US 200803 06098A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0306098 A1 Mutz et al. (43) Pub. Date: Dec. 11, 2008

(54) PHARMACOKINETICS OF PROTEASE Publication Classification INHIBITORS AND OTHER DRUGS (51) Int. Cl. A 6LX 3L/505 (2006.01) (76) Inventors: Mitchell W. Mutz, La Jolla, CA A63L/4353 (2006.01) (US); Jason E. Gestwicki, Ann C07D 49/12 (2006.01) Arbor, MI (US) C07D 239/04 (2006.01) A6IP3 L/18 (2006.01) Correspondence Address: (52) U.S. Cl...... 514/274: 514/291; 546/90; 54.4/316 MINTZ, LEVIN, COHN, FERRIS, GLOVSKY (57) ABSTRACT AND POPEO, PC 5 Palo Alto Square - 6th Floor,3000 El Camino Real A method for modulating at least one pharmacokinetic prop PALO ALTO, CA 94306-2155 (US) erty of a protease inhibitor upon administration to a host is provided. One administers to the host an effective amount of a bifunctional compound of less than about 5000 daltons (21) Appl. No.: 12/151,329 comprising the protease inhibitor or an active derivative thereof and a pharmacokinetic modulating moiety. The phar (22) Filed: May 5, 2008 macokinetic modulating moiety binds to at least one intrac ellular protein. The bifunctional compound has at least one (30) Foreign Application Priority Data modulated pharmacokinetic property upon administration to the hostas compared to a free drug control that comprises the Nov. 6, 2006 (US) ...... PCT/US2006/043400 protease inhibitor.

FKBP-binding FKBP-binding interface interface

Conjugate

Target Binding Calcineurin-binding interface

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PHARMACOKINETCS OF PROTEASE administers to the host an effective amount of a bifunctional INHIBITORS AND OTHER DRUGS compound of less than about 5000 daltons comprising the protease inhibitor or an active derivative thereof and a phar CROSS-REFERENCE TO RELATED macokinetic modulating moiety. The pharmacokinetic modu APPLICATIONS lating moiety binds to at least one intracellular protein. The 0001. This application claims priority to PCT/US2006/ bifunctional compound has at least one modulated pharma 043400 filed Nov. 6, 2006, which claims priority to U.S. cokinetic property upon administration to the host as com provisional application Ser. No. 60/734,197, filed Nov. 5, pared to a free drug control that comprises the protease inhibi 2005. Both priority documents are incorporated by reference tOr. 0009. In a further embodiment of this invention, a bifunc in their entireties. tional compound comprising protease inhibitor functionality TECHNICAL FIELD and a pharmacokinetic modulating moiety are provided. 0010. In a further aspect of the invention, a bifunctional 0002 This invention relates generally to pharmacology compound is provided in a pharmaceutical formulation which and more specifically to the modification of known active is designed to have a controlled release mechanism in addi agents to give them more desirable properties. tion to that provided by the bifunctional compound. The bifunctional compound may comprise a drug moiety that is a BACKGROUND ART protease inhibitor or some other drug. 0003. When HIV was first discovered, it was feared that all persons infected with HIV would eventually develop full BRIEF DESCRIPTION OF THE DRAWINGS blown AIDS. However, drugs were developed and approved (0011 FIG. 1A depicts the structure of FK506 linked to a which could slow the proliferation of the HIV virus. The most modular linker and target binding moiety, for example a pro usual therapies over the last decade for HIV-infected people tease inhibitor. Due to the modular nature of the synthesis, the have been three-drug cocktails, in which one of the drugs is an linker group and target-binding group have been altered. FIG. HIV protease inhibitor and the other two are reverse-tran 1B illustrates how the steric bulk of an FKBP protein can scriptase inhibitors. The introduction of three-drug cocktails confer protection from P450 enzymes. in the mid-1990s has allowed many HIV-infected people to survive for long periods of time without developing AIDS and 0012. In FIG. 2, the left side depicts the bimodal binding has allowed some AIDS patients to experience a notable character of FK506 whereby it binds both FKBP and cal remission. cineurin. The schematic on the right depicts how the cal 0004 Patient compliance and drug toxicity have always cineurin-binding mode can be eliminated by Substituting a been major issues with HIV protease inhibitors due to the linker and target binding moiety. In this manner, FK506 can frequency of dosage and co-administration of other HIV simultaneously target FKBP and bind a second protein. therapeutics. A reduction in dosage frequency would repre (0013. In FIG. 3, (A) shows the structure of FK506 bound sent an improvement in quality of life for the patient and a to curcumin. (B) illustrates that FK506-curcumin is protected lower chance of toxic side effects due to decreased production from CYP3a4, a P450 enzyme, in the presence of FKBP. (C) of secondary metabolites, especially in the liver. Although gives a schematic of the Invitrogen assay used. recent HIV protease inhibitors require a reduced dosage bur 0014. In FIG. 4, the left side illustrates an exemplary syn den compared with earlier drugs, there is still significant thetic scheme for bifunctional compounds of the invention. opportunity for improving PIs by reducing first pass clear The right side shows the application of this scheme to ance via cytochrome P450 enzymes and increasing the drug , , and . half-life in the circulation. 0015 FIG. 5 depicts useful linkers which have amine and 0005 Examples of HIV protease inhibitors which have (alkyl-protected) carboxyl moieties. diminished half lives due to poor pharmacokinetics are 0016 FIG. 6 sets out a synthetic scheme for the synthesis amprenavir, lopinavir, and ritonavir, among others. of an amprenavir-based SLF-PI conjugate. The amprenavir 0006 Previous methods to improve pharmacokinetics like moiety is shown at the right in FIG. 6. (PK) of HIV protease inhibitors include: medicinal chemis (0017 FIGS. 7A-7B depict a reaction schema for ritonavir try-based analog synthesis, employing pro-drug strategies, and lopinavir respectively. Note the schema proceeds via improved formulation, and co-administration with P450 and analogous chemistry using the well-characterized Boc (t-bu P-glycoprotein inhibitors. Regardless of the methods toxycarbonyl) leaving group. employed, these approaches share a desired outcome: improve pharmacokinetics to make treatment easier for DETAILED DESCRIPTION OF PREFERRED patients. However, these methods have not yielded an inhibi EMBODIMENTS tor that requires a Substantially lower dose (e.g., once or twice 0018. Before describing the present invention in detail, it per week) over the prior inhibitors and is relatively non-toxic is to be understood that this invention is not limited to specific compared with prior inhibitors. Solvents, materials, or device structures, as Such may vary. It 0007. There is therefore still a need in the art for protease is also to be understood that the terminology used herein is for inhibitors and associated dosage forms which have reduced the purpose of describing particular embodiments only, and is first pass clearance and/or improved pharmacokinetics. not intended to be limiting. 0019. As used in this specification and the appended DISCLOSURE OF THE INVENTION claims, the singular forms “a,” “an and “the include both 0008. In an embodiment of this invention, a method for singular and plural referents unless the context clearly dic modulating at least one pharmacokinetic property of a pro tates otherwise. Thus, for example, reference to “an active tease inhibitor upon administration to a host is provided. One ingredient' includes a plurality of active ingredients as well US 2008/0306098 A1 Dec. 11, 2008 as a single active ingredient, reference to “a temperature' bifunctional compound has at least one modulated pharma includes a plurality oftemperatures as well as single tempera cokinetic property upon administration to the host as com ture, and the like. pared to a free drug control that comprises the protease inhibi 0020. The term “bifunctional compound” refers to a non tOr. naturally occurring compound that includes a pharmacoki 0033 Bifunctional compound in general have aroused netic modulating moiety and a drug moiety, where these two considerable interest in recent years. See, for example, U.S. components may be covalently bonded to each other either Pat. No. 6,270,957, U.S. Pat. No. 6,316,405, U.S. Pat. No. directly or through a linking group. The term “drug” refers to 6,372,712, U.S. Pat. No. 6,887,842, and U.S. Pat. No. 6,921, any active agent that affects any biological process. Bifunc 531. ConjuChem (Montreal, Canada) scientists have shown tional compounds may have more than two functionalities. that covalent coupling of insulinto human serum albumin can 0021. The pharmacokinetic modulating moiety may be a improve the half-life from 8 hours to over 48 hours. Xenoport peptide or protein and may also be an enzyme or nucleic acid. (Santa Clara, Calif.) has pioneered attachment of receptor Similarly, the drug moiety may also be peptide, protein, ligands to improve druguptake and distribution. Human trials enzyme, or nucleic acid. of methotrexate-albumin conjugates revealed that the modi 0022 Active agents which are considered drugs for pur fied methotrexate had half-lives of up to two weeks compared poses of this application are agents that exhibit a pharmaco with 6 hours for unmodified methotrexate. Other examples logical activity. Examples of drugs include active agents that include PEGylation of growth factors and attachment of are used in the prevention, diagnosis, alleviation, treatment or folate groups that “target' anti-cancer drugs. All these strat cure of a disease condition. egies use modification of a “parent drug to provide new 0023. By “pharmacologic activity” is meant an activity binding profiles or enhanced protection from degradation. that modulates or alters a biological process so as to result in 0034 More recently, a team including one of the inventors a phenotypic change, e.g. cell death, cell proliferation etc. attached SLF to ligands for amyloid beta. Amyloid beta oli 0024. By “pharmacokinetic property' is meant a param gomers are believed to underlie the neuropathology of Alzhe eter the describes the disposition of an active agent in an imer's disease. Therefore, methods to decrease amyloid organism or host. Representative pharmacokinetic properties aggregation are of therapeutic interest. Amyloid ligands, Such include: drug half-life, hepatic first-pass metabolism, Volume as congo red or curcumin (above), can be synthetically of distribution, degree of blood serum protein, e.g. albumin, coupled to FK506 or SLF. The resulting bifunctional com binding, etc. degree of tissue targeting, cell type targeting. pound binds both FKBP and amyloid beta. These molecules 0025 By “half-life” is meant the time for one-half of an are potent inhibitors of amyloid aggregation and they block administered drug to be eliminated through biological pro neurotoxicity. See Jason E. Gestwicki et al., “Harnessing cesses, e.g. metabolism, excretion, etc. Chaperones to Generate Small-Molecule Inhibitors of Amy 0026. By “hepatic first-pass metabolism' is meant the pro loid? Aggregation. Science 306:865-69 (2004). pensity of a drug to be metabolized upon first contact with the 0035 Bifunctional compounds of the type employed in liver, i.e. during its first pass through the liver. the present invention are generally described by the formula: 0027. By “volume of distribution” is meant the distribu tion and degree of retention of a drug throughout the various compartments of an organisms, e.g. intracellular and extra wherein: cellular spaces, tissues and organs, etc. 003.6 X is a drug moiety; 0028. The term “efficacy” refers to the effectiveness of a 003.7 L is a bond or linking group; and particular active agent for its intended purpose, i.e. the ability 0038 Z is a pharmacokinetic modulating moiety, of a given active agent to cause its desired pharmacologic with the proviso that X and Z are different. Thus, as may be effect. seen, a bifunctional compound is a non-naturally occurring or 0029. The term “host” refers to any mammal or mamma synthetic compound that is a conjugate of a drug orderivative lian cell culture or any bacterial culture. thereof and a pharmacokinetic modulating moiety, where 0030. Where the term HIV is used, it is understood that the these two moieties are optionally joined by a linking group. invention may be employed on relative protease inhibitors 0039. In bifunctional compounds used in the invention the Such as found in other immunodeficiency viruses found in pharmacokinetic modulating and drug moieties may be dif non-human species or human variants (HIV II, SIV. FIV, etc). ferent, such that the bifunctional compound may be viewed as 0031 Where FK506 is used, variants or analogs of FK506 a heterodimeric compound produced by the joining of two are included. Such as rapamycin, pimecrolimus, or synthetic different moieties. In many embodiments, the pharmacoki ligands of FK506 binding proteins (SLFs) such as those dis netic modulating moiety and the drug moiety are chosen Such closed in U.S. Pat. Nos. 5,665,774, 5,622,970, 5,516,797, that the corresponding drug target and any binding partner of 5,614,547, and 5,403,833 or described by Holt et al., “Struc the pharmacokinetic modulating moiety, e.g., a pharmacoki ture-Activity Studies of Synthetic FKBP Ligands as Peptidyl netic modulating protein to which the pharmacokinetic Prolyl Isomerase Inhibitors. Bioorganic and Medicinal modulating moiety binds, do not naturally associate with each Chemistry Letters, 4(2):315-320 (1994). other to produce a biological effect. 0032. In an embodiment of this invention, a method for 0040. The bifunctional compounds are typically small. As modulating at least one pharmacokinetic property of a pro Such, the molecular weight of the bifunctional compound is tease inhibitor upon administration to a host is provided. One generally at least about 100 D, usually at least about 400 D administers to the host an effective amount of a bifunctional and more-usually at least about 500D. The molecular weight compound of less than about 5000 daltons comprising the may be less than about 800D, about 1000D, about 1200 D, or protease inhibitor or an active derivative thereof and a phar about 1500 D, and may be as great as 2000 D or greater, but macokinetic modulating moiety. The pharmacokinetic modu usually does not exceed about 5000 D. The preference for lating moiety binds to at least one intracellular protein. The small molecules is based in part on the desire to facilitate oral US 2008/0306098 A1 Dec. 11, 2008

administration of the bifunctional compound. Molecules that tion. Thus, for example, drugs having a strong first-pass effect are orally administrable tend to be small. may be candidates for incorporation into a bifunctional com 0041. The pharmacokinetic modulating moiety modulates pound. a pharmacokinetic property, e.g. half-life, hepatic first-pass 0045. In general, the pharmacokinetic modulating moiety metabolism, Volume of distribution, degree of albumin bind Z will be one which is capable of reversible attachment to a ing, etc., upon administration to a host as compared to free common protein, meaning one which is abundant in the body drug control. By modulated pharmacokinetic property is or in particular compartments of the body or particular tissue types. Common proteins include, for example, FK506 bind meant that the bifunctional compound exhibits a change with ing proteins, cyclophilin, tubulin, actin, heat shock proteins, respect to at least one pharmacokinetic property as compared and albumin. Common proteins are present in concentrations to a free drug control. For example, a bifunctional compound of at least 1 micromole, preferably at least 10 micromoles, of the Subject invention may exhibit a modulated, e.g. longer, more preferably at least 100 micromoles, and even more half-life than its corresponding free drug control. Similarly, a preferably 1 millimole in the body or in particular compart bifunctional compound may exhibit a reduced propensity to ments or tissue types. The pharmacokinetic modulating moi be eliminated or metabolized upon its first pass through the ety should, like the drug, have a moiety which is capable of liver as compared to a free drug control. Likewise, a given reacting with Suitable linkers. bifunctional compound may exhibit a different volume of 0046. It is desirable for at least some embodiments of the distribution that its corresponding free drug control, e.g. a present invention that the binding of the pharmacokinetic higher amount of the bifunctional compound may be found in modulating moiety Z to a common protein be such as to the intracellular space as compared to a corresponding free sterically hinder the activity of common metabolic enzymes drug control. Analogously, a given bifunctional compound such as CYP450 enzymes when the bifunctional compound is may exhibit a modulated degree of albumin binding Such that so bound. Persons of skill in the art will recognize that the the drug moiety's activity is not as reduced, if at all, upon effectiveness of this steric hindrance depends, among other binding to albumin as compared to its corresponding free factors, on the conformation of the common protein in the drug control. In evaluating whether a given bifunctional com vicinity of the pharmacokinetic modulating moiety's binding pound has at least one modulated pharmacokinetic property, site on the protein, as well as on the size and flexibility of the as described above, the pharmacokinetic parameter of interest linker. The choice of a suitable linker and pharmacokinetic is typically assessed at a time at least 1 week, usually at least modulating moiety may be made empirically or it may be 3 days and more usually at least 1 day following administra made by means of molecular modeling of some sort if an tion, but preferably within about 6 hours and more preferably adequate model of the interaction of candidate pharmacoki within about 1 hour following administration. netic modulating moieties with the corresponding common 0042. The linker L, if not simply a bond, may be any of a proteins exists. variety of moieties chosen so that they do not have an adverse 0047. The attachment point and linker characteristics are effect on the desired operation of the two functionalities of the preferably selected based on structural information such that molecule and also chosen to have an appropriate length and the inhibitory potency of the protease inhibitor is preserved, flexibility. The linker may, for example, have the form F giving the desired Superior pharmacokinetic characteristics. (CH2), F where F and F are suitable functionalities. A 0048. Where the pharmacokinetic modulating moiety linker of this sort comprising an alkylene group of Sufficient operates by binding a protein, it may be referred to as a length may allow, for example, for the free rotation of the drug “presenter protein ligand’ and the protein which it binds to moiety even when the pharmacokinetic modulating moiety is may be referred to as a “presenter protein.” bound. Alternatively, a stiffer linker with less free rotation 0049. The pharmacokinetic modulating moiety may be, may be desired. The hydrophobicity of the linker is also a for example, a derivative of FK506, which has high affinity relevant consideration. FIG.5 depicts some precursors which for the FK506-binding protein (FKBP), as depicted for may be used for the linker (with the carboxyl functionality example in FIG.1. The abundance of FKBP (millimolar) in protected). blood compartments, such as red blood cells and lympho 0043. The drug moiety X may, in certain embodiments of cytes, makes it likely that a significant proportion of a dose of the invention, preferably be a protease inhibitor. The drug bifunctional compounds comprising FK506 would wind up moiety may be derived from a known protease inhibitor, bound in the blood. A mechanism that tends to increase the which is preferably effective against HIV and/or against portion of the protease dose that winds up in red blood cells another prevalent virus such as hepatitis B. The drug moiety and CD4+ lymphocytes will have a favorable effect on anti preferably has a functionality which may readily and control HIV activity, as these sites are prime targets of HIV lably be made to react with a linker precursor. HIV protease and viral load. The steric bulk conferred by FKBP would inhibitors include, for example, , , hinder a protease inhibitor moiety from fitting into the bind ritonavir, indinavir, , amprenavir, , ing pocket of CYP450 enzymes and so would prevent degra mozenavir, TMC114 (), , and lopinavir. dation via this class of enzymes. The known HIV protease inhibitors are generally susceptible 0050. An inactive form of FK506 may be preferable in to metabolism and Subsequent deactivation by hepatic first some applications to avoid the possibility of side effects due pass clearance mechanisms. Protease inhibition is an active to the possible interaction of the active FK506-FKBP com area of research. plex with calcineurin. it may be advantageous to use FKBP 0044) Certain of the concepts of this invention have appli binding molecules such as synthetic ligands for FKBP (SLFs) cability to other drug moieties besides protease inhibitors. In described by Holt et al., Supra. This class of molecule is lower general, bifunctional compounds may usefully be made with molecular weight than FK506, and that is generally advanta any drug having a Suitable moiety capable of reacting with geous for drug delivery and pharmacokinetics. For illustrative linkers and which has a need for pharmacokinetic modula purposes, the diagrams will show examples of the use of US 2008/0306098 A1 Dec. 11, 2008

FK506, though it should be understood that the same strategy secondary amine is then synthesized by any appropriate tech can apply to other ligands of peptidyl prolyl isomerases Such nique, with the secondary amine in the synthesized molecule as the FKBP proteins. being protected during synthesis by an appropriate protecting 0051. The value of FK506 and other FKBP binding moi moiety Such as Boc. The protecting moiety is then removed, eties as pharmacokinetic modulating moieties of the inven leaving a primary amine which may react with a carboxyl tion is further supported by the following. FK506 (tacroli group through a variety of known chemistries for making a mus) is an FDA-approved immunosuppressant. It has been peptide bond (see, e.g., J. Mann et al., Natural Products. determined that FK506 can be readily modified such that it Their Chemistry and Biological Significance (1994), chapter loses all immunomodulatory activity but retains high affinity 3). for FKBP. FKBP is an abundant chaperone that is particularly 0058. In a further aspect of the invention, a bifunctional prevalent (-millimolar) in red blood cells (rbcs) and lympho compound comprising a protease inhibitor moiety with anti cytes. The complex between FK506-FKBP gains affinity for viral activity is formulated, for example in the form of a tablet, calcineurin and inactivation of calcineurin blocks lympho capsule, parenteral formulation, to make a pharmaceutical cyte activation and causes immunosuppression. preparation The pharmaceutical preparation may be 0052. This interesting mechanism of action is derived employed in a method of treating a patient having a viral from FK506’s chemical structure. FK506 is bifunctional; it infection against which the protease inhibitor moiety is effec has two non-overlapping protein-binding faces. One side tive. For example, if the protease inhibitor moiety is effective binds FKBP, while the other binds calcineurin. This property against the HIV virus, the pharmaceutical preparation may be provides FK506 with remarkable specificity and potency. administered to a patient infected with the HIV virus. Moreover, FK506 has a long half-life in non-transplant 0059 For the preparation of a pharmaceutical formulation patients (21 hrs) and excellent pharmacological profile. In containing bifunctional compounds as described in this appli part, this is because FK506 is unavailable to metabolic cation, a number of well known techniques may be employed enzymes via its high affinity for FKBP, which favors distri as described for example in Remington. The Science and bution into protected cellular compartments (72-98% in the Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.; Mack blood). It can be expected that suitable bifunctional com Publishing Company, 1995). pounds with an FKBP-binding pharmacokinetic modulating 0060. In a further aspect of the invention, a bifunctional moiety will likewise possess some favorable characteristics compound is formulated as part of a controlled release for of inactive FK506, namely, good pharmacokinetics and blood mulation in which an additional controlled release mecha cell distribution. nism besides the effect of the pharmacokinetic modulating 0053. In general, the pharmacokinetic modulating moiety moiety is employed to achieve desirable release characteris will have a molecular weight less than about 2000 D, less than tics. The bifunctional compound is as above, comprising a about 1600D, less than about 1300 D, less than about 1100D, drug moiety, a linker, and a pharmacokinetic modulating or less than about 900 D. moiety. In this aspect of the invention, a drug moiety may be 0054. It is also possible to coadminister the common pro a protease inhibitor or a different type of drug. tein to which the pharmacokinetic modulating moiety binds 0061. As discussed above, bifunctional compounds have with the bifunctional compound in order to modify the phar the advantage that they can favorably improve the pharmaco macokinetics to a greater degree than would be possible with kinetic characteristics of existing or new drugs. However, in just the native concentration of the common protein. spite of this important advantage, bifunctional compounds 0055. In a further embodiment of this invention, a method used in conventional dosage formulations may not provide is provided for synthesizing a bifunctional compound com enough of an advantage over the mono-functional drug mol prising protease inhibitor functionality and the ability to bind ecules to overcome the additional expense of regulatory to a common protein. approval, drug manufacture, and cost incurred to make the 0056. The synthesis of the bifunctional compound starts more complex bifunctional compound. with a choice of Suitable pharmacokinetic modulating and 0062. Many mono-functional drugs are not amenable to drug moieties. It is desirable to identify on each of these controlled release technologies due to: 1) short biological half moieties a suitable attachment point which will not result in a life, 2) potent drug with narrow therapeutic index, 3) require loss of biological function for either one. This is preferably large doses, 4) poor adsorption, 5) poor targeting, 6) low done based on the existing knowledge of what modifications solubility, 7) extensive first-pass metabolism, 8) active result or do not result in a biological function. On that basis, adsorption, 9) the time course of the circulating drug does not it may reliably be conjectured that certain attachment points agree with pharmacological response. on the pharmacokinetic and drug moieties do not affect bio 0063. Since the addition of a protein targeting moiety may logical function. Thus, for example, in FK506 there is a alter the half life, first-pass metabolism, solubility, targeting, carboxylic acid function which is Suitable as an attachment and other properties of monofunctional drugs, the bifunc point. Likewise, in FIG. 4 one sees secondary amine functions tional strategy can greatly expand the number of drugs which on three protease inhibitors which are believed not to signifi can be used in a controlled release formulation. At the same cantly affect biological function. time, a controlled release formulation of the bifunctional 0057. A general synthetic strategy is to locate a secondary compound can provide enough additional value that may amine on the drug moiety at which the drug moiety can be overcome the disadvantage of the extra cost of obtaining split (so that the secondary amine does not form part of any regulatory approval for and manufacturing bifunctional drugs cycle in the drug moiety). The secondary amine is chosen compared to mono-functional drugs. Such that, from experimental or other considerations, it is 0064. By combining controlled release technologies with believed that the drug will retain its efficacy if only the portion bifunctional drugs, it may be possible to 1) enable the use of of the drug moiety to one side of the secondary amine is a wider repertoire of monofunctional drugs in controlled present. The portion of the drug moiety to that side of the release formats by the addition of a targeting moiety; 2) US 2008/0306098 A1 Dec. 11, 2008

enhance the value of bifunctional molecules by lowering the desipramine. The formation of bifunctional compounds is required dose and increasing the efficacy of the bifunctional particularly appropriate for these drugs. compound relative to a free drug control; 3) alter the pharma 0070. It is to be understood that while the invention has cokinetic properties of mixtures of drugs where one or both been described in conjunction with the preferred specific drugs in the mixture is bifunctional. embodiments thereof, the foregoing description is intended to 0065 For example, in a mixture of methotrexate and illustrate and not limit the scope of the invention. Other clomethiazole, it may be useful to increase the first-pass aspects, advantages, and modifications within the scope of the metabolism of methotrexate using a conjugate to a pharma invention will be apparent to those skilled in the art to which cokinetic altering moiety such as FK506 or other protein or the invention pertains. nucleic acid binding ligand. However, it may be undesirable 0071 All patents, patent applications, and publications to alter the pharmacokinetics of clomethiazole where reduc mentioned herein are hereby incorporated by reference in ing drug clearance might lead to toxic side effects if the their entireties. However, where a patent, patent application, patient's liver function is damaged. or publication containing express definitions is incorporated 0066. In principle, almost any controlled release technol by reference, those express definitions should be understood ogy may be applied to a bifunctional compound Such as one to apply to the incorporated patent, patent application, or containing a protease inhibitor. A wide variety of controlled publication in which they are found, and not to the remainder release technologies are known. See, e.g., Encyclopedia of of the text of this application, in particular the claims of this controlled drug delivery (Edith Mathiowitz ed., 1999), and application. Modern Pharmaceutics (Gilbert S. Banker & Christopher T. Rhodes eds., 4th ed. 2002), especially chapter 15. Exemplary Experimental methods of controlled drug delivery include slow erosion, (0072. The general method for testing P1-FK506 conju erosion core, pellets in capsules, pellets in tablets, leaching, gates is to synthesize the bifunctional compound and test ion-exchange resins, complexation, microencapsulation, flo whether the bifunctional version maintains activity against tation-diffusion, and osmotic pumps. HIV protease. Then, the P450 susceptibility of the bifunc 0067. The drugs that are candidates for bifunctionalization tional compound was tested in a series of fluorescence-based and then the application of other controlled release technolo assays. An important aspect of these experiments was the gies will generally be those in which other controlled release addition of FKBP sources, such as recombinant protein, red technologies by themselves do not produce a satisfactory blood cells or lymphocytes. The desired outcome is pro release profile, and bifunctionalization is both possible (e.g., longed drug lifetime in the presence of an FKBP source there are suitable linkage points in the drug which do not combined with potent anti-HIV protease activity. The syn affect function) and yet does not produce a release profile thetic schemes and methods for determining drug lifetime in which is fully adequate. the presence of P450s will be discussed. 0068 Drugs which are candidates for bifunctionalization 0073. The FDA-approved PIs amprenavir, lopinavir and followed by application of other controlled release technolo ritonavir are models for generating FK506-coupled deriva gies may belong to a wide variety of therapeutic categories tives. These drugs are chosen based on their known defects in including, but not limited to: analeptic agents; analgesic pharmacological characteristics. Like all FDA-approved PIs. agents; anesthetic agents; antiarthritic agents; respiratory these compounds are based on peptide Substrate sequences. drugs, including antiasthmatic agents; anticancer agents, Therefore, we developed a modular amide-based approach to including antineoplastic drugs; anticholinergics; anticonvul the synthesis of bifunctional compounds (see below). Spe sants; antidepressants; antidiabetic agents; antidiarrheals; cifically, an amine in the P2 region (shaded boxes, FIG. 4) is antihelminthics; antihistamines; antihyperlipidemic agents; targeted for attachment to a pendant carboxylate on FK506. antihypertensive agents; anti-infective agents such as antibi This region is selected based on structure activity relation otics and antiviral agents; antiinflammatory agents; antimi ships that show the P2 site as a region in which modifications graine preparations; antinauseants; antiparkinsonism drugs; are permitted without affecting biological activity. This antipruritics; antipsychotics; antipyretics; antispasmodics; approach is amenable to high throughput, Solid-phase synthe antitubercular agents; antiulceragents; antiviral agents; anxi sis if large-scale diversification becomes necessary. These olytics; appetite Suppressants; attention deficit disorder conjugates between FK506 and a PI-derivative are expected (ADD) and attention deficit hyperactivity disorder (ADHD) to have better activity than the FDA-approved drug on which drugs; cardiovascular preparations including calcium chan they are based. nel blockers, antianginal agents, central nervous system 0074 Another feature of the synthesis is that linker bear (CNS) agents, beta-blockers and antiarrhythmic agents; cen ing an amine on one end and a protected carboxylate on the tral nervous system stimulants; cough and cold preparations, other can be readily installed as shown in FIG. 6. This capa including decongestants; diuretics; genetic materials; herbal bility is designed to allow facile synthesis of analogs with a remedies; hormonolytics; hypnotics; hypoglycemic agents; variety of linker properties (length, flexibility, solubility, etc). immunosuppressive agents; leukotriene inhibitors; mitotic For example, longer linkers can be installed if a shorter linker inhibitors; muscle relaxants; narcotic antagonists; nicotine; reduces protease inhibition. nutritional agents, such as vitamins, essential amino acids and fatty acids; ophthalmic drugs such as antiglaucoma agents; Example 1 parasympatholytics; peptide drugs; psychostimulants; seda tives; steroids, including progestogens, estrogens, corticos FKBP Protection of Curcumin Conjugates teroids, androgens and anabolic agents; Smoking cessation 0075 An amyloid ligand, curcumin, is known to be a good agents; sympathomimetics; tranquilizers; and vasodilators substrate for CYP3.a4 (a common P450 enzyme). We inves including general coronary, peripheral and cerebral. tigated whether conjugates between curcumin and FK506 0069 Exemplary drugs presently known to have high would also be substrates for the enzyme. To test this possibil first-pass metabolism include HIV protease inhibitors as dis ity, we utilized a well-known fluorescence-based CYP3.a4 cussed above, paclitaxel, methotrexate, vinblastine, Vera assay. This assay, marketed by Invitrogen (Carlsbad, Calif.), pamil, morphine, lidocaine, acebutolol, isoproterenol, and under the name VIVID probes, relies on cytochrome-medi US 2008/0306098 A1 Dec. 11, 2008

ated production of a fluorescent marker from a model sub FK506 moiety, a common pool of FK506-linker molecules strate. When a compound, such as curcumin, binds to the may be used on all three synthetic Schemes. P450, it displaces the substrate and reduces the rate of pro duction of the fluorescent product. When we tested curcumin Example 5 FK506 conjugates in this assay, we found that both curcumin and the conjugate were good Substrates for the enzyme. Thus, Test of Efficacy of Bifunctional Compounds Against attachment of FK506 did not appear to significantly alter HIV Protease curcumin’s susceptibility to degradation by CYP3.a4. How 0080. To analyze efficacy of the conjugates of Example 2 ever, when we supplied a source of human FKBP (in this case, against HIV protease, a commercial biochemical assay was recombinant bacterially-expressed protein), we observed used. The AnaSpec (www.anaspec.com) 71126 HIV-1 pro very different results as shown in FIG. 3. In the presence of tease assay was used according to the manufacturer's direc FKBP, the curcumin-FK506 conjugate is protected from deg tion, except that 1 uM recombinant FKBP was used in some radation. FKBP was unable to protect unmodified curcumin, wells. The assay uses a quenched fluorophore substrate. Pro which suggests that the ability to bind FKBP is required for teolytic cleavage of the Substrate reverses the quench and FKBP to have a protective effect. It is believed that FKBP releases a fluorescent substrate. Experiments were performed protects the curcumin conjugates from degradation by steri in 96-well black Costar plates in real time using a Molecular cally hindering CYP3.a4binding. In the presence of cellular Devices M5 plate reader. In all cases, fluorescence correc FKBP sources, this effect would be predicted to be increased tions were made for all drugs and proteins. Linear portions of because the compartmentalization of the conjugate further the curve were used to predict drug lifetime and relative Ki reduces availability to P450 enzymes. values. I0081. The AnaSpec assay kit shows amprenavir bifunc Example 2 tional retains inhibitory activity against HIV protease. It is twice as effective as the monofunctional TMC114, showing Synthesis of Amprenavir Conjugate that linker choice has resulted in optimized activity of bifunc 0076. The synthesis of a conjugate based on amprenavir tional relative to monofunctional. proceeded as outlined below. FIG. 6 depicts the overall syn I0082 Similarly, a commercial Invitrogen P2856 assay thesis. Briefly, a commercially available Phe-derived epoxide was used to test for degradation via CYP3.a4 in accordance is opened with a valine isostere. The resulting compound is with the manufacturer's directions. coupled to Boc-protected aminobenzenesulfonyl chloride. I0083. In the presence of 1 uM FKBP, the bifunctional is Deprotection of the Boc groups is followed by coupling to an completely protected from degradation via the CYP3a4. The activated acid derivative of SLF using 1-ethyl-3-(3-dimethy monofunctional TMC114 compound is >70% degraded laminopropyl) carbodiimide hydrochloride (EDC) and N-hy under the same condition. In the absence of FKBP, the bifunc droxylsuccinimide (NHS) (10 equivalents EDC to 1 equiva tional is over 70% degraded by the CYP450 enzyme. lent NHS). The coupling takes place in dimethylformamide 1. A method for modulating at least one pharmacokinetic (DMF) at room temperature over four hours. Relative nucleo property of a protease inhibitor upon administration to a host, philicity of the two amines is used to direct amide formation; the method comprising: the benzyl amino group is believed to have diffuse electron administering to the host an effective amount of a bifunc density and lowered reactivity. We carried out water work-up tional compound of less than about 5000 daltons com and flash chromatography on silica gel using 1:1 ethyl prising the protease inhibitor or an active derivative, acetate:MeOH. Overall yield was very roughly 15%. fragment or analog thereof and a pharmacokinetic 0077. The linkers shown in FIG. 5 may be coupled to modulating moiety, FK506 or SLF via EDC-mediated amide formation followed by deprotection of the newly installed carboxylate. This acid wherein the pharmacokinetic modulating moiety binds to is then used for conjugation to the amprenavir-based mol at least one intracellular protein and wherein the bifunc ecule as above. The linker can be readily altered to enhance tional compound has at least one modulated pharmaco solubility or other physical characteristics of the bifunctional kinetic property upon administration to the host as com compound. pared to a free drug control that comprises the protease 0078. The amprenavir conjugate of this example may also inhibitor. be regarded as a TMC114 conjugate, because TMC114 shares 2. The method according to claim 1, wherein the pharma with amprenavir the structure to the right of the attachment cokinetic property is selected from the group consisting of point used in this example. half-life, hepatic first-pass metabolism, volume of distribu tion, and degree of blood protein binding. EXAMPLES 3-4 3. The method according to claim 1, wherein the bifunc tional compound is administered as a pharmaceutical prepa Synthesis of Lopinavir and Ritonavir Conjugates ration. 4. The method according to claim 1, wherein the host is a 0079. The syntheses of two additional P1-FK506 conju mammal. gates may proceed in a fashion generally similar to that employed for the amprenavir-based molecule, as shown in 5. The method according to claim 1, wherein the pharma FIGS. 7A-7B.. An advanced “Phe-Phe intermediate 1 can cokinetic property is half-life. serve as a common precursor for both the lopinavir- and 6. The method of claim 1, wherein the pharmacokinetic ritonavir-based compounds. Amide formation with one of property is hepatic first-pass metabolism. two carboxylates provides the branch point for the two 7. The method of claim 1, wherein the at least one intrac schemes. In both cases, Boc deprotection provides a handle ellular protein bound comprises an FK506 binding protein, for creation of the amide with FK506. Various linkers of FIG. tubulin, actin, a heat shock protein, or albumin. 5 may be employed to provide additional diversity and desir 8. The method of claim 1, wherein the at least one intrac able characteristics. Because the linkers are installed on the ellular protein bound comprises an FK506 binding protein. US 2008/0306098 A1 Dec. 11, 2008

9. The method of claim 1, wherein the pharmacokinetic 19. The method according to claim 17 where the presenter modulating moiety has a molecular weight less than about protein ligand has a molecular weight of less than 5000 dal 1100 daltons. tOnS. 10. A bifunctional compound comprising a protease inhibi 20. The method of claim 17, wherein the drug is a protease tor moiety, a linker, and a pharmacokinetic modulating moi inhibitor. ety, wherein the linker is attached to the protease inhibitor 21. The method according to claim 17, wherein the host is moiety and the pharmacokinetic modulating moiety, and the a mammalian host. pharmacokinetic modulating moiety gives the bifunctional 22. The method according to claim 17, wherein the mam compound a different pharmacokinetic behavior from that of malian host is human. the protease inhibitor moiety in the absence of the linker and 23. The method of claim 17 where the presenter protein pharmacokinetic modulating moiety, wherein the molecular ligand is FK506. weight of the bifunctional compound is less than about 5000 daltons and the molecular weight of the pharmacokinetic 24. The method of claim 17 where the presenter protein modulating moiety is less than about 1100 daltons. ligand target is a peptidyl prolyl isomerase. 11. The bifunctional compound according to claim 10 25. The method of claim 17 where the presenter protein where the attachment point of the linker to the protease inhibi ligand targets an intracellular protein. tor moiety has been optimized for best drug activity relative to 26. The method of claim 17 where the presenter protein other bifunctional compounds having the same protease ligand binds to a derivative of cyclosporin. inhibitor moiety and pharmacokinetic modulating moiety. 27. The method of claim 17 where the presenter protein 12. The bifunctional compound according to claim 10 ligand binds to FKBP. where the attachment point of the linker to the pharmacoki 28. The method of claim 17 where the presenter protein netic modulating moiety has been optimized for best drug ligand binds to albumin. activity relative to other bifunctional compounds having the 29. The method according to claim 17, wherein the con same protease inhibitor moiety and pharmacokinetic modu trolled release mechanism is slow erosion. lating moiety. 30. The method according to claim 17, wherein the con 13. The bifunctional compound according to claim 10 trolled release mechanism is erosion core only. where the attachment point of the linker to the pharmacoki 31. The method according to claim 17, wherein the con netic modulating moiety has been optimized to improve phar trolled release mechanism is pellets in capsules. macokinetics relative to other bifunctional compounds hav ing the same protease inhibitor moiety and pharmacokinetic 32. The method according to claim 17, wherein the con modulating moiety. trolled release mechanism is pellets in tablets. 14. Abifunctional compound according to claim 10, where 33. The method according to claim 17, wherein the con the linker comprises at least three carbons. trolled release mechanism is leaching. 15. Abifunctional compound according to claim 10, where 34. The method according to claim 17, wherein the con the efficacy of the bifunctional compound in the presence of trolled release mechanism is ion-exchange resins. a suitable protein to which the pharmacokinetic modulating 35. The method according to claim 17, wherein the con moiety couples is increased relative to the efficacy of the trolled release mechanism is complexation. protease inhibitor moiety. 36. The method according to claim 17, wherein the con 16. Abifunctional compound according to claim 15, where trolled release mechanism is microencapsulation. the efficacy of the bifunctional compound in the presence of 37. The method according to claim 17, wherein the con a suitable protein to which the pharmacokinetic modulating trolled release mechanism is flotation-diffusion. moiety couples is increased by a factor of at least about 2 38. The method according to claim 17, wherein the con relative to the efficacy of the protease inhibitor moiety. trolled release mechanism is an osmotic pump. 17. In a method of administering a drug to a host in need of 39. A method for modulating at least one pharmacokinetic said drug, the improvement comprising: property of a drug upon administration to a host, the method administering to said host an effective amount of a bifunc comprising: tional compound comprising said drug or a derivative, administering to the host an effective amount of a bifunc fragment or analog thereoflinked to a ligand for a pre tional compound of less than about 5000 daltons com senter protein endogenous to said host, wherein said prising the drug oran active fragment, analog, or deriva drug binds to a drug target and said ligand binds to a tive thereof and a pharmacokinetic modulating moiety, presenterprotein that is not said drug target, wherein the wherein the pharmacokinetic modulating moiety binds bifunctional compound is administered in a controlled to at least one intracellular protein and wherein the release formulation that operates according to a con bifunctional compound modulates at least one pharma trolled release mechanism in addition to whatever con cokinetic property and one efficacy property upon trolled release is provided by the ligand. administration to the host as compared to a free drug 18. The method according to claim 17 where the bifunc control. tional compound has a molecular weight of less than 5000 daltons.