intracellular or extracellular protein that will serve to, when bound to the bifunctional molecule, i.e. when present as a binary complex with the bifunctional molecule, protect or shield the drug moiety from biotransformation or excretion, e.g. by the liver or the kidney. These intracellular pharmacokinetic modulating proteins are generally proteins that are highly abundant in cells and thus the intracellular space of the host, e.g. the interior or erythrocytes, etc. These proteins serve as a protective reservoir for the bifunctional molecule and drug component thereof, thereby extending the half-life of the drug. Representative intracellular proteins for which the modulating moiety may serve as a ligand in this embodiment mclude the peptidyl prolyl isomerases, heat shock protems (hsp's), tubulms, and the like, where additional potential mtracellular pharmacokmetic modulating protems are descnbed supra Extracellular pharmacokmetic modulating proteins are also of interest, where the protems should be long-lived protems that impart their long half-life to the bifunctional molecule and the drug component thereof upon formation of a binary complex with the bifunctional molecule Representative extracellular pharmacokmetic modulating protems of mterest mclude albumin, αl-acid glycoprotem, and the like In the above embodiments, the half-life the drag component of the bifunctional molecule is generally prolonged as compared to the corresponding free drag control by a factor of about 1 5, usually by a factor of about 3 and more usually by a factor of about 6 In those embodiments where it is desired to modulate, and specifically, reduce the hepatic first-pass metabolism of a drag, the bifunctional molecule generally mcludes a ligand for an abundant mtracellular protein, and more specifically an abundant blood cell mtracellular protem, and m certain embodiments an abundant red blood cell or ervthrocyte mtracellular protem The binary complex formed between the bifunctional molecule and the mtracellular pharmacokmetic modulating protein should be transient, e g lasting on average from about 1 to 5, usually 1 to 3 rmnutes and in many embodiments around two minutes While a vanety of mtracellular protems are of mterest, m many embodiments, peptidyl prolyl isomerases, e g FKBP and cyclophilin, are prefened as the pharmacokmetic modulating protem In these embodiments, the amount of drug that is eliminated via hepatic first-pass metabolism is decreased by a factor of at least about 2 , usually by at least about 5 and more usually by at least about 10 In those embodiments where it is desired to modulate the volume of distnbution of a drug, the bifunctional molecule may include a ligand for a intracellular or extracellular pharmacokmetic modulating protein Thus, where one wishes to change the distnbution of a drug so that the drug is distnbuted m greater amounts m the mtracellular space as compared to the extracellular space, the modulating moiety of the bifunctional molecule is a ligand for an mtracellular pharmacokinetic modulating protem, e g peptidyl prolyl isomerase, where suitable ligands are descnbed above Alternatively, where it is desired to enhance the amount of drag located m the extracellular space as compared to that which is present in the mtracellular space, the ligand of the bifunctional molecule is a ligand for an extracellular protein, e g albumin, where representative ligands and extracellular pharmacokinetic modulating protems are further descnbed supra

In certain embodiments, a drag is administered as a bifunctional molecule m order to reduce the degree of blood protem or serum protem, e g albumin, bmdmg of the drag By degree of serum protem bmdmg is meant the propensity of a drug to expenence an serum protem effect, l e to be bound by serum protem, such as albumin (or another serum protem such as α-1 acid glycoprotem) and be rendered mactive In this embodiments, the drugs of interest are those drags which have a certain affinity for the serum protem, e g albumin, m their free drug form, where this affinity is generally at least about 10³, usually at least about 10 ⁵and more usually at least about 10 ⁶ In certain embodiments where modulation of serum protem bmdmg effect is desired, the affinity of the modulating ligand for the serum protem will be greater than the affinity of the drug moiety for the serum protein, usually at least about 2 fold greater, m certain embodiments at least about 3 fold greater and m other embodiments at least about 5 fold greater The linker moiety of the bifunctional molecule is chosen such that the drag moiety of the bifunctional is displayed in manner that retains its desired activity, e g target bmdmg activity, when a binary complex is formed with the serum protein, e g albumm In addition, the linker is chosen such that the drug moiety cannot bind to a second serum protem, e g albumm molecule, to produce a tnpartite complex of two serum portem molecules and a bifunctional molecule In these embodiments, the degree of albumm bmdmg of the drug or albumm bmdmg effect is reduced by a factor of about 2, usually by a factor of about 3 and more usually by a factor of about 4 As such, the amount of drag that must be administered m order to be effective is generally at least about 2 fold less, in certain embodiments at least about 3 fold less and in other embodiments at least about 4 fold less than the amount of conesponding free drug that must be administered

METHODS OF USE, PHARMACEUTICAL PREPARATIONS AND KITS

The subject bifunctional molecules find use m the pharmacological treatment of a host condition, e g a disease condition In the methods of the subject invention, an effective amount of the bifunctional molecule is admimstered to the host, where "effective amount" means a dosage sufficient to produce the desired result, e g an improvement m a disease condition or the symptoms associated therewith In many embodiments, the amount of drag in the form of the bifunctional molecule that need be admimstered to the host m order to be an effective amount will vary from that which must be admimstered m free drug form, where by free drag is meant drug that is not conjugated with another moiety, e g as is found m the subject bifunctional molecules The difference in amounts may vary, and m many embodiments may range from 2 fold to 10 fold In certain embodiments, e g where the resultant modulated pharmacokmetic property(s) results m enhanced activity as compared to the free drug control, the amount of drag that need be administered to be an effective amount is less than the amount of corresponding free drag that needs to be administered, where the amount may be 2-fold, usually about 4-fold and more usually about 10-fold less than the amount of free drag that is administered

The bifimctional molecule may be administered to the host usmg any convenient means capable of producing the desired result Thus, the bifunctional molecule can be incorporated into a vaπety of formulations for therapeutic administration More particularly, the bifunctional molecule of the present invention can be formulated mto pharmaceutical compositions by combmation with appropnate, pharmaceutically acceptable carriers or diluents, and may be formulated mto preparations m solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositones, injections, inhalants and aerosols As such, administration of the bifunctional molecule can be achieved in vanous ways, mcludmg oral, buccal, rectal, parenteral, mtrapentoneal, lntradermal, transdermal, mtracheal, etc , administration In pharmaceutical dosage forms, the bifunctional molecule may be admimstered alone or in combmation with other pharmaceutically active compounds The following methods and excipients are merely exemplary and are m no way limiting For oral preparations, the bifunctional molecules can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The bifunctional molecules can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The bifunctional molecules can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. Furthermore, the bifunctional molecules can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonfiil, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing active agent. Similarly, unit dosage forms for injection or intravenous administration may comprise the active agent in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

The subject methods find use in the treatment of a variety of different disease conditions. In certain embodiments, of particular interest is the use of the subject methods in disease conditions where an active agent or drug having desired activity has been previously identified, but which active agent or drag does not bind to its target with desired affinity and or specificity. With such active agents or drugs, the subject methods can be used to enhance the binding affinity and/or specificity of the agent for its target. The specific disease conditions treatable by with the subject bifunctional compounds are as varied as the types of drag moieties that can be present in the bifunctional molecule. Thus, disease conditions include cellular proliferative diseases, such as neoplastic diseases, autoimmune diseases, cardiovascular diseases, hormonal abnormality diseases, infectious diseases, and the like.

By treatment is meant at least an amelioration of the symptoms associated with the disease condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as inflammation and pain associated therewith. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition. A variety of hosts are treatable according to the subject methods. Generally such hosts are

"mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans. Kits with unit doses of the bifunctional molecule, usually in oral or injectable doses and often in a storage stable formulation, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating pathological condition of interest. Preferred compounds and unit doses are those described herein above.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

I. Modulating Half-Life

A. Introduction The liver and kidney are the two organs where most of the biotransformation and secretion of drag molecules takes place. Both organs can take up free drag molecules but the uptake of protein-bound drug is much reduced Therefore, drugs, that cannot associate with mtracellular protems and other molecules like hpids or which cannot bmd plasma protems, are more likely to be metabolized and excreted quickly As a result, such molecules tend to have a short half-life

FK506 and rapamycin are two molecules that display a long half-life, 30 hours, and 60 hours, respectively This relatively long half-life is surpnsmg smce the molecular weights of FK506 (800 D) and rapamycin (900 D) are high relative to most drags, and large molecular weights usually lead to rapid metabolic modification, inactivation, and excretion The basis for the extended half-life of these two molecules is the fact that both bmd with high affinity to a very abundant mtracellular protem, called FKBP There are about one million FKBP molecules in every cell of the human body When FK506 and rapamvcin enter human cells, they are retained by this large pool of FKBP This underlying mechanism for the extension of a molecule's half-life can be used to prolong the half-lives of drags of interest

B Extension of Drag Half-Life

Intracellularly acting drags with an unsatisfactory half-life are covalentiy linked to ligands of FKBP When the resultmg bifunctional molecule enters a cell, it will be bound by the large FKBP pool via its FKBP ligand For example, AG 337 is a thymidylate synthase inhibitor which is undergoing clinical tnals as an anti-cancer agent Its half-life is 2-3 hours which requires a 24 hour continuous IV infusion to achieve the desired therapeutic concentration By coupling AG 337 to a ligand for FKBP, the drag is bound and retained by the FKBP pool inside of cells so that the half-life is of the drag is extended As a result, the administration of the drag may be reduced to a shorter time penod or may be administered usmg the oral route

There are natural FKBP ligands like FK506 and rapamycin as well as many synthetic FKBP ligands They all have somewhat different affinities for FKBP which will determine to what degree the molecule is retained and, therefore, to what extent the half-life of the drag of mterest will be prolonged Creating a senes of bifunctional molecules usmg different FKBP ligands can allow the selection of a molecule that displays the desired pharmacokmetic properties

The extension of half-lives of mtracellularly acting drugs is not restncted to the use of ligands for FKBP Other abundant and widely expressed proteins are cyclophilin, tubulm, actin or the heat shock proteins (hsp's) Cyclophilin has high affinity ligands called cylosponns Tubuhn's high affinity ligands are molecules like nocodazole, vinblastme and colchicme, whereas cytochalasms and latrunculms are ligands for actin Geldanamycin is a high affinity ligand for the abundant hsp90 Like FKBP ligands, the listed ligands are coupled to drugs of mterest in order to change then half-lives One important aspect m the choice of ligand is its expected toxicity at the concentrations one anticipates to use In this respect, FKBP and cyclophilin ligands are of great interest because they display little toxicity even at high concentrations if they are denvatized so that they cannot inhibit the drag targets of FK506, rapamycin or cyclosporm A

Two important drug bmdmg molecules in plasma are albumm and αl acid glycoprotem Albumm has two bmdmg sites for small molecule ligands Site I shows a preference for heterocyclic compounds with a negative charge and site II seems to prefer small aromatic carboxylic acids In addition to small molecule bmdmg sites, albumm also has bmdmg sites for lipids αl acid glycoprotem has only one bind g site for small molecules In contrast to albumin, αl acid glycoprotem binds neutral or basic small molecules like propanolol, chlorpromazine, dipynmadole, metoclopramide, apπndine or verapamil

In order to prolong the half-lives of drugs, bifunctional molecules are synthesized which contam an albumm bmdmg ligand or an αl acid glycoprotem ligand covalentiy linked to the drug of mterest For example, naproxen is a small molecule that shows extensive bmdmg to albumm The half-life of naproxen is 14 hours By linking it to a drag like hdocame, which has a half life of only two hours, one can extend the half life of hdocame

Another albumm ligand of mterest is warfarm In plasma, warfarm is almost completely bound by albumm and it has a half-life of 25-60 hours The volume of distribution of warfarin is equivalent to the space that albumm can reach These properties make warfarm a good ligand to link to a drug of mterest that has an extracellular target and whose half-life needs to be prolonged Aside from vanous candidate small molecule drags, such drags can be peptides Many peptides are agomsts or antagonists for extracellular receptors like opiate peptides (e g enkaphalrns and endorphins), orexins, leptin, insulin, vasopressm, growth hormone, erythropoietin, cytokmes, renm and thrombm inhibitors and many more In general, peptide drugs have very short half-lives because the free peptide is rapidly cleared by the liver Couplmg peptide drugs to warfarin will lead to bifunctional molecules that are avidly bound by albumin Due to the bmdmg to albumm, the rate of clearance of the peptide is much reduced and the half-life is prolonged

There are many well charactenzed albumm ligands that can be used to extend the half-life of drugs by makmg bifunctional molecules However, additional albumm ligands with potentially higher affinity than above mentioned ligands can be found by screening small molecule hbranes Every chosen albumin ligand may have different properties with respect to the half-life and the volume of distnbution it confers onto the bifunctional molecule Hence, if vanous ligands with different albumm bmdmg properties are used in combmation with one drug of mterest, a senes of bifunctional molecules can be produced with different pharmacokmetic properties for distinct therapeutic requirements

II Hepatic first-pass metabolism

Many drags expenence extensive metabolism during their first pass through the liver In order to avoid hepatic first-pass clearance, the drag of interest is covalentiy linked to an FKBP ligand like FK506 or rapamycin FKBP is highly expressed in red blood cells After the uptake from the gut, the bifunctional molecule can diffuse mto red blood cells where it is retained by bmdmg to the large FKBP pool In contrast to the free drug, which is accessible by endothelial cells m the liver, the bifunctional molecule can pass the liver inside red blood cells As a result, the drag of mterest is not taken up by the liver where it would be metabolized or excreted into the bile The interaction between FKBP and the bifunctional molecule is transient The bifunctional molecule will diffuse out of red blood cells m less than two minutes It will then enter the next red blood cell or a tissue cell Drags that show extensive hepatic first-pass clearance are mercaptopurrne, naloxone, verapamil, nifedipine, diltiazem, lovastatm, simvastatin, pravastatin, fluorostatin, propanolol, or propafenone III Volume of distnbution

One important charactenstic of a drug is its volume of distnbution which reflects its distnbution and degree of retention m tissues throughout the body Highly lipophilic drugs like many dihydrofolate reductase inhibitors (e g pintrexim, tnmetrexate) and thynudylate synthase inhibitors (e g AG331, AG337) often pass qmckly through cells without reachmg the therapeutic concentration required in the cytoplasm To improve the distribution of such molecules and to improve their retention in the cytoplasm of cells, they are linked to FKBP ligands to form bifunctional molecules All cells m the human body express high levels of FKBP Bifunctional molecules that are based on FKBP ligands are slowed down m their passage through tissues and show a good volume of distnbution like the drags FK506 and rapamycin do These drags are found m all tissues despite the fact that they are quite lipophilic

Similarly, drugs directed against extracellular targets should not enter mto cells smce this would reduce their effective concentration The ideal volume of distnbution of these drags would equal the volume accessible by extracellular fluids Sulfisoxazole and warfarm are two molecules which are found almost exclusively m the extracellular space because they are extensively bound to albumm which is found all extracellular fluids If a drag of interest, which tends to have a large volume of distribution, is bound to sulfisoxazole or warfarm, it will be preferentially retained in the extracellular space Its volume of distnbution will be reduced

IV Excessive albumm bmdmg Albumin is a very abundant plasma protem that bmds many small molecules m two distinct bmdmg pockets For example, the non-steroidal anti-inflammatory drag lbuprofen has a Kd of 4 0x10^"'M for one bmdmg pocket and the Kd for the second bindmg pocket is 5 0x10"' M Another albumm ligand is salicylic acid which is the core molecule of many analgesic-antipyretic and antiinflammatory agents Its affinity for albumin's first bmdmg pocket is 5x10"^M and it binds the second bindmg pocket with an affinity of 6x10^{" 4}M

When small molecule drags are bound by albumm they are not accessible for their drag targets Sometimes drags bmd albumm so well that large doses of the drug have to be used in order to saturate albumm bmdmg sites and to create a pool of free drag For example, chlorpromazme is an antipsychotic drag that acts at dopaminergic synapses Chlorpromazine is extensively bound to albumm and its affinity for albumin is 5x10"^M If chlorpromazme is linked to a ligand that has an affinity for albumm which is higher than chlopromazine's affinity for albumm like ιbuprofen(4xl 0" ' M), then, the bifunctional molecule will be bound by albumm through its lbuprofen moiety so that chlorpromazme sticks out of the albumm surface Bemg presented at the albumm surface will allow chlorpromazme to interact with its target at the synapse Administering chlorpromazme as a bifunctional molecule makes it possible to achieve a much higher concentration of target accessible drug m the plasma Therefore, the amount of admimstered bifunctional drag can be sharply reduced with respect to free drag so that general and liver toxicities can be reduced and the efficacy of the drag can be enhanced Good efficacy of a drag is especially important if the drag of interest is to be administered transdermally The skm has only a limited capacity to absorb drags so that molecules with high potency and efficacy are required for this route of drug administration The linker connecting a ligand for a plasma protem like albumin and a drag of interest has to be designed m such a way that the drug target can still bmd the drug of mterest However, the linker should position the drag of mterest with respect to the protem surface so that a second albumm molecule cannot bmd

It is evident from the above results and discussion that the subject invention provides a powerful tool for improvmg drug efficacy by modulating one or more pharmacokmetic properties of the drug Specifically, the subject invention provides a convenient way to modulate the pharmacokmetic properties of a small molecule drug while retaining the small molecule nature of the drug As such, the subject method provides for the improvement of drags currently m use Furthermore, the subject methods can be used to improve drugs that have, until now, been clinically useless due to certain factors, e g short half-life, unacceptable volume of distnbution, etc Therefore, the invention provides for the potential usefulness of the vanety of previously discovered and discarded biologically active compounds Accordingly, the invention provides an important advancement m pharmacological science

All publications and patent applications cited in this specification are herem incorporated by reference as if each individual publication or patent application were specifically and individually mdicated to be mcorporated by reference The citation of any publication is for its disclosure pnor to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of pnor invention

Although the foregomg invention has been descnbed m some detail by way of illustration and example for purposes of clanty of understanding, it is readily apparent to those of ordinary skill m the art m light of the teachmgs of this invention that certain changes and modifications may be made thereto without departing from the spint or scope of the appended claims

Claims

WHAT IS CLAIMED IS

1 A non-naturally occurring bifunctional molecule of less than about 5000 daltons consisting of a drag moiety and a pharmacokinetic modulatmg moiety, wherem said drag moiety and said pharmacokmetic modulating moiety are optionally jomed by a lmkmg group and said bifunctional molecule exhibits at least one modulated pharmacokmetic property upon administration to a host as compared to a free drug control

2 The bifunctional molecule according to Claim 1 , wherem said bifunctional molecule compnses a linking group

3 The bifunctional molecule according to Claim 1 , wherem said bifunctional molecule does not mclude a lmkmg group

4 The bifunctional molecule according to Claim 1 , wherem said pharmacokmetic property is selected from the group consisting of half-life, hepatic first-pass metabolism, volume of distnbution and degree of blood protem bmdmg

5 The bifunctional molecule according to Claim 1 , wherem said pharmacokinetic modulating moiety binds to a protem

6 The bifunctional molecule according to Claim 5, wherem said protem is an extracellular protein

7 The bifunctional molecule according to Claim 5 , wherem said protem is an intracellular protem

8 A synthetic bifunctional molecule of less than about 5000 daltons of the formula

Z-L-X wherem

X is a drug moiety, L is a bond or a linking group, and Z is a pharmacokmetic modulatmg moiety, wherem X and Z are different and bifunctional molecule exhibits at least one modulated pharmacokmetic property upon administration to a host as compared to a free drag control 9 The bifunctional molecule according to Claim 8, wherem said pharmacokmetic property is selected from the group consisting of half-life, hepatic first-pass metabolism, volume of distnbution and degree of blood protein binding

10 The bifunctional molecule according to Claim 8, wherem said drug moiety has a molecular weight of from about 50 to 2000 D

11 The bifunctional molecule according to Claim 8, wherem said drag moiety binds to a protem target

12 The bifunctional molecule according to Claim 8, wherem said pharmacokinetic modulating moiety bmds to an extracellular protem

13 The bifunctional molecule according to Claim 8, wherem said pharmacokinetic modulating moietv bmds to an mtracellular protem

14 The bifunctional molecule according to Claim 8, wherem said bifunctional molecule compnses a lmkmg group

15 The bifunctional molecule according to Claim 8, wherem said pharmacokinetic modulating moiety has substantially no pharmacologic activity apart from bmdmg to an endogenous protem of said host

16 A method for modulating at least one pharmacokinetic property of a drag upon administration to a host, said method compnsmg administering to said host an effective amount of a bifunctional molecule of less than about 5000 daltons consistmg of said drag or an active denvative thereof and a pharmacokinetic modulating moiety optionally jomed by a lmkmg group, wherein said bifunctional molecule has at least one modulated pharmacokmetic property upon administration to said host as compared to a free drag control, whereby at least one pharmacokmetic property of said drag upon administration to said host is modulated as compared to a free drag control

17 The method accordmg to Claim 16 , wherem said pharmacokmetic property is selected from the group consisting of half-life, hepatic first-pass metabolism, volume of distnbution and degree of blood protein bmdmg

18 The method accordmg to Claim 16„ wherem said bifunctional molecule compnses a lmkmg group 19 The method accordmg to Claim 16, wherem pharmacokmetic modulating moiety binds to an mtracellular protem

20 The method accordmg to Claim 16, wherem said pharmacokmetic modulating moiety binds to an extracellular protem

21 The method accordmg to Claim 16, wherem said drag target is a protem

22 The method accordmg to Claim 16, wherem said bifunctional molecule is administered as a pharmaceutical preparation

23 A method for modulating the half life of a drug upon admmistration to a host, said method compnsmg administering to said host an effective amount of a bifunctional molecule of less than about 5000 daltons consistmg of said drag or an active denvative thereof and a half-life modulating moiety optionally jomed by a linkmg group, wherem said bifunctional molecule has a modified half-life upon admmistration to said host as compared to a free drug control, whereby the half life of said drug upon administration to said host is modulated as compared to a free drug control

24 The method accordmg to Claim 23, wherem said half-life modulating moiety bmds to an mtracellular protem

25 The method accordmg to Claim 23, wherein said half-life modulating moiety bmds to an extracellular protem

26 The method accordmg to Claim 23, wherem said drag target is a protem

27 The method according to Claim 23 , wherem said bifunctional molecule is administered as a pharmaceutical preparation

28 A method for modulating the hepatic first-pass metabolism of a drag upon administration to a host, said method compnsmg administering to said host an effective amount of a bifunctional molecule of less than about 5000 daltons consisting of said drag or an active denvative thereof and a hepatic first-pass metabolism modulating moiety optionally jomed by a linkmg group, wherem said bifunctional molecule has a modified hepatic first- pass metabolism upon admmistration to said host as compared to a free drug control, whereby the hepatic first-pass metabolism of said drag upon administration to said host is modulated as compared to a free drag control

29 The method accordmg to Claim 28, wherem said hepatic first-pass metabolism modulating moiety bmds to an mtracellular protem

30 The method accordmg to Claim 28, wherem said hepatic first-pass metabolism modulating moiety bmds to an extracellular protem

31 The method accordmg to Claim 28, wherem said drag target is a protem

32 The method accordmg to Claim 28, wherem said bifunctional molecule is administered as a pharmaceutical preparation

33 A method for modulating the volume of distnbution of a drag upon admmistration to a host, said method compnsmg administering to said host an effective amount of a bifunctional molecule of less than about 5000 daltons consistmg of said drag or an active denvative thereof and a volume of distnbution modulating moiety optionally jomed by a linkmg group, wherein said bifunctional molecule has a modified volume of distnbution upon admmistration to said host as compared to a free drag control, whereby the volume of distnbution of said drag upon admmistration to said host is modulated as compared to a free drag control

34 The method accordmg to Claim 33 , wherem said volume of distnbution modulatmg moiety bmds to an mtracellular protem

35 The method accordmg to Claim 33, wherem said volume of distnbution modulating moiety bmds to an extracellular protem

36 The method accordmg to Claim 33 , wherem said drag target is a protem 37 The method accordmg to Claim 33, wherem said bifunctional molecule is administered as a pharmaceutical preparation

38 A method for modulating the blood protem bindmg effect on a drag upon admmistration to a host, said method compnsmg administering to said host an effective amount of a bifunctional molecule of less than about 5000 daltons consistmg of said drag or an active denvative thereof and albumin effect modulating moiety optionally jomed by a lmkmg group, wherem said bifunctional molecule exhibits a modified blood protem bmdmg effect upon admmistration to said host as compared to a free drug control, whereby the blood protem bmdmg effect on said drag upon admimstration to said host is modulated as compared to a free drag control

39 The method accordmg to Claim 38, wherem said blood protem bmdmg effect modulating moiety is a ligand for albumm

40 The method according to Claim 39, wherem said bifunctional molecule compnses a linking group

41 The method according to Claim 40, wherem said linkmg group is sufficient to display said drag moiety in a manner such that it is available for bmdmg to its target but not to a second albumm molecule

42 The method according to Claim 38, wherem said bifunctional molecule is admimstered as a pharmaceutical preparation

43 In a method of administering a drag to a host m need of said drug, the improvement compnsmg administering to said host an effective amount of a bifunctional molecule of less than about 5000 daltons consistmg of said drag or a denvative thereof covalentiy linked, either directly or through an optional lmkmg group, to a pharmacokmetic modulating moiety

44 The method accordmg to Claim 43 , wherem said host is a mammalian host

45 The method according to Claim 44, wherem said mammalian host is human

46 The method accordmg to Claim 43, wherem said drag is a small molecule 47 The method accordmg to Claim 43, wherein said pharmacokmetic modulating moiety binds to an extracellular protein

48 The method accordmg to Claim 43, wherem said pharmacokmetic modulating moiety binds to an mtracellular protem

49 A pharmaceutical preparation compnsmg a bifunctional molecule according to Claim 1

50 A kit compnsmg the pharmaceutical preparation according to Claim 49 and mstractions for use m a therapeutic method