US006955888B2 (12) United States Patent (10) Patent N0.: US 6,955,888 B2 Zerangue et al. (45) Date of Patent: Oct. 18, 2005

(54) METHODS OF SCREENING AGENTS, Rieger R. et al. Glossary of GeneticszClassical and Molecu CONJUGATES OR CONJUGATE MOIETIES lar. 5”1 ed. p 16—17, Springer—Verlag, NeW York, 1991.* FOR TRANSPORT BY A PEPT2 Liu W. et al. Biochimica et Biophysica Acta 1235:461—466, TRANSPORTER 1995 .* Liang R, et al. J. Biol. Chem. 270(12), 6456—6463, 1995.* (75) Inventors: Noa Zerangue, San Carlos, CA (US); Doring et al., “Delta—aminolevulinic Acid Transport by Tracy Dias, Pleasanton, CA (US); Intestinal and Renal Peptide Transporters and Its Physiologi William J Dower, Menlo Park, CA cal and Clinical Implications,” J. Clin. Invest. (Us) 101(12):2761—2767 (1998). (73) Assignee: XenoPort, Santa Clara, CA (US) Fei et al., Nature 368: 563—566 (1994). Miyamoto et al., Biochimica et Biophysica Acta 1305:34—38 ( * ) Notice: Subject to any disclaimer, the term of this (1996). patent is extended or adjusted under 35 Liu et al., Biochimica et Biophysica Acta 1235:461—466 U.S.C. 154(b) by 58 days. (1995). Boll et al., Prod. Natl. Acad. Sci. USA 93:284—289 (1996). (21) Appl. No.: 10/170,217 Saito et al., Biochimica et Biophysica Acta 1280:173—177 (1996). (22) Filed: Jun. 11, 2002 Iseki et al., Intestinal Absorption of Several [3—Lactam (65) Prior Publication Data Antiobiotics, J. Pharm. Dyn., 7, 768—775 (1984). Ganapathy et al., Differential Recognition of [3—Lactam US 2003/0017964 A1 Jan. 23, 2003 Antibiotics by Intestinal and Renal Peptide Transporters, PEPT 1 and PEPT 2, The Journal of Biological Chemistry, Related US. Application Data vol. 270, No. 43, Issue of Oct. 27, pp. 25672—25677, 1995. (60) Provisional application No. 60/361,002, ?led on Mar. 1, 2002, and provisional application No. 60/297,732, ?led on Jun. 11, 2001. (Continued) (51) Int. Cl.7 ...... G01N 33/53; G01N 33/567; Primary Examiner—Robert S. Landsman C12P 21/06; C12N 1/20 (74) Attorney, Agent, or Firm—ToWnsend and ToWnsend (52) US. Cl...... 435/72; 435/721; 435/691; and CreW LLP 435/252.3 (57) ABSTRACT (58) Field of Search ...... 435/7.21, 7.1, 435/7.2, 69.1, 252.3, 471; 530/350 The invention provides methods of screening agents, con jugates or conjugate moieties, linked or linkable to agents, (56) References Cited for capacity to be transported as substrates through the PEPT2 transporter. The invention also provides methods of U.S. PATENT DOCUMENTS treatment involving delivery of agents that either alone, or as 5,710,018 A 1/1998 DantZig et al. a result of linkage to a conjugate moiety, are substrates of the PEPT2 transporter. The invention also provides conjugates OTHER PUBLICATIONS comprising a pharmaceutical agent Which is linked to a Ayala F] and Kiger Jr JA. Modern Genetics. p 45—48. James conjugate moiety that is a substrate for a PEPT2 transporter. W. Behnke, ed. The Benjamin/Cummings Publishing Co., MenloW Park, CA, 1980* 12 Claims, 7 Drawing Sheets

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OTHER PUBLICATIONS Teuscher et al., Functional Evidence for Presence of PEPT2 in Rat Choroid Plexus: Studies with Glycylsarcosine’, The Siarnak A. Adibi, The Oligopeptide Transporter (PEPT—1) in Journal of Pharmacology and Experimental Therapeutics, Human Intestine: Biology and Function, Special Reports vol. 294, No. 2 (2000). and RevieWs, The American Gastroenterological Associa tion, 1997. Terada et al., CharacteriZation of Stably Transfected Kidney Randall J. Mrsny, Oligopeptide Transporters as Putative Epithelial Cell Line Expressing Rat H+/Peptide Contrans Therapeutic Targets for Cancer Cells, Pharmaceutical porter PEPT1: Localization of PEPT1 and Transport of Research, vol. 15, No. 6, 1998. [3—Lactarn Antibiotics 1, The Journal of Pharmacology and Terada et al., Recognition of [3—Lactarn antibiotics by rat Experimental Therapeutics, vol. 281, No. 3 (1997). peptide transporters, PEPT1 and PEPT2, in LLC—PK Cells, GonZales et al., An Oligopeptide Transporter is Expressed at 1997, The American Physiological Society. High Levels in the Pancreatic Carcinorna Cell Lines AsPc—1 Iseki et al., Multiplicity of the H+ —Dependent Transport and Capan—2 1, Cancer Research 58, 519—525. Feb. 1, 1998. Mechanism of Dipeptide and Anionic [3—Lactarn Antibiotic Inui et al., Physiological and Pharrnacological Implications Ceftibuten in Rat Intestinal Brush—Border Mernbrane, The of Peptide Transporters, PEPT1 and PEPT2, Nephrology Journal of Pharmacology and Experimental Therapeutics, Dialysis Transplantation, Nephrol Dial Transplant (2000) 15 vol. 289, No. 1 (1999). (Suppl 6): 11—13. KitagaWa and Sugaya, Chaacteristics of Uptake of Cefroxa Tarnai et al., Functional Expression of Transporter for dine by Rabbit Srnall Intestinal Brush Border Mernbrane [3—Lactarn Antibiotics and Dipeptides in Xenopus Laevis Vesicles, Biol. Pharrn. Bull. 19(2) 268—273 (1996). Oocytes Injected With Messenger RNA from Human, Rat Saitoh et al., Restricted Intestinal Absorption of Some and Rabbit Srnall Intestines 1, The Journal of Pharmacology [3—Lactarn Antibiotics by an Energy—Dependent Ef?ux Sys and Experimental Therapeutics, vol. 273, No.1 (1995). tern in Rat Intestine, Pharmaceutical Research, vol. 14, No. Naasani et al., Comparison of the Transport Characteristics 5, 1997. of Ceftibuten in Rat Renal and Intestinal Brush—Border Otto and Bauer, Dipeptide Uptake: A Novel Marker for Mernbranes, Biochirnica et Biophysica Acta 1231 (1995) Testicular and Ovarian Macrophages, The Anatornical 163—168. Record 245:662—667 1996. Ganapathy and Liebach, Peptide Transporters, Department DantZig et al., Association of Intestinal Peptide Transport of Bochernistry and Molecular Biology, Medical College of With a Protein Related to the Cadherin Superfarnily, Science, Augusta, Georgia, pp. 395—400 (1996). vol. 264, Apr. 15, 1994. Zhu et al., Differential Recognition of ACE Inhibitors in Yuasa et al., Peptide Carrier—Mediated Transport in Intesti Xenopus Laevis Oocytes Expressing Rat PEPT1 and nal Brush Border Mernbrane Vesicles of Rats and Rabbits: PEPT2, Pharmaceutical Research, vol. 17, No. 5, 2000. Cephradine Uptake and Inhibition, Pharmaceutical Ganapathy et al., Interaction of Anionic Cephalosporins With Research, vol. 10, No. 3, 1993. the Intestinal and Renal Peptide Transporters PEPT 1 and Inui et al., Transepithelial Transport of oral Cephalosporins PEPT2, Biochirnica et Biophysica Acta 1324 (1997) by Monolayers of Intestinal Epithelial Cell Line Caco—2: 296—308. Speci?c Transport Systems in Apical and Basolateral Mern Fei et al., Preferential Recognition of ZWitterionic Dipep branes: The Journal of Pharmacology and Experimental tides as Transportable Substrates by the High—Af?nity Pep Therapeutics, vol. 261, No. 1 (1992). tide Transporter PEPT2, Biochirnica et Biophysica Acta Iseki et al., Comparison of Transport Characteristics of 1418 (1999) 344—351. Amino [3—Lactarn Antibiotics and Dipeptides Across Rat Dixon and MiZen, Absorption of Amino Penicillins frorn Intestinal Brush Border Mernbrane, J. Pharrn. Pharrnacol. Everted Rat Intestine, J. Physiol. (1977), 269, pp. 549—559. 1989, 41:628—632. Lee et al., Biopharrnaceutics of Transrnucosal Peptide and DantZig and Bergin, Carrier—Mediated Uptake of Cephal Protein Drug Administration: Role of Transport Mecha exin in Human Intestinal Cells, Biochemical and Biophysi nisrns With a Focus on The Involvement of PEPT1, Journal cal Research Communications, pp. 1082—1087, vol. 155, of Controlled Release 62 (1999) 129—140. No. 2, 1988. Han et al., CHO/HPEPT1 Cells Overexpressing the Human Peptide Transporter (HPEPT1) as an Alternative in Vitro Meredith and Boyd, Structure and Function of Eurkryotic Model for Peptidornirnetic Drugs, Journal of Pharmaceutical Peptide Transporters, CMLS Cellular and Molecular Life Sciences/vol. 88, No. 3, Mar. 1999. Sciences, CMLS, Cell. Mol. Life Sci. 57 (2000) 754—778. Tarnai et al., The Predorninant Contributions of Oligopep Ogihara et al., Irnrnuno—LocaliZation of H +/Peptide tide Transporter PEPT1 to Intestinal Absorption of [3—Lac Cotransporter in Rat Digestive Tract, Biochemical and Bio tarn Antiobiotics in the Rat Srnall Intestine, J. Pharrn. physical Research Cornrnunications 220 848—852 (1996). Pharrnacol. 1997, 49; 796—801. Leibach and Ganapathy, Peptide Transporters in the Intes Ming et al., Human Dipeptide Transporter, Hpept1, Stably tine and the Kidney, Annu. Rev. Nutr. 1996. 16:99—119. Transfected into Chinese Harnster Ovary Cells, Pharrnaceu tical Research, vol. 13, No. 11, 1996. * cited by exarniner U.S. Patent Oct.18,2005 Sheet 1 0f 7 US 6,955,888 B2

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US 6,955,888 B2 1 2 METHODS OF SCREENING AGENTS, KnoWn examples of solute carrier systems include tWo CONJUGATES OR CONJUGATE MOIETIES peptide transporters, PEPT1 and PEPT2. The endogenous FOR TRANSPORT BY A PEPT2 substrates for these transporters are small peptides consist TRANSPORTER ing of tWo or three amino acids. These transporters function CROSS-REFERENCE TO RELATED in the absorption of peptides arising from the digestion of APPLICATIONS dietary proteins (small intestine) and in the reabsorption of peptides present in the glomerular ?ltrate. The present application derives priority from US. Ser. The human intestinal peptide transporter (PEPT1) and the No. 60/361,002, ?led Mar. 1, 2002 and US. Ser. No. 10 human kidney peptide transporter (PEPT2) exhibit about 60/297,732 ?led on 11 Jun. 2001, both incorporated by 48% identity at the amino acid level. Neither peptide trans reference in their entirety for all purposes. porter shoWs signi?cant sequence identity to other knoWn BACKGROUND OF THE INVENTION mammalian sequences- they are both about 20% identical to PHT1 and PHT2. The tWo transporters shoW some differ Recent advances in the pharmaceutical industry have 15 resulted in the formation of an increasing number of poten ences in the recognition of [3-lactam antibiotics as substrates tial therapeutic agents. HoWever, formulating the com as Well as their marked differences in af?nity for many pounds for effective oral bioavailability has proven dif?cult substrates. As such, PEPT1 is a high capacity, loW-affinity because of problems associated With uptake and high sus transporter and PEPT2 is a high af?nity transporter. Both ceptibility to metabolic enZymes. 20 transporters accept small peptides as substrates and are driven by a transmembrane electrochemical H+ gradient. Natural transporter proteins are involved in the uptake of various molecules into and/or through cells. In general, tWo PEPT1 and PEPT2 have been reported to shoW different major transport systems exist: solute carrier-mediated sys patterns of expression in different human tissues. PEPT1 has been reported to be expressed predominantly in the intestine, tems and receptor mediated systems. Carrier-mediated sys 25 tems use transport proteins that are anchored to the cell and also in the kidney (pars convoluta), and liver, With small membrane, typically by a plurality of membrane-spanning amounts of expression in the brain and pancreas. Fei et al., loops and function by transporting their substrates via an Nature 386:563—566 (1994) and Miyamoto et al., Bio energy-dependent ?ip-?op or other mechanism, exchange chimica er BiophysicaActa 1305:34—38 (1996). By contrast, and other facilitative or equilibrative mechanisms. Carrier 30 PEPT2 has been reported to be expressed in the kidney and mediated transport systems are involved in the active or brain, With loWer expression reported in the lung, liver and non-active, facilitated transport of many important nutrients heart and no expression reported in the small intestine. Liu such as vitamins, sugars, and amino acids. The carrier et al., Biochimica et Biophysica Acta 1235:461—466 (1995) and Boll et al., Proc. Natl. Acad. Sci. USA 93:284—289 systems result in transport into the enterocytes from blood or 35 lumen, and across the epithelial cell layer from lumen into (1996) and Saito et al., Biochimica et Biophysica Acta blood (absorption) or blood to lumen (secretion). Carrier 1280:173—177 (1996). Because of the vieW that PEPT1 and mediated transporters are also present in organs such as the not PEPT2 is expressed in the intestine, existing efforts to liver and kidney, in Which the proteins are involved in the improve oral delivery of drugs via peptide transporters have excretion or re-absorption of circulating compounds. 40 focused on identifying pharmacological agents that are, or can be modi?ed to be, substrates for PEPT1. Receptor-mediated transport systems differ from the carrier-mediated systems in that these systems usually uti SUMMARY OF THE CLAIMED INVENTION liZe proteins that span the cell membrane only a single time. The present invention provides a conjugate comprising a Furthermore, substrate binding triggers an invagination and 45 encapsulation process that results in the formation of various pharmaceutical agent Which is linked to a conjugate moiety transport vesicles to carry the substrate (and sometimes that is a substrate for a PEPT2 transporter. The conjugate has other molecules) into and through the cell. This process of a Vmax of at least 1% of Gly-Sar for the PEPT2 transporter. membrane deformations that result in the internaliZation of The conjugate has a greater Vmax for PEPT2 than the certain substrates and their subsequent targeting to certain 50 pharmaceutical agent alone, i.e., Without the conjugate moi locations in the cytoplasm is generally referred to as endocy ety. tosis. Preferably, the conjugate has a Vmax for the PEPT2 Polar or hydrophilic compounds are typically poorly transporter of at least 5%, more preferably at least 10%, even more preferably at least 20%, still more preferably at least absorbed through an animal’s intestine as there is a substan 55 tial energetic penalty for passage of such compounds across about 50%, and most preferably at least 100%, respectively, the lipid bilayers that constitute cellular membranes. Many of the Vmax of substrate Gly-Sar for PEPT2. nutrients that result from the digestion of ingested foodstuffs Preferably, the pharmaceutical agent Without the conju in animals, such as amino acids, di- and tripeptides, gate moiety has a Vmax for the PEPT2 transporter of less monosaccharides, nucleosides and Water-soluble vitamins, 60 than 1% of the Vmax of substrate Gly-Sar for PEPT2. are polar compounds Whose uptake is essential to the Preferably, the ratio of Vmax betWeen the conjugate and viability of the animal. For these substances there exist Gly-Sar is greater for the PEPT2 transporter than for the speci?c mechanisms for active transport of the solute mol PEPT1 transporter, more preferably the ratio for the PEPT2 ecules across the intestinal epithelia. This transport is fre 65 transporter is at least tWice, even more preferably at least 10 quently energiZed by co-transport of ions doWn a concen times, and most preferably at least 100 times, respectively, tration gradient. of the ratio for the PEPT1 transporter. US 6,955,888 B2 3 4 The present invention also provides a method of treatment A “transport protein” is a protein that has a direct or comprising administering a pharmacologically effective indirect role in transporting a molecule into and/or through amount of the conjugate to a patient as Well as a method of a cell. The term includes, for example, membrane-bound making a pharmaceutical composition comprising formulat proteins that recogniZe a substrate and effects its entry into, ing the conjugate With a pharmaceutically acceptable carrier. or exit from a cell by a carrier-mediated transporter or by The present invention further provides a method of receptor-mediated transport. These proteins are sometimes referred to as transporter proteins. The term also includes screening pharmaceutical agents, conjugates and/or conju intracellularly expressed proteins that participate in traf?ck gate moieties for pharmacological administration. The ing of substrates through or out of a cell. The term also method includes providing cell(s) expressing PEPT2 includes proteins or glycoproteins exposed on the surface of transporter, contacting the cell(s) With the agent, conjugate a cell that do not directly transport a substrate but bind to the or moiety and determining Whether the agent, conjugate, or substrate holding it in proximity to a receptor or transporter moiety passes into and/or through the cell by Way of the protein that effects entry of the substrate into or through the transporter. Preferably, the cell(s) is transfected With DNA cell. Examples of carrier proteins include: the intestinal and encoding the PEPT2 transporter. More preferably, the cell(s) liver bile acid transporters, dipeptide transporters, oligopep is an oocyte injected With nucleic acid encoding the PEPT2 tide transporters, simple sugar transporters (e.g., SGLTl), transporter. Even more preferably, the cell(s) exhibits no phosphate transporters, monocarboxcylic acid transporters, detectable PEPTl receptor. transporters comprising P-glycoproteins, organic anion The invention also provides methods of manufacturing a transporters (OATP), and organic cation transporters. pharmaceutical composition. Such method include linking Examples of receptor-mediated transport proteins include: an agent to a conjugate moiety to form a conjugate Wherein viral receptors, immunoglobulin receptors, bacterial toxin the conjugate is transported by the PEPT2 transporter With receptors, plant lectin receptors, bacterial adhesion a Vmax of at least 1% of the Vmax of the substrate Gly-Sar. receptors, vitamin transporters and cytokine groWth factor 25 The conjugate is then formulated With a carrier as a phar receptors. maceutical composition. A “substrate” of a transport protein is a compound Whose uptake into or passage through a cell is facilitated by the BRIEF DESCRIPTIONS OF THE FIGURES transport protein. The term “ligand” of a transport protein includes sub FIG. 1 shoWs uptake of cephradine by Caco-2 cells. strates and other compounds that bind to the transport FIG. 2 shoWs uptake of cefadroxil by Caco-2 cells. protein Without being taken up or transported through a cell. FIG. 3 shoWs F moc-amino acids. Some ligands by binding to the transport protein inhibit or antagoniZe uptake of the substrate or passage of substrate FIG. 4 shoWs Boc-Alloc amino acids. 35 through a cell by the transport protein. Some ligands by FIG. 5 shoWs carboxylic acid. binding to the transport protein promote or agoniZe uptake FIG. 6 shoWs Fmoc-alloc-amino acids. or passage of the compound by the transport protein or FIG. 7 shoWs Boc-amino acids. another transport protein. For example, binding of a ligand to one transport protein can promote uptake of a substrate by DEFINITIONS 40 a second transport protein in proximity With the ?rst trans The phrases “speci?cally binds” When referring to a port protein. protein or “speci?cally immunoreactive With” When refer The term “agent” is used to describe a compound that has ring to an antibody, refers to a binding reaction Which is or may have a pharmacological activity. Agents include determinative of the presence of the protein in the presence 45 compounds that are knoWn drugs, compounds for Which of a heterogeneous population of proteins and other biolog pharmacological activity has been identi?ed but Which are ics. Thus, under designated conditions, a speci?ed ligand undergoing further therapeutic evaluation, and compounds binds preferentially to a particular protein and does not bind that are members of collections and libraries that are to be in a signi?cant amount to other proteins present in the screened for a pharmacological activity. sample. A molecule such as an antibody that speci?cally An agent is “orally active” if it can exert a phamalogical binds to a protein often has an association constant of at least activity When administered via an oral route. 106 M-1 or 107 M_1, preferably 108 M-1 to 109 M_1, and A “conjugate moiety” refers to a compound or part of a more preferably, about 1010 M“1 to 1011 M“1 or higher. compound that does not itself have pharmacological activity HoWever, some substrates of transporter, PEPTl in 55 but Which can be linked to an agent to form a conjugate that particular, have much loWer af?nities of the order of 10—103 does have pharmacological activity. Typically, the agent has M-1 and yet the binding can still be shoWn to be speci?c. A pharmacologic activity Without the conjugate moiety. The variety of immunoassay formats may be used to select conjugate moiety facilitates therapeutic use of the agent by antibodies speci?cally immunoreactive With a particular promoting uptake of the agent via a transporter. A conjugate protein. For example, solid-phase ELISA immunoassays are moiety can itself be a substrate for a transporter or can routinely used to select monoclonal antibodies speci?cally become a substrate When linked to a compound (e.g., immunoreactive With a protein. See, e.g., HarloW and Lane valacyclovir). Thus, a conjugate moiety formed from a (1988) “Antibodies, A Laboratory Manual”, Cold Spring compound and a conjugate moiety can have higher uptake Harbor Publications, NeW York, for a description of immu 65 activity than either the compound or moiety alone. noassay formats and conditions that can be used to deter A “pharmacological” activity means that an agent at least mine speci?c immunoreactivity. exhibits an activity in a screening system that indicates that US 6,955,888 B2 5 6 the agent is or may be useful in the prophylaxis or treatment the parameters M (reWard score for a pair of matching of a disease. The screening system can be in vitro, cellular, residues; alWays >0) and N (penalty score for mismatching animal or human. Agents can be described as having phar residues; alWays <0). For amino acid sequences, a scoring macological activity notwithstanding that further testing matrix is used to calculate the cumulative score. Extension may be required to establish actual prophylactic or thera of the Word hits in each direction are halted When: the peutic utility in treatment of a disease. cumulative alignment score falls off by the quantity X from Vmax and Km of a compound for a transporter are de?ned its maximum achieved value; the cumulative score goes to in accordance With convention. Vmax is the number of Zero or beloW, due to the accumulation of one or more molecules of compound transported per second at saturating 10 negative-scoring residue alignments; or the end of either concentration of the compound. Km is the concentration of sequence is reached. For identifying Whether a nucleic acid the compound at Which the compound is transported at half or polypeptide is Within the scope of the invention, the of Vmax. In general, a high value of Vmax is desirable for default parameters of the BLAST programs are suitable. The a substrate of a transporter. A loW value of Km is desirable BLASTN program (for nucleotide sequences) uses as for transport of loW concentrations of a compound, and a 15 defaults a Word length of 11, an expectation of 10, high value of Km is desirable for transport of high concen M=5, N=—4, and a comparison of both strands. For amino trations of a compound. Vmax is affected both by the acid sequences, the BLASTP program uses as defaults a intrinsic turnover rate of a transporter (molecules/transporter Word length of 3, an expectation of 10, and the protein) and transporter density in plasma membrane that BLOSUM62 scoring matrix. The TBLATN program (using depends on expression level. For these reasons, the intrinsic protein sequence for nucleotide sequence) uses as defaults a capacity of a compound to be transported by a particular Word length of 3, an expectation of 10, and a transporter is usually expressed as the ratio Vmax of the BLOSUM 62 scoring matrix. (see Henikoff & Henikoff, compound/Vmax of a control compound knoWn to be a Proc. Natl. Acad. Soc. USA 89110915 (1989)). substrate for the transporter. 25 In addition to calculating percent sequence identity, the For sequence comparison, typically one sequence acts as BLAST algorithm also performs a statistical analysis of the a reference sequence, to Which test sequences are compared. similarity betWeen tWo sequences (see, e.g., Karlin & When using a sequence comparison algorithm, test and Altschul, Proc. Nat’l. Acad. Sci. USA 9015873—5787 reference sequences are input into a computer, subsequence 30 (1993)). One measure of similarity provided by the BLAST coordinates are designated, if necessary, and sequence algo algorithm is the smallest sum probability (P(N)), Which rithm program parameters are designated. The sequence provides an indication of the probability by Which a match comparison algorithm then calculates the percent sequence betWeen tWo nucleotide or amino acid sequences Would identity for the test sequence(s) relative to the reference occur by chance. For example, a nucleic acid is considered sequence, based on the designated program parameters. 35 similar to a reference sequence if the smallest sum prob Optimal alignment of sequences for comparison can be ability in a comparison of the test nucleic acid to the conducted, e.g., by the local homology algorithm of Smith reference nucleic acid is less than about 0.1, more preferably & Waterman, Adv. Appl. Math. 21482 (1981), by the homol less than about 0.01, and most preferably less than about ogy alignment algorithm of Needleman & Wunsch, J. Mol. 0.001. Biol. 481443 (1970), by the search for similarity method of 40 A transporter is expressed in a particular tissue, e.g., the Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 8512444 jejunum, When expression can be detected by mRNA (1988), by computeriZed implementations of these algo analysis, protein analysis, antibody histochemistry, or func rithms (GAP, BESTFIT, FASTA, and TFASTA in the Wis tional transport assays. Typically, detectable mRNA expres consin Genetics SoftWare Package, Genetics Computer 45 sion is at a level of at least 0.01% of that of beta actin in the Group, 575 Science Dr., Madison, Wis.), or by visual same tissue. Preferred transporters exhibit levels of expres inspection (see generally Ausubel et al., supra). sion in the desired tissue of at least 0.1, or 1 or 10% of that Another example of algorithm that is suitable for deter of beta actin. Conversely a transporter is not expressed in a mining percent sequence identity and sequence similarity is particular tissue (e.g., the descending colon) if expression is the BLAST algorithm, Which is described in Altschul et al., not detectable above experimental error by any of the above J. Mol. Biol. 2151403—410 (1990). SoftWare for performing techniques. Thus, transporters that are not expressed in BLAST analyses is publicly available through the National particular tissue exhibit express levels less than 0.1% of beta Center for Biotechnology Information (http1// actin, and usually less than 0.01% of beta actin. WWW.ncbi.nlm.nih.gov/). This algorithm involves ?rst iden 55 tifying high scoring sequence pairs (HSPs) by identifying Sustained release refers to release of a therapeutic or prophylactic amount of the drug or an active metabolite short Words of length W in the query sequence, Which either thereof into the systemic blood circulation over a prolonged match or satisfy some positive-valued threshold score T period of time relative to that achieved by oral administra When aligned With a Word of the same length in a database tion of a conventional formulation of the drug. sequence. T is referred to as the neighborhood Word score 60 threshold (Altschul et al., supra.). These initial neighbor DETAILED DESCRIPTION OF THE hood Word hits act as seeds for initiating searches to ?nd INVENTION longer HSPs containing them. The Word hits are then 1. Introduction extended in both directions along each sequence for as far as 65 The invention provides methods of screening agents, the cumulative alignment score can be increased. Cumula conjugates or conjugate moieties, linked or linkable to tive scores are calculated using, for nucleotide sequences, agents, for capacity to be transported as substrates through US 6,955,888 B2 7 8 the PEPT2 transporter. The invention also provides methods encoded a 729-amino acid protein of a molecular mass of 81 of treatment involving oral delivery of agents that either kDa. The overall amino acid identity Was 48% identical to alone, or as a result of linkage to a conjugate moiety, are the rat PEPT1. The rat PEPT2 has tWelve putative substrates of the PEPT2 transporter. The present methods are membrane-spanning ot-helices and four potential N-linked premised, in part, on the inventors’ results shoWing that glycosylation sites at a predicted large extracellular loop PEPT2 is expressed in the human intestine, particularly the betWeen ot-helices 9 and 10. The rat PEPT2 shoWed 83% stomach, jejunum, ileum, the ileo-caecal valve, the cecum amino acid identity to the human PEPT2. The experiments and the ascending colon. Previous Workers have reported described in the Examples shoW that human PEPT2 is that PEPT2 is present in the brain and kidney but is absent expressed in the kidney, pancreas, liver, brain, lungs, ileum, from the intestine. It is believed that the discrepancy jejunum and duodenum among other tissues. PEPT2 is also betWeen the present results and previous Work may be expressed in the CaCo2 cell line that derives from intestinal because most previous Work to determine tissues in Which cells. PEPT2 is expressed Was performed on the rat rather than the 3. Methods of Identifying Agents, Conjugates or Conjugate Moieties that are Substrates of the PEPT2 Receptor human, and because of the greater sensitivity of detection of 15 quantitative PCR employed in the present examples. Agents knoWn or suspected to have pharmacological The insight that PEPT2 is expressed in the human intes activity can be screened directly for their capacity to act as tine opens up neW strategies for design and delivery of drugs substrates of the PEPT2 transporter. Alternatively, conjugate through this transporter. Because of the different substrate moieties can be screened as substrates, and the conjugate speci?cities of PEPT1 and PEPT2, some agents, conjugates 20 moieties linked to agents having knoWn or suspected phar or conjugate moieties that are poor substrates for PEPT1 are macological activity. In such methods, the conjugate moi transported to a greater extent by PEPT2. Therefore, the eties can be linked to an agent or other molecule as a availability of PEPT2 as an alternative transporter to PEPT1 conjugate during the screening process. If another molecule broadens the range of agents, conjugates and conjugate is used, the molecule is sometimes chosen to resemble the moieties that can pass through or facilitate passage through 25 structure of an agent ultimately intended to be linked to the the intestine. Therefore, agents, conjugates or conjugate conjugate moiety for pharmaceutical use. The screening is moieties that are found to be poorly transported by PEPT1 typically performed on cells expressing the PEPT2 trans in screening assays should not necessarily be discarded but porter. In some methods, the cells are transfected with DNA can be retested for transport via PEPT2. Agents, conjugates 30 encoding the PEPT2 transporter. In other methods, natural or conjugate moieties can also be designed or screened With cells expressing the PEPT2 transporter are used. In some PEPT2 as the intended target. Expression of PEPT2 in the methods, PEPT2 is the only transporter or the only peptide kidney can result in recirculation of agents or conjugates for transporter expressed. In other methods, cells express PEPT2 from the kidney back into the systemic circulation. PEPT2 in combination With other transporters. For example, Reuptake increases the half-life of a drug or conjugate 35 in some methods, cells expressing both PEPT1 and PEPT2 moiety and hence reduces the dosage that need be admin are used. In still other methods, agents, conjugates or istered. An advantage of PEPT2 is that its higher affinity for conjugate moieties are screened on different cells expressing substrates alloWs testing of candidate substrates at loWer different transporters. For example, agents or conjugates can concentrations of the candidate substrates than PEPT1. For be screened on cells expressing PEPT2 and on cells express 40 candidate substrates that are available in only small amounts ing PEPT1. Methods of screening agents, conjugates or or Which have loW solubilities, the ability to determine conjugate moieties for passage through cells bearing a substrate properties at loW concentration of substrate is a transporter are described in WO 01/20331. signi?cant advantage. InternaliZation of a compound evidencing passage 2. PEPT1 and PEPT2 45 through transporters can be detected by detecting a signal Human PEPT1 has been cloned as a cDNA of 2263 bp from Within a cell from any of a variety of reporters. The With an open reading frame of 2127 bp encoding a protein reporter can be as simple as a label such as a ?uorophore, a of 708 amino acids (SEQ ID NO:1) by Liang, Journal of chromophore, a radioisotope, Confocal imagining can also Biological Chemistry 270: 6456—6463 (1995). Reference to be used to detect internaliZation of a label as it provides PEPT1 includes the amino acid sequence of Liang, allelic, 50 sufficient spatial resolution to distinguish betWeen ?uores cognate and induced variants thereof. Usually such variants cence on a cell surface and ?uorescence Within a cell; shoW at least 90% sequence identity to the exemplary alternatively, confocal imaging can be used to track the sequence of Liang. Cognate forms of the human PEPT1 movement of compounds over time. In another approach, sequence have been cloned from rabbit, and rat tissues. Fei, 55 internaliZation of a compound is detected using a reporter Nature 368: 563—566 (1994), and Miyamoto, Biochimica et that is a substrate for an enZyme expressed Within a cell. Biophysica Acta 1305: 34—38 (1996), respectively. Once the complex is internalized, the substrate is metabo Human PEPT2 has been cloned by Liu et al., Biochimica liZed by the enZyme and generates an optical signal or et Biophysica Acta 1235:461—466 (1995). Reference to radioactive decay that is indicative of uptake. Light emission PEPT2 includes the amino acid sequence of Liu et al. (SEQ 60 can be monitored by commercial PMT-based instruments or ID NO:2), allelic cognate and induced variants thereof. by CCD-based imaging systems. In addition, assay methods Usually such variants shoW at least 90% sequence identity to utiliZing LCMS detection of the transported compounds or the exemplary sequence of Liu. Saito et al., Biochimica et electrophysiological signals indicative of transport activity Biophysica Acta 1280, 173—177 (1996) have described the 65 are also employed. isolation of cDNA encoding the rat H+-coupled peptide In some methods, multiple agents, conjugates or conju transporter PEPT2. The PEPT2 cDNA had 3938 bp, Which gate moieties are screened simultaneously and the identity of US 6,955,888 B2 9 10 each agent or conjugate moiety is tracked using tags linked based on our detection of signi?cant PEPT2 expression in to the agents, conjugates or conjugate moieties. In some human intestine, compounds transported by PEPT2 can be methods, a preliminary step is performed to determine recogniZed and optimiZed for oral delivery through PEPT2 binding of an agent or conjugate moiety to PEPT2. Although transporters in the human intestine). not all agents or conjugates that bind PEPT2 are substrates 4. Agents, Conjugates and Conjugate Moieties to be of the transporter, observation of binding is an indication Screened Compounds constituting agents, conjugates or conjugate that alloWs one to reduce the number of candidate substrates moieties to be screened can be naturally occurring or syn from an initial repertoire. In some methods, substrate capac thetic molecules. Natural sources include sources such as, ity of an agent or conjugate moiety is tested in comparison 10 e.g., marine microorganisms, algae, plants, and fungi. With a reference substrate of PEPT2. The arti?cial dipeptide Alternatively, compounds to be screened can be from com Gly-Sar has often been used as a reference for PEPT1, and binatorial libraries of agents, including peptides or small can also be used as a reference for PEPT2. The comparison molecules, or from existing repertories of chemical com can either be performed in separate parallel assays in Which pounds synthesiZed in industry, e.g., by the chemical, an agent or conjugate moiety under test and Gly-Sar are 15 pharmaceutical, environmental, agricultural, marine, compared for uptake on separate samples of the same cells. cosmeceutical, drug, and biotechnological industries. Com Alternatively, the comparison can be performed in a com pounds can include, e.g., pharmaceuticals, therapeutics, petition format in Which an agent or conjugate moiety under environmental, agricultural, or industrial agents, pollutants, test and Gly-Sar are applied to the same cells. Typically, the 20 cosmeceuticals, drugs, organic compounds, lipids, agent or conjugate moiety and Gly-Sar are differentially glucocorticoids, antibiotics, peptides, sugars, carbohydrates, labeled in such assays. and chimeric molecules. In such comparative assays, the Vmax of an agent, A variety of methods are available for producing peptide conjugate moiety, or conjugate comprising an agent and libraries (see, e.g., Lam et al., Nature, 354: 82,1991 and WO conjugate moiety tested can be compared With that of 25 92/00091; Geysen et al, J Immunol Meth, 102: 259, 1987: Gly-Sar. If an agent, conjugate moiety or conjugate has a Houghten et al, Nature, 354: 84, 1991 and WO 92/09300 and Vmax of at least 1%, preferably at least 5%, more preferably Lebl et al., Int J Pept Pr0t Res, 41, 201, 1993). Peptide at least 10%, even more preferably at least 20%, and most libraries can also be generated by phage display methods. preferably at least 50% of Gly-Sar for the PEPT2 transporter 30 See, e.g., DoWer, US. Pat. No. 5,723,286. then the agent, conjugate moiety or conjugate can be con Combinatorial libraries can be produced for many types sidered to be a substrate for PEPT2. In general, the higher of compounds that can be synthesized in a step-by-step the Vmax of the agent, conjugate moiety or conjugate fashion (see eg Ellman & Bunin, J Amer Chem Soc, relative to that of Gly-Sar the better. Therefore, agents, 114:10997, 1992 (benZodiaZepine template), WO 95/32184 conjugate moieties or conjugates having Vmax’s of at least 35 (oxaZolone and aminidine template), WO 95/30642 50%, 100%, 150% or 200% of the Vmax of Gly-Sar for (dihydrobenZopyran template) and WO 95/35278 PEPT2 are screened in some methods. The agents to Which (pyrrolidine template). Libraries of compounds are usually conjugate moieties are linked can by themselves shoW little synthesiZed by solid phase chemistry on particle. HoWever, or no detectable substrate activity for PEPT2 (e.g., Vmax solution-phase library synthesis can also be useful. Strate 40 relative to that of Gly-Sar of less than 0.1 or 1%). gies for combinatorial synthesis are described by Dol+ Le & In some methods, the Vmax of an agent, conjugate moiety Nelson, J. Combinatorial Chemistry 1. 235—282 (1999)) or conjugate is also determined relative to Gly-Sar for the (incorporated by reference in its entirety for all purposes). transporter PEPT1. Such screening may reveal that the Synthesis is typically performed in a cyclic fashion With a agent, conjugate moiety or conjugate is a better substrate for 45 different monomer or other component being added in each PEPT2 than PEPT1. The relative capacities of a substrate for round of synthesis. Some methods are performed by suc PEPT2 and PEPT1 can be compared by a comparison of the cessively fractionating an initial pool. For example, a ?rst ratios of Vmax of the agent, conjugate moiety or conjugate round of synthesis is performed on all supports. The sup and Gly-Sar for the respective transporters. For example, if ports are then divided into tWo pools and separate synthesis 50 the ratio of Vmax’s for the agent, conjugate moiety or reactions are performed on each pool. The tWo pools are then conjugate to Gly-Sar is greater for PEPT2 than for PEPT1 further divided, each into a further tWo pools and so forth. then the agent, conjugate moiety or conjugate is a better Other methods employ both splitting and repooling. For substrate for PEPT2 than for PEPT1. In some methods, the example, after an initial round of synthesis, a pool of ratio of Vmax’s is at least 2, 10, 20, 50, or 100 times greater 55 compounds is split into tWo for separate syntheses in a for PEPT2 than for PEPT1. In some methods, the ratio of the second round. Thereafter, aliquots from the separate pools agent, conjugate moiety or conjugate to Gly-Sar for PEPT1 are recombined for a third round of synthesis. Split and pool is less than 0.1, 1 or 10% In other methods, the agent, methods result in a pool of mixed compounds. These meth conjugate moiety or conjugate is a substrate for PEPT1 ods are particularly amenable for tagging as described in (Vmax of at least 10%, 50%, 100%, 150% or 200% of the 60 more detail beloW. The siZe of libraries generated by such Vmax of Gly-Sar for PEPT1. Robust assays are available for methods can vary from 2 different compounds to 104, 106, both PEPT1 and PEPT2, alloWing design and characteriZa 108, or 1010, or any range therebetWeen. tion of compounds With substrate (or inhibitor) activities for Preparation of encoded libraries is described in a variety either PEPT1 or PEPT2, or both. Based on the conventional 65 of publications including Needels, et al., Proc. Natl. Acad. Wisdom, compounds lacking substrate activity on PEPT1 Sci. USA 1993, 90, 10700; Ni, et al.,J. Med. Chem. 1996, 39, Would be rejected as candidates for oral delivery; hoWever, 1601, WO 95/12608, WO 93/06121, WO 94/08051, WO US 6,955,888 B2 11 12 95/35503 and WO 95/30642 (each of Which is incorporated moiety. Such complementary reactive groups are Well by reference in its entirety for all purposes). Methods for knoWn in the art as illustrated beloW: synthesizing encoded libraries typically involve a random combinatorial approach and the chemical and/or enZymatic assembly of monomer units. For example, the method typically includes steps of: (a) apportioning a plurality of COMPLEMENTARY BINDING CHEMISTRIES solid supports among a plurality of reaction vessels; (b) First Reactive Group Second Reactive Group Linkage coupling to the supports in each reaction vessel a ?rst hydroxyl carboxylic acid ester monomer and a ?rst tag using different ?rst monomer and hydroxyl haloformate carbonate tag combinations in each different reaction vessel; (c) pool thiol carboxylic acid thioester ing the supports; (d) apportioning the supports among a thiol haloformate thiocarbonate amine carboxylic acid amide plurality of reaction vessels; (e) coupling to the ?rst mono hydroxyl isocyanate carbamate mer a second monomer and coupling to either the solid hydroxyl haloformate carbamate amine isocyanate urea support or to the ?rst tag a second tag using different second 15 monomer and second tag combinations in each different carboxylic acid carboxylic acid anhydride hydroxyl phosphorus acid phosphonate or reaction vessel; and optionally repeating the coupling and phosphate ester apportioning steps With different tags and different mono mers one to tWenty or more times. The monomer set can be In addition to the complementary chemistry of the func expanded or contracted from step to step; or the monomer tional groups on the linker to both the agent and conjugate set could be changed completely for the next step (e.g., moiety, the linker (When employed) is also selected to be amino acids in one step, nucleosides in another step, carbo cleavable in vivo. Cleavable linkers are Well knoWn in the hydrates in another step). A monomer unit for peptide art and are selected such that at least one of the covalent synthesis, for example, can include single amino acids or bonds of the linker that attaches the agent to the conjugate 25 larger peptide units, or both. moiety can be broken in vivo thereby providing for the agent Compounds synthesiZable by such methods include or active metabolite thereof to be available to the systemic polypeptides, beta-turn mimetics, polysaccharides, blood circulation. The linker is selected such that the reac phospholipids, hormones, prostaglandins, steroids, aromatic tions required to break the cleavable covalent bond are compounds, heterocyclic compounds, benZodiaZepines, oli favored at the physiological site in vivo Which permits agent gomeric N-substituted glycines and oligocarbamates. Pre (or active metabolite thereof) release into the systemic blood pared combinatorial libraries are also available from com circulation. The selection of suitable cleavable linkers to mercial sources (e.g., ChemRx, South San Francisco, provide effective concentrations of the agent or active Calif.). metabolite thereof for release into the systemic blood cir Some compounds to be screened are variants of knoWn 35 culation can be evaluated using endogenous enZymes in transporter substrates. The natural function of these trans standard in vitro assays to provide a correlation to in vivo porters is to transport peptides arising from the digestion of cleavage of the agent or active metabolite thereof from the dietary proteins (small intestine) and prevent loss of peptides conjugate, as is Well knoWn in the art. It is recogniZed that the exact cleavage mechanism employed is not critical to the in the glomerular ?ltrate (kidney). Some compounds to be 40 screened are peptides, variants of amino acids, ZWitterionic methods of this invention provided, of course, that the antibiotics, sugars or nucleosides, or structural variants of conjugate cleaves in vivo in some form to provide for the any of these. Compounds to be screened also include vari agent or active metabolite thereof for sustained release into ants of knoWn substrates, such as [3-lactam antibiotics, the the systemic blood circulation. anti-cancer agent Bestatin, and angiotensin converting 45 In another approach, a conjugate moiety and agent are enZyme (ACE) inhibitors. Orally bioavailable antibiotics each attached to moieties having mutual af?nity for each interact differently With PEPT1 and PEPT2. In general, (e.g., avidin or streptavidin and biotin, or hexahistidine and although not invariably, [3-lactam antibiotics having an Ni”). In another approach, both agent and conjugate moiety ot-amino group (cefadroxil, cephradine, amoxacillin, and are linked to a solid phase. Examples of such supports cyclacillin) are better substrates for PEPT2 than PEPT1. include nanoparticles (see, e.g., US. Pat. Nos. 5,578,325 [3-lactam antibiotics Without ot-amino groups (ceftibuten, and 5,543,158), molecular scaffolds, liposomes (see, e.g., ce?xime, and cefdinir) are not as good substrates for PEPT2, Deshmuck, D. S., et al., Life Sci. 28:239—242 (1990), and but are moderate substrates for PEPT1. Aramaki, Y., et al., Pharm. Res. 10:1228—1231 (1993), 5. Linkage of Agents to Conjugate Moieties 55 protein cochleates (stable protein-phospholipid-calcium pre Conjugate moieties that are substrates for PEPT2 or other cipitates; see, eg Chen et al., J. Contr. Rel. 42:263—272 transporter can be attached to or incorporated into agents (1996), and clathrate complexes. These supports can be used having pharmacological activity by a variety of means. to attach other active molecules. Certain supports such as Conjugates of this invention can be prepared by either direct nanoparticles can also be used to encapsulate desired com conjugation of an agent to a conjugate moiety, Wherein the pounds. An agent can be linked to a support via a cleavable resulting covalent bond is cleavable in vivo, or by covalently linkage alloWing separation of the agent after uptake through coupling a difunctionaliZed linker precursor With an agent to a transporter. a conjugate moiety. The linker precursor is selected to Examples of cleavable linkers suitable for use as contain at least one reactive functionality that is comple 65 described above include nucleic acids With one or more mentary to at least one reactive functionality on the agent restriction sites, or peptides With protease cleavage sites and at least one reactive functionality on the conjugate (see, e.g., US. Pat. No. 5,382,513). Other exemplary linkers US 6,955,888 B2 13 14 that can be used are available from Pierce Chemical Com nileprost, prostacyclin, prostaglandins E1, E2, or F20‘, and pany in Rockford, Ill.; suitable linkers are also described in thrornboXane A2; quinolone antibiotics such as acrosoXacin, EPA 188,256; US. Pat. Nos. 4,671,958; 4,459,839; 4,414, cinoXacin, cipro?oXacin, enoXacin, ?urnequine, naladiXic 148; 4,699,784; 4,680,338, 4,569,789 and 4,590,071; and in acid, nor?oXacin, o?oXacin, oXolinic acid, pe?oXacin, pipe EggenWeiler, H. M, Drug Discovery Today, 3: 552 (1998), rnidic acid, and pirornidic acid; other antibiotics such as each of Which is incorporated in its entirety for all purposes. aZtreonarn, irnipenern, rneropenern, and related carbopenern There are many existing drugs for Which uptake can be antibiotics. improved through the intestine. Drugs suitable for conver Representative drugs containing amine groups include: sion to prodrugs that are capable of uptake from the intestine 10 acebutalol, albuterol, alprenolol, atenolol, bunolol, typically contain one or more of the following functional bupropion, butoparnine, butoXarnine, carbuterol, cartelolol, groups to Which a prornoiety may be conjugated: primary or colterol, deterenol, deXpropanolol, diacetolol, dobutarnine, secondary amino groups, hydroXyl groups, carboXylic acid eXaprolol, eXprenolol, fenoterol, fenyripol, labotolol, groups, phosphonic acid groups, or phosphoric acid groups. levobunolol, rnetolol, rnetaproterenol, rnetoprolol, nadolol, Examples of drugs containing carboXyl groups include, 15 parnatolol, penbutalol, pindolol, pirbuterol, practolol, for instance, angiotensin-converting enzyme inhibitors such prenalterol, prirnidolol, priZidilol, procaterol, propanolol, as alecapril, captopril, 1-[4-carboXy-2-rnethyl-2R,4R quinterenol, rirniterol, ritodrine, solotol, soterenol, pentanoyl]-2,3-dihydro-2S-indole-2-carboXylic acid, enala sul?niolol, sul?nterol, sulictidil, taZaolol, terbutaline, prilic acid, lisinopril, N-cyclopentyl-N-[3-[(2,2-dirnethyl-1 20 tirnolol, tiprenolol, tipridil, tolarnolol, thiabendaZole, oXopropyl)thio]-2-rnethyl-1-oXopropyl]glycine, pivopril, albendaZole, albutoin, alendronate, alinidine, aliZapride, quinaprilat, (2R, 4R)-2-hydroXyphenyl)-3-(3 arniloride, arninoreX, aprinocid, carnbendaZole, cirnetidine, rnercaptopropionyl)-4-thiaZolidinecarboXylic acid, (S) cisapride, clonidine, cyclobenZadole, delavirdine, efegatrin, benZarnido-4-oXo-6-phenylheXenoyl-2-carboXypyrrolidine, etintidine, fenbendaZole, fenrnetaZole, ?ubendaZole, [2S-1[R*(R*))]]2(X, 3ot[3, 7ot[3]-1[2-[[1-carboXy-3 25 ?udoreX, gabapentin, icadronate, lobendaZole, phenylpropyl]-arnino]-1-oXopropyl]octahydro-1H-indole-2 rnebendaZole, rnetaZoline, rnetocloprarnide, carboXylic acid, [3S-1[R*(R*))]], 3R*]-2-[2-[[1-carboXy-3 rnethylphenidate, rneXiletine, neridronate, nocodaZole, phenylpropyl]-arnino]-1-oXopropyl]-1,2,3,4-tetrahydro-3 oXfendaZole, oXibendaZole, oXrnetidine, parnidronate, isoquinolone carboXylic acid, and tiopronin; cephalosporin 30 parbendaZole, prarnipeXole, praZosin, pregabalin, antibiotics such as cefaclor, cefadroXil, cefarnandole, procainarnide, ranitidine, tetrahydraZoline, tiarnenidine, cefatriZine, cefaZedone, cefaZu?ur, cefaZolin, tinaZoline, tiotidine, tocainide, tolaZoline, trarnaZoline, cefbuperaZone, ce?Xirne, cefrnenoxirne, cefrnetaZole, XylornetaZoline, dirnethoXyphenethylarnine, N-[3(R)-[2 cefodiZirne, cefonicid, cefoperaZone, ceforanide, piperidin-4-yl)ethyl]-2-piperidone-1-yl]acetyl-3(R)-rnethyl cefotaXirne, cefotefan, cefotiarn, cefoXitin, cefpirniZole, 35 [3-alanine, adrenolone, aletarnine, arnidephrine, cefpirorne, cefpodoXirnc, cefroXadine, cefsulodin, arnphetarnine, aspartarne, barnethan, betahistine, carbidopa, cefpirarnide, ce?aZidirne, cefteZole, ceftiZoXirne, clorprenaline, chlorterrnine, doparnine, L-Dopa, ephrineph ceftriaXone, cefuroXirne, cephacetrile, cephaleXin, rine etryptarnine, fen?urarnine, rnethyldoparnine, cephaloglycin, cephaloridine, cephalosporin, cephanone, norepincphrine, tocainide, enviroXirne, nifedipine, 40 cephradine, and latarnoXef; penicillins such as arnoXycillin, nirnodipine, triarnterene, nor?oXacin, and similar corn anpicillin, apalcillin, aZidocillin, aZlocillin, benZylpencillin, pounds such as pipedernic acid, 1-ethyl-6-?uoro-1, carbenicillin, carfecillin, carindacillin, cloXacillin, 4dihydro-4-oXo-7-(1-piperaZinyl)-1,8-napthyridine-3 cyclacillin, dicloXacillin, epicillin, ?ucloXacillin, hetacillin, carboXylic acid, 1-cyclopropyl-6-?uoro-1, and 4-dihydro-4 rnethicillin, rneZiocillin, nafcillin, oXacillin, phenethicillin, 45 oXo-7-(piperaZinyl)-3-quinolinecarboXylic acid. piperaZillin, sulbenicllin, ternocillin, and ticarcillin; throrn Representative drugs containing hydroXy groups include: bin inhibitors such as argatroban, rnelagatran, and napsagat steroidal horrnones such as allylestrenol, cingestol, ran; in?uenza neurarninidase inhibitors such as Zanarnivir dehydroepiandrosteron, dienostrol, diethylstilbestrol, and perarnivir; non-steroidal antiin?arnrnatory agents such dirnethisteron, ethyneron, ethynodiol, estradiol, estron, ethi as acarnetacin, alclofenac, alrninoprofen, aspirin nyl estradiol, ethisteron, lynestrenol, rnestranol, rnethyl (acetylsalicylic acid), 4-biphenylacetic acid, bucloXic acid, testosterone, norethindron, norgestrel, norvinsteron, carprofen, cinchofen, cinrnetacin, clornetacin, cloniXin, oXogeston, quinestrol, testosterone, and tigestol; tranquiliz diclenofac, di?unisal, etodolac, fenbufen, fenclofenac, fen ers such as dofeXaZeparn, hydroXyZin, loraZeparn, and closic acid, fenoprofen, ferobufen, ?ufenarnic acid, 55 oXaZeparn; neuroleptics such as acetophenaZine, ?ufenisal, ?urbipro?n, ?uprofen, ?utiaZin, ibufenac, carphenaZine, ?uphenaZine, perphenyZine, and pipera ibuprofen, indornethacin, indoprofen, ketoprofen, ketorolac, etaZine; cytostatics such as aclarubicin, cytarabine, lonaZolac, loXoprofen, rneclofenarnic acid, rnefenarnic acid, decitabine, daunorubicin, dihydro-5-aZacytidine, 2-(8-rnethyl-10,11-dihydro-11-oXodibenZ[b,f]oXepin-2-yl) doXorubicin, epirubicin, estrarnustin, etoposide, ?udarabine, propionic acid, naproXen, ni?urninic acid, 60 gerncitabine, 7-hydroXychlorprornaZin, nelarabine, neplano O-(carbarnoylphenoXy)acetic acid, oXoproZin, pirprofen, cin A, pentostatin, podophyllotoXin, teZacitabine, prodolic acid, salicylic acid, salicylsalicylic acid, sulindac, troXacitabine, vinblastin, vincristin, and vindesin; hormones suprofen, tiaprofenic acid, tolfenarnic acid, tolrnetin and and hormone antagonists such as buserilin, gonadoliberin, Zopernirac; prostaglandins such as ciprostene, 16-deoXy-16 65 icatibrant, and leuprorelin acetate; antihistarnines such as hydroXy-16-vinyl prostaglandin E2, 6,16 terphenadine; analgesics such as di?unisal, naproXol, dirnethylprostaglandin E2, epoprostostenol, rneteneprost, paracetarnol, salicylarnide, and salicyclic acid; antibiotics US 6,955,888 B2 15 16 such as aZidarnphenicol, aZithrornycin, carnptothecin, (antiprotoZoals), nandrolon (anabolic), propafenon and qui cefarnandol, chlorarnphenicol, clarithrornycin, clavulanic nadine (antiarythrnics), quetiapine (CNS), serotonin acid, clindarnycin, derneclocyclin, doXycyclin, (neurotransrnitter), and silybin (hepatic disturbance). erythrornycin, gentarnycin, irnipenern, latarnoXef, Representative drugs containing phosphonic acid rnoi rnetronidaZole, neornycin, novobiocin, oleandornycin, eties include: adefovir, alendronate, (N6-[2-rnethylthio) oXytetracyclin, tetracycline, thiarnenicol, and tobrarnycin; ethyl]-2-[3,3,3-tri?uoropropylthio]-5‘-adenylic acid, BMS antivirals such as acyclovir, dideoXydidehydrocytidine, 187745 (a squalene synthase inhibitor frorn Bristol-Meyers dideoXycytosine, 1-(2-deoXy-2-rnethylene-beta-D-erythro Squibb Inc.), ceronapril, CGP-24592 (Novartis, Inc.), DL pentofuranosyl)cytidine, ?uoro-dideoXydidehydrocytidine, (E)-2-arnino-4-rnethyl-5-phosphono-3-pentenoic acid; ?uorodideoXycytosine, FMAU (1-(2-deoXy-2-?uoro-beta 4-rnethyl-APPA, CGP-39551 (ethyl esters of (DL-[E]-2 D-arabinofuranosyl)thyrnine), deoXy-5-?uoro-3‘ arnino-4-rnethyl-5-phosphono-3-pentenoic acid)), CGP thiacytidine, 2‘-?uoro-ara-dideoXyinosine, ganciclovir, 40116 (a competitive NMDA antagonist by Novartis Inc.), larnivudine, penciclovir, SddC, stavudine, cidofovir, clodronate, EB-1053 (1-hydroXy-3-(1 5-tri?uorornethyl-2‘-deoXyuridine, Zalcitabine, and Zidovu 15 pyrrolidinyl)propylidene-1,1-bisphosphonate), etidronate, dine; bisphosphonates such as EB-1053 (1-hydroXy-3-(1 fanapanel, foscarnet, fosfornycin, fosinopril, fosinoprilat, pyrrolidinyl)propylidene-1,1-bisphosphonate), etidronate, ibandronate, rnidafotel, neridronate, olpadronate, ibandronate, olpadronate, residronate, 1-hydroXy-2 parnidronate, residronate, tenofovir, tiludronate, [2-(8,9 (irnidaZo[1,2-a]pyridin-3-yl)ethylidene]-bisphosphonic dioXo-2,6-diaZabicyclo[5.2.0]non-1(7)-en-2-yl)ethyl] acid, and Zolendronate; protease inhibitors such as phosphonic acid, 1-hydroXy-2-(irnidaZo[1,2-a]pyridin-3-yl) ciprokiren, enalkiren, ritonavir, saquinavir, and terlakiren; ethylidene]-bisphosphonic acid, and Zolendronate. prostaglandins such as arbaprostil, carboprost, rnisoprostil, Representative drugs containing phosphoric acid rnoieties and prostacydin; antidepressives such as include: bucladesine, choline alfoscerate, citocoline, ?u 8-hydroXychlorirniprarnine and 2-hydroXyirniprarnine; anti 25 darabine phosphate, fosoparnine, GP-668, perifosine, tricir hypertonics such as sotarol and fenoldoparn; anticholinero ibine phosphate, and phosphate derivatives of nucleoside genics such as biperidine, procyclidin and triheXyphenidal; analogs Which require phophorylation for activity, such as antiallergenics such as crornolyn; glucocorticoids such as larnivudine, acyclovir, aZidothyrnidine, E-5-(2 betarnethasone, budenosid, chlorprednison, clobetasol, brornovinyl)-2‘-deoXyuridine, dideoXycytosine, clobetasone, corticosteron, cortisone, cortodeXon, dideoXyinosine, FMAU (1-(2-deoXy-2-?uoro-beta-D dexarnethason, ?ucortolon, ?udrocortisone, ?urnethasone, arabinofuranosyl)thyrnine), deoXy-5-?uoro-3‘-thiacytidine, ?unisolid, ?uprednisolon, ?urandrenolide, ?urandrenolon ganciclovir, gerncitabine, (R)-9-[4-HydroXy-2 acetonide, hydrocortisone, rneprednisone, (hydroXyrnethy)butyl]guanine, larnivudine, penciclovir and rnethylpresnisolon, pararnethasone, prednisolon, prednisol, 35 the like. triarncinolon, and triarncinolon acetonide; narcotic agonists Preferred drugs for rnodi?cation to prodrugs capable of and antagonists such as apornorphine, buprenorphine, intestional absorption and incorporation into sustained butorphanol, codein, cyclaZocin, hydrornorphon, release forrnulations include the folloWing cornpounds: ketobernidon, levallorphan, levorphanol, rnetaZocin, analgesics and/or antiin?arnrnatory agents selected from the morphine, nalbuphin, nalrnefen, naloXon, nalorphine, 40 group consisting of acetaminophen, buprenorphine, naltreXon, oXycodon, oXyrnorphon, and pentaZocin; stirnu diclofenac, di?unisal, fenoprofen, ibuprofen, indornethacin, lants such asrnaZindol and pseudoephidrine; anaesthetics ketoprofen, rnefenarnic acid, rneptaZinol, rnorphine, such as hydroXydion and propofol; [3-receptor blockers such oXycodone, pentaZocine, pethidine, tolrnetin, and trarnadol; as acebutolol, albuterol, alprenolol, atenolol, betaZolol, 45 antihypertensive agents selected from the group consisting bucindolol, cartelolol, celiprolol, cetarnolol, labetalol, of captopril, diltiaZern, rnethyldopa, rnetoprolol, praZosin, levobunelol, rnetoprolol, rnetipranolol, nadolol, propranolol, quinapril, sotalol, and tirnolol; antibiotic agents oXyprenolol, pindolol, propanolol, and tirnolol; selected from the group consisting of arnoXicillin, ot-syrnpathornirnetics such as adrenalin, rnetararninol, arnpicillin, aZtreonarn, cefaclor, cefadroXil, ce?Xirne, rnidodrin, norfenefrin, octaparnine, oXedrin, oXilofrin, cefotaXirne, cefoXitin, cefpodoXirne, ceftiZoXirne, oXirnetaZolin, and phenylefrin; [3-syrnpathornirnetics such as ceftriaXone, cefuroxirne, cephaleXin, cipro?aXacin, barnethan, clenbuterol, fenoterol, heXoprenalin, isoprenalin, clindarnycin, erythrornycin, irnipenern, rnandol, isoXsuprin, orciprenalin, reproterol, salbutarnol, and terbuta rneropenern, rnetronidaZole, and tobrarnycin; antiviral lin; bronchodilators such as carbuterol, dyphillin, etophyllin, 55 agents selected from the group consisting of acyclovir, fenoterol, pirbuterol, rirniterol and terbutalin; cardiotonics delavirdine, didanosine, foscarnet, ganciclovir, indinavir, such as digitoXin, dobutarnin, etilefrin, and prenalterol; larnivudine, nel?navir, penciclovir, ritonavir, saquinavir, antirnycotics such as arnphotericin B, chlorphenesin, stavudine, Zalcitabine, and Zidovudine; bronchodilator and nystatin, and perirnycin; anticoagulants such as or anti-asthrnatic agents selected from the group consisting acenocournarol, dicournarol, phenprocournon, and Warfarin; of salbutarnol and terbutaline; antiarrhythrnic agents vasodilators such as barnethan, dipyrirnadol, diprophyllin, selected from the group consisting of rneXiletine, isoXsuprirn, vincarnin and Xantinol nicotinate; antihypocho procainarnide, and tocainide; centrally acting substances lesterernics such as cornpactin, eptastatin, rnevinolin, and selected from the group consisting of baclofen, benseraZide, sirnvastatin; rniscellaneous drugs such as brornperidol 65 bupropion, carbidopa, gabapentin, levodopa, (antipsychotic), dithranol (psoriasis) ergotarnine (migraine) rnethylphenildate, prarnipeXole, pregabalin, quetiapine, iverrnectin (antihelrninthic), rnetronidaZole and secniZadole ropinirole, and vigabatrin; cytostatics and metastasis inhibi US 6,955,888 B2 17 18 tors selected from the group consisting of cytarabine, compositions can provide quick, sustained or delayed decitabine, docetaxal, ?utamide, gemcitabine, paclitaxel, release of the active ingredient after administration to the and pentostatin; and, agents for treatment of gastrointestinal patient. The tablets or pills of the present invention may be disorders selected from the group consisting of cisapride, coated or otherWise compounded to provide a dosage form metoclopramide, and misoprostol. affording the advantage of prolonged action. For example, 6. Pharmaceutical Compositions and Methods of Treatment the tablet or pill can comprise an inner dosage and an outer Agents that are themselves substrates for PEPT2 or Which dosage component, the latter being in the form of an are linked to conjugate moieties that are substrates for envelope over the former. The tWo components can be PEPT2 can be can be incorporated into pharmaceutical compositions. Usually, although not necessarily, such phar 10 separated by an enteric layer Which serves to resist disinte gration in the stomach and permit the inner component to maceutical compositions are designed for oral administra tion. Oral administration of such compositions results in pass intact into the duodenum or to be delayed in release. A uptake through the intestine via the PEPT2 and entry into the variety of materials can be used for such enteric layers or systemic circulation. The pharmaceutical composition can coatings, such materials including a number of polymeric 15 thus be ef?ciently delivered to a Wide range of tissues in the acids and mixtures of polymeric acids With such materials as body. The speci?city of compositions for PEPT2 renders the shellac, cetyl alcohol, and cellulose acetate. compositions susceptible to uptake by the brain (including For preparing solid compositions such as tablets, the the choroid plexus) and kidney that express PEPT2 at high principal active ingredient is mixed With a pharmaceutical levels. HoWever, the methods are also useful for treating a excipient to form a solid preformulation composition con Wide variety of diseases in patients Who are free of diseases taining a homogeneous mixture of a compound of the of the brain, kidney, lung, and spleen in Which PEPT2 is present invention. When referring to these preformulation expressed to a signi?cant extent. In such methods, the compositions as homogeneous, it is meant that the active expression of PEPT2 in the kidney increase reabsorption of ingredient is dispersed evenly throughout the composition the pharmaceutical composition into the systemic circula 25 so that the composition may be readily subdivided into tion thereby increasing its half life and thereby reducing the equally effective unit dosage forms such as tablets, pills and dosage necessary. In some methods, the agent or conjugate capsules. This solid preformulation is then subdivided into moiety is a substrate for both PEPT2 and PEPT1. In some unit dosage forms of the type described above containing methods, the agent or conjugate moiety is a substrate for from, for example, 0.1 mg to about 2 g of the active agent. PEPT2 and is not a substrate, or is a poor substrate, for The compositions can be administered for prophylactic PEPT1. and/or therapeutic treatments. A therapeutic amount is an Agents optionally linked to a conjugate moiety are com amount sufficient to remedy a disease state or symptoms, or bined With pharmaceutically-acceptable, non-toxic carriers otherWise prevent, hinder, retard, or reverse the progression of diluents, Which are de?ned as vehicles commonly used to 35 of disease or any other undesirable symptoms in any Way formulate pharmaceutical compositions for animal or human Whatsoever. In prophylactic applications, compositions are administration. The diluent is selected so as not to adversly administered to a patient susceptible to or otherWise at risk affect the biological activity of the combination. Examples of a particular disease or infection. Hence, a “prophylacti of such diluents are distilled Water, buffered Water, physi cally effective amount” is an amount suf?cient to prevent, 40 ological saline, PBS, Ringer’s solution, dextrose solution, hinder or retard a disease state or its symptoms. In either and Hank’s solution. In addition, the pharmaceutical com instance, the precise amount of compound contained in the position or formulation can also include other carriers, composition depends on the patient’s state of health and adjuvants, or non-toxic, nontherapeutic, nonimmunogenic Weight. stabiliZers, excipients and the like. The compositions can 45 An appropriate dosage of the pharmaceutical composition also include additional substances to approximate physi is readily determined according to any one of several ological conditions, such as pH adjusting and buffering Well-established protocols. For example, animal studies agents, toxicity adjusting agents, Wetting agents, detergents (e.g., mice, rats) are commonly used to determine the and the like (see, e.g., “Remington’s Pharmaceutical maximal tolerable dose of the bioactive agent per kilogram Sciences”, Mace Publishing Company, Philadelphia, Pa., of Weight. In general, at least one of the animal species 17th ed. (1985); for a brief revieW of methods for drug tested is mammalian. The results from the animal studies can delivery, see, Langer, Science 249:1527—1533 (1990); each be extrapolated to determine doses for use in other species, of these references is incorporated by reference in its such as humans for example. entirety). 55 The pharmaceutical compositions can be administered in Pharmaceutical compositions for oral administration can a variety of different Ways. Examples include administering be in the form of e.g., tablets, pills, poWders, loZenges, a composition containing a pharmaceutically acceptable sachets, cachets, elixirs, suspensions, emulsions, solutions, carrier via oral, intranasal, rectal, topical, intraperitoneal, or syrups. Some examples of suitable excipients include intravenous, intramuscular, subcutaneous, subdermal, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum transdermal, intrathecal, and intracranial methods. The route acacia, calcium phosphate, alginates, tragacanth, gelatin, of administration depends in part on the chemical compo calcium silicate, microcrystalline cellulose, sition of the active compound and any carriers. polyvinylpyrrolidone, cellulose, sterile Water, syrup, and The components of pharmaceutical compositions are methyl cellulose. Preserving agents such as methyl- and 65 preferably of high purity and are substantially free of propylhydroxy-benZoates; sWeetening agents; and ?avoring potentially harmful contaminants (e.g., at least National agents can also be included. Depending on the formulation, Food (NF) grade, generally at least analytical grade, and US 6,955,888 B2 19 20 more typically at least pharmaceutical grade). To the extent factor derived from ampli?cation of knoWn plasmid copy that a given compound must be synthesized prior to use, the numbers. To compare different tissues, all data are expressed resulting product is typically substantially free of any poten as a fraction of [3-actin transcript levels. tially toxic agents, particularly any endotoxins, Which may be present during the synthesis or puri?cation process. Compositions for parental administration are also sterile, Table 1 shoWs expression levels of PEPT1 and PEPT2 substantially isotonic and made under GMP conditions. Compositions for oral administration need not be sterile or mRNA expressed as a percentage of the expression level of substantially isotonic but are usually made under GMP beta actin mRNA in the same tissue. It can be seen that conditions. 10 substantial expression of PEPT2 is obtained in the human jejunWn, ileum, ileocecum, and cecum and detectable EXAMPLES 1. PCR Analysis of Transporter Expression expression in several other intestinal tissues. Levels of Oligonucleotide primers Were designed to amplify spe expression in the rat duodenumn, jejunum and ileum and ci?c sequences in either human PEPT1 (2 sets using colon Were barely detectable.

TABLE 1

Expression of PEPT1 and PEPT2 in Various Tissues and Cell lines

Sto Eso Duo Jej Ile Il-Ce Cec Acol Tcol Dcol Hea Bra Lun SMus Kid Pan

PEPT1 U13173 18.96 2.97 4.92 4.52 3.74 1.13 0.28 0.31 5.27 12.12 5.27 8.25 0.00 16.68 5.62 7.26 PEPT2 NMi 0.05 0.01 0.10 0.77 0.52 0.29 0.11 0.07 0.01 0.01 0.01 0.09 0.08 0.01 1.14 0.46 021082

Caco-2 HCT28 HI‘29

Liv Thy Spl Leu Pla Pros Test Ova Diff. Undiff. Diff. Undiff. Diff

PEPT1 U13173 4.08 12.12 21.54 24.48 12.12 0.00 0.00 10.00 3.31 30.24 16.63 3.06 2.78 9.18 77.43 PEPT2 NMi 0.00 0.03 0.09 0.02 0.01 0.53 0.11 0.00 0.09 0.76 0.26 0.01 0.18 0.01 0.64 021082

Genbank) or PEPT2 (2 sets using Genbank). The forWard GB#=GenBank accession number and reverse primer sequences Were (PEPT1#1 Sto=stomach F-catgcaccaccacgcccagctatttt (SEQ ID N013) and 35 Eso=esophogus R-gcgcggtagctcaagcctgtaatccc (SEQ ID NO:4) Which ampli Duo=duodenum ?es 147 base pairs in the 3‘UTR, PEPT1#2 Jej =jejunum F-ccgcgttgcttctggtcgtctgtgta (SEQ ID N015) and Ile=ileum R-tccatcctccacttgcctcctgacct (SEQ ID NO:6) Which ampli Il-Ce=ileum-cccum valve. 40 Cec=cecum ?ers 197 base pairs across the stop codon; PEPT2#1 Acol=ascending colon F-acaaccaatgggatgacaaccgtgag (SEQ ID NO:7) and Tcol=total colon R-aggcagatcaccagcaggaggcagga (SEQ ID NO:8) Which Dcol=descending colon ampli?ers 533 base pairs in the PEPT2 open reading frame; Hea=heart PEPT2#2 F-caatgttggtgaagactatggtgtgt (SEQ ID NO:9) and 45 Bra=Brain R-aacaagcacgatgatattcccaactg (SEQ ID NO:10) Which Lun=lung ampli?ers the least 376 base pairs in the PEPT2 open SMus=smooth muscle reading frame). All primers had annealing temperatures Kid=kidney above 55° C. and products Were sequenced to verify speci Pan=pancreas ?city. 50 Liv=liver Transporter expression Was quantitated by PCR thy=thymus spl=spleen (polymerase chain reaction) ampli?cation using real-time Leu=leukocytes PCR (Cepheid Smartcycler PCR instrument and Perkin Pla=platelets Elmer SYBR-green reagents; all protocols per manufactur 55 Pros=prostate ers speci?cations). Single-stranded cDNA Was prepared test=testes from human mRNA (purchased from Clontech, BioChain, Ova=ovaries and Stratagene) or differentiated Caco-2 cells (Qiagen RNA 2. Functional Analysis of PEPT1 and PEPT2 puri?cation columns) using Thermoscript (Stratagene) The complete open reading frame Was cloned into a reverse transcriptase kit. Real-time PCR Was performed Xenopus oocyte expression plasmid, lineariZed, and cRNA using the primer sets listed above to amplify fragments of Was generated by run-off transcription using the T7 poly human PEPT1 or PEPT2. In addition, total mRNA abun merase. Xenopus oocytes Were prepared and maintained as dance Was normaliZed by measurement of [3-Actin levels in previously described each tissue (Clontech primer set). Transcript abundance Was 65 (Collins, et al., 1997) and injected With 10—30 ng RNA. measured by determining the threshold cycle for PEPT1 or Transport currents Were measured 2—4 days after injection PEPT2 and calculating transcript number using a calibration using tWo-electrode voltage-clamp (Axon Instruments). All US 6,955,888 B2 21 22 experiments Were performed using a modi?ed oocyte Ring ers solution (90 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 TABLE 2 mM MgCl2, and 10 mM NaHEPES, pH 6.8). The membrane PEPT1 (% Glv-Sar) PEPT2 (% Glv-Sar) potential of oocytes Was held at —60 mV and current traces acquired using PoWerLab softWare. Responses to com Drug drug only +XP10973 drug only +XP10973 pounds Were measured in the presence and absence of a Gly-Sar 100 16 100 12 speci?c non-transported inhibitor (XP10973) for PEPT1 and Cephradine 3.5 1.9 159 37 Cephadroxil 18 2.1 96 27 PEPT2. Data are expressed as the currents that are blocked Cefaclor 4 1.6 148 22 by XP10973 Amoxacillin 1.3 1.1 85 15 Stable clones of CHOK1 cells Were obtained by electroporation, selection in G418, and sorting into single 3. Analysis of Uptake in Differentiated Caco-2 Cells clones using ?oW-activated cell sorting (Cytomation). Stable Caco-2 cells Were plated on Millipore transWell ?lters and alloWed to differentiate for 18—22 days. Integrity of the clones expressing PEPT1 or PEPT2 Were identi?ed by 15 enhanced uptake of radiolabeled Gly-Sar. For cell uptake monolayers Was con?rmed by lack of radiolabeled inulin studies, CHOK1 clones Were seeded into polylysine coated transport across the monolayer. Compounds Were added to 96-well microtitre plates and groWn for 2—3 days. Cells Were the apical chamber, and the appearance of compounds in the incubated With experimental solutions (combinations of basolateral chamber Were measured at various timepoints by radiolabeled and unlabeled compounds) for 30 minutes, scintillation counting or LC/MS/MS. Washed four times, and either lysed in scintillation solution FIGS. 1 and 2 shoW uptake of cephradine and cefadroxil by Caco-2 cells in the presence and absence of XP10973 or Water. Uptake of unlabeled compounds Was quantitated by LC/MS/MS. inhibitor. As shoWn in Table 1, Caco-2 cells express both the PEPT1 and PEPT2 transporters. HoWever, as described Table 2 shoWs the Vmax of several commercial com 25 above, cephradine and cefadroxil are poor substrates for pounds in oocytes transfected With PEPT1 or PEPT2 com PEPT1 and good substrates for PEPT2. The ?gures shoW pared With Vmax of control Gly-Sar. The table also shoWs that both cephradine and cefadroxil are taken up by the Vmax in the presence of the inhibitor XP10973. Caco-2 cells and that uptake is inhibited by XP10973. It can be inferred from these results that the cephradine and XP10973 cefadroxil are taken up via the PEPT2 transporter. 4. Procedure for Preparing a Library to Explore for PEPT2 O O Speci?c Substrates Into tWenty-one 50 ml Altech tubes is added Polystyrene /N s\// NH\)k 35 0// g HZN 5 OH chlorotritylchloride resin (5 g to each), dichloromethane (25 mL), and 3 equivalents of F moc-amino acids (see FIG. 3 for structures), and 6 equivalents of diethylisopropylamine. The O Y reactions are shaken at room temperature for 30 minutes. The resins are drained and Washed With methanol (2><), 40 dichloromethane (3x), and N,N-dimethylformamide (3x). XP10973 is a speci?c inhibitor of both PEPT1 and The resins are then treated With 20% piperidine in N,N PEPT2. It can be seen that cephradine, cephadroxil, cefaclor dimethylformamide for 1 hour. The resins are drained and and amoxacillin are relatively poor susbstrates relative to Washed With methanol (2><), dichloromethane (3x), and Gly-Sar for cells transfected With PEPT1. HoWever, these 45 N,N-dimethylformamide (3x). Each resin is divided into compounds have Vmax’s comparable to or greater than that four 25 mL Alltech tubes, and N,N-dimethylformamide (10 of Gly-Sar for cells transfected With PEPT2 indicating that mL) Was added. To each of the four different tubes Was the compounds are relatively good substrates for PEPT2. added a mixture of 5 equivalents of Boc-Alloc amino acids This conclusion is reinforced by the data in the presence of (FIG. 4), 5 equivalents of HATU, and 10 equivalents of the XP10973 inhibitor. The result that XP10973 inhibits diethylisopropylamine, in 10 mL of N,N transport in both PEPT1 and PEPT2 transfected cells indi dimethylformamide. The reactions are shaken at ambient cates that transport in such cells is at least in part due to the temperature for 20 hours. The resins are drained and Washed PEPT1 and PEPT2 transporters respectively. The smaller With methanol (2><), dichloromethane (3x), and N,N extent of inhibition for most substrates for PEPT1 relative to 55 dimethylfonnamide (3x). Each resin is treated With 0.1 PEPT2 indicates that nonspeci?c transport mechanisms equivalents of tetrakis(triphenylphosphine)palladium(0) in make a more signi?cant relative contribution in the cells N,N-dimethylformamide (10 mL) for 20 hours to effect the transfected With PEPT1. Except for cephadroxil, treatment alloc deprotection. The resins are drained and Washed With With XP1097 results in a lesser percentage decrease in Vmax methanol (2><), dichloromethane (3x), and N,N for oocytes transfected With PEPT1 relative to oocytes dimethylformamide (3x). Each resin is divided into tWelve transfected With PEPT2. In short, the experiment shoWs that 4 mL Alltech tubes, and dichloromethane (1 mL) Was added. cefaclor, cefadroxil and cephradine are better substrates for To each of the tWelve different tubes is added a mixture of PEPT2 than they are for PEPT1. Because it is knoWn that the 5 equivalents of carboxylic acid (FIG. 5), 5 equivalents of commercial compounds are orally available, it is probable 65 HATU, and 10 equivalents of diethylisopropylamine, in 1 that they are taken up through the mechanism by an alternate mL of N,N-dimethylformamide. The reactions are shaken at transporter, such as PEPT2. ambient temperature for 20 hours. The resins are drained and