CA2189978A1 - Compounds for reversing drug resistance - Google Patents

Abstract

The invention features novel peptide derivatives called Reversins, and provides for their use in a method of reducing the activity of the multi-drug transporter protein MDR1 in order to overcome multidrug resistance in a mammal. The peptide derivatives are of the formula (I) X1n-X2-X3(X4)n-X5, wherein n is 0 or 1, and each n is the same or different; X1 is BOC, BOC-Asu, Z-Asu, benzyloxycarbonyl, Glu(OBzl)-OBzl, Trp-OMe, Trp-Phe-OMe, Phe-Trp-OMe, Phe-Phe-OtBu, Trp-Trp-OtBu, indoloacetyl, benzoyl, an alkylamine of 1-4 carbons, dibenzylamide, tryptamide, 1-amino-adamantine, aminomethylcyclohexane, indoline, phenylethylamide or dicyclohexylamide; X2 is Glu(OBzl), Asp(OBzl), succinyl, O,O-dibenzoyltartaroyl, diphenoyl, muconyl, Thx, Cpa, Asu, Nal, Pen, Phg, Dbt, Lys(BOC), Lys(Z), Cys(Bzl), Thr(Bzl), Glu(OtBu), tert.-Leu, Leu, Nle, Pro, Phe, Tyr(Bzl), or Ser(Bal); X3 is Asp, Asu, Lys, Glu, Trp, Thx, Cpa, Nal, Pen, Phg, Dbt, Glu(OtBu), tert.-Leu, Leu, Nle, Pro, Tyr, Phe, or Tyr(Bzl); X4 is BOC-Glu(OBzl), Glu(OBzl), Asu, OBzl, Bzl, BOC, BOC-Lys(BOC), Z-Glu(OtBu), Asp(OBzl), Asp(OBz)-OBzl, benzyloxycarbonyl, O-(cyclo-hexyl), fluorenylmethyl ester, Glu(OtBu), Glu(OtBu)-OBzl, 1-amino-adamantine, aminomethylcyclohexane, indoline, phenylethylamide, or dicyclohexylamide; and X5 is OMe, OBzl, OtBu, Phe-OMe, -O- (cyclohexyl), Trp-OMe, (chlorophenyl)-isobutylamide, fluorenylmethyl ester, ONp, 1-aminoadamantane, aminomethylcyclohexane, indoline, phenylethylamide, or dicyclohexylamide.

Description

~ W0 95131474 2 1 8 9 ~ 7 8 P~,l/~ L'~ 144 ÇQ~O~ A FOR R~v _ _ DRUG RE8I8~N~l Ba~h~L~ of the Invention This invention relates to '~ for ~v~:L~' in~
resistance that a patient may build to theri-rP~It; r.
Treatment of many rl;l::PJIo:P~: can be severely limited by resistance to the chosen therapeutic drug. For example, chemotherapy, while generally an effective 10 L-e~, L against human cancerous ~a~;cP~Pc, is hampered when a patient becomes resistant to the chemotherapeutic.
In one special fcrm of drug resistance, called "Multidrug Resistance, " the cell becomes resistant not only to the chemotherapeutic being administered, but to a wide range 15 of ~L-u.Lu-..lly and flm~tjt~n~lly unrelated drugs simulf~nPo~c:ly (see Ford et al., Pharmacological Reviews, 42: 155-199, 1992 ) .
The cause of multidrug resistance is the appearance of an integral glycoprotein in the plasma 20 membrane of the targeted cell, e.g., a tumor cell (Fig.
1). The protein functions as a multidrug LL~..r~.,-Ler, and is variously called MUltiDLU ~ Resistance 1 protein (MDR1), P-glycoprotein (pleiotropic-glycoprotein), Pgp, or P-170. MDR1 consists of 1280 amino acid residues, and 25 contains 12 L- ane s- and two nucleotide-binding domains . It strongly rPcpmh1 Pq prokaryotic and eukaryotic members of the so-called ABC (ATP Binding rlc~cette) transporters, or traffic ATPases (see Endicott et al., Annu. Rev. Biochem. 58:137-171, 198g; Higgins, 30 Annu. Rev. Cell. Biol. 8:67-113, 1992).
MDRl naturally functions to, and is highly ~ Le~d in tissues normally rP~pnn~;hle for, extruding toxic materials and waste-~ lu~ L~, from cells (e.g., lung, kidney, and liver), and secretes l~y lLu~l~obic 35 _ _ a from exocrine or endocrine glands (Gottesman et SUBSTITUTE SHEET ~RULE 261

2 1 8 9 9 7 8 F~ 44 al., J. Biol. Chem. 263:12163-12166, 1988; Higgins et al., supr~). Consistent with its natural function, MDR1 catalyses an ATP-~l~r~n~ nt extrusion of various cytotoxic drugs from the cell, e.g., vinca alkaloids, 5 anthracyclines, and other natural antibiotics, thereby maintaining their c~l 1 ul Ar level at a subtoxic .ullc~.lLL<ltion. Thus, when expressed by tumor cells, MDR1 expels cytotoxic chemotherapeutic agents, and thus allows the tumor cell to survive ant~rAnrC-r LLeai ~s even at 10 high drug doses. At the same time, "ordinary" cells, having no such extrusion '~n;c-n~ may receive a lethal drug ~x~o~uLe. Tumors developing from tissues normally expressing the MDR1 protein often show a primary drug resistance, while in other tumors a secondary drug 15 resistance may develop after chemotherapy.
The rhr- of multidrug resistance is not limited to tumor cells. l~DR1 and its h~ lo~lc~c are e~lJLe~ed in a wide variety of cell-types, inrlllrl;n~
parasitic protozoa. C ~e~ ly, ~Jverel~LèS~iOn of a 20 member of the NDR1 family of proteins creates obstacles to a wide variety of parasitic li;Cl:-AC~lC, ;nr3ll11;
malaria, African sleeping sirkn~Cs, and others (C
et al., Chemotherapy of Parasitic Diseases, Plenum Press:NY, 1986; Henderson et al., Nol. Cell. Biol.
25 12:2855-65, 1992). ~DR1 is also e,.~L~ssed by endothelial cells of human capillary blood vessels at the blood-brain barrier and b3 ood-testis barrier (Ford et al. supr~, at 159) .
It is known that verapamil, a drug that blocks 30 voltag~ r-nt calcium rhAnn~lc, stimulates the activity of MDR1-bound ATPase at a c-,..cel.LLc.tion of 1 to 20 ,uM but inhibits it as a concentration a_ove 100 ~LM
(Sarkadi et al. J. Biol. Chem. 267:4854-4858, 1992).
While between these C.,l. ellLL~-tions verapamil blocks the 35 extrusion of antitumor drugs, its high toxicity severely ~ 2189q78 ~ -- 3 limits its clinicaL use (Solary et al. ~eukemia 5:592-597, 1991; Dalton et al. J. Clin. Oncology 7:415-418, 1989) .
In SU-A-1544778, Golovina, T. N. et al describe the prep-aration of different peptides one of which, BOC-Leu-Tyr-OMe 5 is structurally close to the peptides provided by the present invention. Nevertheless, no hints on the possible biological activity of said peptide are disclosed.
Summary of the Invention The invention generally features chemical compositions 10 which reduce or overcome multidrug resistance in a mammal, e . g ., ~ human, and in rnicroorganisms causing disease in a mammal. The compounds, called 'iFormula (I)" compounds, or " Reversins", are hydrophobic peptide derivatives which effectively compete with cytostatic drugs on the MDRl 15 protein, thus reducing or eliminating drug resistance.
" Multi-drug resistance", as used herein, refers to the ability of cells to develop resistance to a broad range of structurally or functionally unrelated drugs. This occurs by outward transport of the drug from the cell, the transport 20 being mediated by the MDR1 glycoprotein or its homologues.
The term "multidrug resistance" also applies to the cross-resistance between drugs which is adversely affected by the Reversin compounds of the invention (see below) . Preferably, " multidrug resistance" refers to the state which is dependent 25 on expression or overexpression of the= MDRl protein or its homologues, and/or on gene amplification of human mdrl or its homologues. Both primary and secondary multidrug resistance are included. ~here the drug resistance is "primary" the cell has experienced no previous exposure to a member of the group 30 of drugs, yet exhibits inherent resistance to them. Where drug resis~Lnce is " secondary", the cell has been exposed to only one drug, or to only a subset of two or more, but not necessarily to the whole, group of drugs affected by cross-resistance .
/~MENDED SI~EET

~ 2 i 89q7~

The compounds of the invention, hereafter called "Reversins", are of~ formula (I):
xln-x2-x3 (X4)n-X5 ( I ) wherein 5 n is 0 or l, and each n is the same or different;
xl is BOC, BOC-Asu, Z-Asu, benzyloxycarbonyl, Glu (OBzl) -OBzl, Trp-OMe, Trp-Phe-OMe, Phe-Trp-OMe, Phe-Phe-OtBu, Trp-Trp-OtBu, indoloacetyl, benzoyl, an alkylamine of 1-4 carbons, dibenzylamide, tryptamide, 1-amino-adamantine, alrLino:~ethylcyclohexane, indoline, phenylethylamide or dicyclohexylamide;
x2 is Glu(OBzl), Asp(OBzl), succinyl, O,O-dibenzoyltarta-ro.yl, diphenoyl, muconyl, Thx, Cpa, Asu, Nal, Pen, Phg, Dbt, Lys (BOC), Lys (Z), Cys (Bzl), Thr (Bzl), Glu (OtBu), 15 = tert.-Leu, Lau, Nle, Pro, Phe, Tyr (Bzl), or Ser (Bzl);
X3 is Asp, Asu, L~ys, Glu, Trp, Thx, Cpa, Nal, Pen, Phg, Dbt, Glu (OtBu), tert. -Leu, Leu, Nle, Pro, Tyr, Phe, or Tyr (Bzl);
X4 is BOC-Glu (OBzl), Glu (OBzl), Asu, OBzl, Bzl, BOC, BOC-Lys (BOC), Z-Glu (OtBu), Asp (OBzl), Asp (OBz) -OBzl, benzyl-oxycarbonyl, O- (cyclo-hexyl), fluorenylmethyl ester, Glu (OtBu), Glu (~7tBu) -OBzl, l-amino-adamantine, amino-methylcyclohexane, indoline, phenylethylamide, or dicyc-lohexylamide; and 25 X5 is OMe, OBzl, OtBu, Phe-OMe, -O- (cyclohexyl), Trp-OMe, (chlorophenyl)-isobutylamide, fluorenylmethyl ester, ONp, l-aminoadamantane, aminomethylcyclohexane, indoline, phenylethylamide, or dicyclohexylamide, with the proviso that said formula (I) is not BOC-Leu-Tyr-OMe.
Formula (I) compounds containing amino acids with either the L or D configura~ion fall within the scope of the invention .
,~tEND~O SH~

~ WO95/31474 21 8997'~ r~ . 144 Side chain protecting groups of amino acids may be substituted by one or more halogen atoms , e . g ., chloro-Z , or bromo-Z. Such blocking groups are known. A benzyl ester group can be substituted by one or more nitro 5 groups in the second or fourth position of the benzene ring .
me abbreviations used herein are known to those skilled in the art (see, e.g., J. Biol. Chem. 241:527, 1966; J. B~ol. Chem. 247:977 1972; hereby inuuL~uL~-ed by 10 reference). ûther abbreviations used herein are as f ollows:
AM: aceto~methyl ester Asu: Am; nnc~ r,; n; c acid or Ami nr~e;nr, r, i n~yl BOC: tert.--butylu,Ly-~Lbul.y Bzl: benzyl, Cpa: 4-chlorophenylalanyl, CY8: c~steinyl, Dbt: d_bI LYLUYY1~
DBTA: d_benzoyltartaroyl, DCC: d cyclohexylcar~OAi ;mi~D, DCU: d cyclohexylurea, DIC: d isopropylrArl~oA; ;m;A~D
DMF: d methylfrrr~m;AD
HPLC: h gh ~L~ Sc,ULe liquid chromatography, MDRl: product of multidrug resistance gene, Me: methyl, Nal: naphthylalanyl, Nle: norleucyl, m. p .: melting point OPFP: pentailuorphenyl ON: p-nil.- u~ llyl Pe~: pDn;r; 1 1 ;nAlAnyl~
Phe: phenylalanyl, Phg: phenylglycyl, Pro: prolyl, R~: retention f actor, SUC: succinyl, TEA: triethylamine, THF: tetrally-lL ur UL c.l.
Thr: threonyl, Thx: thyroxyl, TLC: thin layer chromatography, Trp: tryptophyl, Z: ben zylu.sy L CI1 bUI Iy 1 .

WO g~/31474 2 1 8 9 9 7 8 r~ 44 ~

As preferred ' 'i- Ls of the invention, X1 can be BOC, Glu(OBzl~-OBzl, Z, or (D-Phe-Trp-OMe); X2 can be Asp(OBzl~, succinyl, Glu(OBzl), or DBTA; X3 can be Lys, Glu, Asp, or Phe; X4 can be Z, OBzl, BOC-Glu(OBzl), BOC-5 Lys (BOC), or Z-Glu (OtBu); or X3 and X4 in combination can be LystBOC-Glu(OBzl) ]; and X5 can be OtBu, OBzl, OMe, or Trp-OMe .
Preferred formula (I) ~ of the invention include, but are not limited to, the peptide deriY2tives 10 BOC-Asp(OBzl)-Lys(Z)-OtBu (Reversin 121); succinyl-bis[Glu(OBzl)-OBzl]; Z--D--Glu(OBzl)--D--Asp(OBzl)--OBzl;
DBTA-bis(D-Phe-Trp-OMe); DBTA-[Glu(OBzl)2]2; Na,N~-bis [ BOC-Glu ( Bz l ) ] -Lys -OMe ( Revers in 2 0 5 ); BOC-Tyr ( B z l ) -Tyr(Bzl)-OMe; BOC-D-Ser(Bzl)-Lys(Z)-OtBu; BOC-Glu(OBzl)-15 Lys(Z)-otBu; BOC-Glu(OBzl)-Lys(Z)--OMe; Na,N~--bis[BOC--Lys(BOC) ]-Lys-OMe; or Na,N~-bis[Z-Glu(OtBu) ]-Lys-OMe.
Any of the various ~- ' of the invention can be ~ ; n~fl with a rh~nr --entically acceptable carrier or an a.lJuv~-L. The various ~ -c of the invention can 2 0 also be combined with a drug , e . g ., a chemotherapeutic , 2ntiparasitic, or antibiotic drug, for convenient co-administration of the Reversin _ _ ' and the drug to a patient. The Reversin molecule can, in addition to inhibiting MDRl activity, also act as a adjuvant to 25 enhance the activity of the drug. As used herein, a "drug", includes a medication, a rh~-re~ltical, or a substance which is intended for use in ~ gn~5i c, cure, mitigation, treatment, or prevention of disease, or which is generally int~nrlc.fl to affect the structure or the 30 function of the body of a mammal.
The invention also i nrl~ C a method of preparing any of the various formula (I) ~ of the invention. The method involves providing a combination of one or more of Xln, X2, X3, X4n, Xs, or X1X2 in a 35 solution, e.g., by dissolving the combination, e.g., one W09~;131474 r~ lt ~4.
21 ~9978 or more of Xln, X2, X3, X4n, Xs, or XlX2, in a solution;
cooling the solution; adjusting the pH of the solution to the neutral range, e.g., a pH of 4-8, preferably pH 6-8, or between pH 7 and 8, inclusive; and purifying the 5 formula (I) ~ _ from the solution. By "purifying"
i8 meant extracting, filtering, ~:va,uu~ating, precipitating, washing, ~ u~ ya~ 7;nlJ~ isolating by chromatography, or any other means of isolating the desired Reversin ' from the reaction mixture. The 10 method can further include an additional purif ication step to remove any impurities fron the final product, e. g ., a gel f iltration step , or a chromatographic step (see Methods, below). The method can also include, or further include, a step of active ester coupling, or a 15 step of dicyclohexylcarbo~i ;m;~lP c~n~lPncation characterized by the following paL P~ 5: cooling by ice-water, 10% excess of DCC, pH adjusted between 7-8 with tertiary base (e.g. triethylamine, N-methylmolrhol;nP, diisopropyl-ethylamine). In certain 20 cases 1-1ly~Lv,-y~el~zotriazole additive is used for activation and to avoid pos5;hlP racemization (W. Lonig et al. Chem Ber. 103:2024, 1970, hereby inc;u~urc.Led by ref erence) .
The invention also ;nrlll~Pc a method of reducing 25 the activity of a multidrug LLa--a~u~ Ler protein or its homologues in a mammal. The method involves administering to the mammal an amount o~ any of the various formula (I) _ of the invention in a therapeutically effective amount. The method of reducing 30 the activity of MDR1 can be used to lower resistance to a drug, where the drug ;nr1ll~1pc one or more, e.g., at least one, two, or three drugs, which are chemotherapeutic drugs, antiparasitic drugs, or antibiotic drugs. By "mammal" is meant a human, a domesticated animal, e.g., a 35 cat or a dog, or an agricultural animal, e.g., a cow, WOgsl3l~7~ 21 8997 8 r~ 4~ --pig, sheep, horse, or poultry. A "chemotheLtl~t uLic drug"
;nr~ P5 any drug intended to target and kill a tumor cell, e.g., neoplastic, r~ nAnt, or benign tumor eell, in a mammal . An "antiparasitic drug" ; nrlll~lPC a drug 5 intended to target the agent of a parasitic infeetion, e.g., asearis, enterobium, hookworm, threadworm, ~O.p_. JLJ~I, sehistosomes, whipworm, protozoa, e.g., intestinal or extraintestinal amebas, giardia, malaria, toxoplasma, or trir' -c. An 'lantibiotie drug"
10 ineludes substances whieh inhibit or kill fungal or baeterial mieroorganisms , e . g ., actinomycin . r 1PC Of drugs within the scope of the invention include, but are not limited to, the substances listed in Table 1, as well as any chemotherapeutic, antiparasitic, and antibiotic 15 drugs which r.l;n;c:~lly elicit, or whose therapeutic effects are limited by, primary or se~ ry multidrug resistance caused by NDRI ~Ford et al. supra; hereby incuL uuL ~ted by ref erence) .
Cytotoxic drugs which are extruded by the MDRl 20 protein or its homologues include, but are not limited to, the ~ s shown in Table 1.

21 89978 P~l/ L'd.~4~
_ g _ Table T.
Drugs exported by the MDRl protein Anti-Cancer Druqs ~y;lmt leS
5 Vinca ~lk~lnifl~ vinblastine, vincristine v; n~ ; ne Anthracyclines doxorubicin, daunorubicin epirubicin Epipodophyllotoxins etnpssi~e 10 Antibiotics actinomycin D
Others mitomycin C, taxol, topotecan, mithramycin Oth~r cvtotoxic a~ents ~y~mr leS
Anti-microtubule drugs colchicine, podophyllotoxin Protein Synthesis inhibitors ~ y~;in~ emetine DNA intercalators eth; 1; tlm bromide Toxic peptides v~l ;r y-,iin, gr;~lm~ ;n D, N-acetyl-leucyl-leucyl-norlell~n:~l (ALLN) The method of reducing the activity of a MDRl protein or lts ~ - log~le~ in a mammal is also used to f~r;l;t~te administration of a drug through ~ s 25 which exclude various substances from a given type of cell or tissue. In particular, Reversins can be used to aid transport of a drug through the blood-brain barrier, or through the blood-testis barrier. By "blood-brain barrier" and "blood-testis barrier" is meant the 30 endothelial lining of cells that are selectively p~ -hl e or ~ -hle to su~La~aes circulating outside o~ the brain or testis, respectively.

WO9~/31474 2~ 8q~78 P~ I44 A "multidrug transporter protein" (NDR1), as used herein, refers to a glycoprotein present on the ~ .e of many cell types which acts to extrude various substances from the cell. In humans, MDRl is commonly 5 referred to as the P-glycoprotein, P-170, or Pgp, and is encoded by the mdrl gene. Also ~ nrl~ d are homologues of MDR1 which are members of the MDRl family of proteins in other organisms , e . g ., prokaryotes or lower eukaryotes, e.g., bacterial, yeast, fungal, parasitic 10 organisms, or other organisms that take up rPci~l~nre within a l; An body. Homologues within the NDR1 îamily of proteins perform the same function as MDR1 for cells of the other organisms , i . e ., by transporting hydrophobic cytotoxic __.lds out of the cell.
15 Generally, the method of the invention is intended to inhibit the activity of members of the MDR1 protein family which are shown by the assays below to be affected by a Reversin __~
A "therapeutically effective amount", as used 20 herein, refers to an amount that is effective at reducing the activity of the multidrug transporter protein; an amount that is effective at lowering resistance of the mammal to a drug or to a group of drugs; or an amount that is effective for facilitating absorption of a drug 25 through the blood brain barrier. An "effective amount"
can be calibrated by the assays below. By "facilitating"
ls meant ~nh;~nrin~ the overall amount of the drug t_at is ~hcnrhed~ or the fraction of the drug that is ;Ihcr~rh~.
As used herein, the term "reducing" means either 30 partially or completely inhibiting MDR1 activity. By "reducing" is also meant decreasing, lowering, or U~/CL~ in~ the effects of drug or multidrug resistance, 80 that less drug is ~LCIII~,,UUL ~ed from the target cell, or so that a greater Cu--ct~ L t-tion of drug 5~ tes 35 within the cell. As used herein, the term "reducing"

~ WO95131474 2 1 89978 r~.,lL L'~ ~41 . .
,er ~ ?~ both treating and preventing the OC-.;ULL-=ll'-e of drug, e.g., multidrug, resistance in a mammal. The level of NDR1 activity present in a cell or in a target tissue is measured by the assays provided below. This in 5 turn permits ~c 1 ~tion of drug at higher c~l-LLc-tions in the cell than would be possible in the absence of the RQversin ~ , '.
other f eatures and advantages of the invention will be ~ from the following detailed description 10 and from the claims.
Detailed Descri~tion We f irst brief ly describe the drawings .
Drawinqs Fig. 1 is a schematic illustration of the MDR1 15 protein in its membrane environment.
Fig. 2 18 a comput-:L-gel.eL~ted illustration of the 8'~'`'".`1i''y ~LLII~ IILa of the Reversin 121 l~cllle, BOC-Asp (OBzl)--Lys (Z)--OtBu.
Fig. 3 is a graph showing drug-stimulation of 20 human MDR1-ATPase activity in isolated insect cell membranes .
Fig. 4 is a graph showing stimulation of the vanadate-sensitive human MDR1-ATPase activity in the isolated - ' c.l~es of Sf9 cells by verapamil and 25 Reversins .
Fig. 5 is a bar graph showing the effects of Reversins on Fura-2 uptake in NIH 3T3 fibroblasts.
Fig. 6 is a bar graph showing the inhibition of Fura-2 AM loading in MDR1 ~Lassing fibroblasts by

3 o agents interacting with MDR1.
Fig. 7 is a photograph showing the effect of verapamil on dye loading in MDR1 ~ Lessing fibroblasts using single-cell imaging for fluuLasc~ dye uptake.
The upper panel shows control NIH 3T3 cells; middle and wo gs/31474 2 1 8 9 9 7 8 P~ 44 ~

lower panels show NDR1-3T3 cells. In the lower panel the medium also contained 25 ~M verapamil.
Fig. 8 is a graphic illustration of the fluu-e:sc~ dye uptake of control cells (A) versus 5 fluoL~sc~ dye uptake in the presence of 5 ~ Reversin 205 (B).
Fig. 9 is a bar graph showing the effect of various pretreatments and washings on f lu~ scc~ dye uptake into 3T3-NDR cells.
Figs. lOA and lOB are graphic illustrations of the effect of Reversin 205 and verapamil on drug-sensitive and multidrug resistant cultured human tumor cells (X562) treated with adriamycin (A) or vincristine (8) . ~R562 control, -+- R652 + 5 ,uNR, ~KK562 + 10 ,uN Verap,~K562 15 MDR, ~NDR + 5 ~NR, n K562 NDR + 10 ,u~V.
Figs. llA and llB are graphic illustrations of the effect of Reversins 121 (A) and 205 (B) on the vinblastine-sensitivity of d-uJ F-~l.sitive (KB3) and multidrug-resistant (K8V1) cultured human tumor cells.
20 KB3 control, -+- KB3 + 2 ug/ml R, ~K83 + 10 ug/ml R,~
KBV1 control, _~KBV1 + 2 ug/ml R, ~KBV1 + 10 ug/ml R.
Reversin C _ _ ' ~
The aim of the present invention is to provide rhPln;CAl reversing agents, or ~ -- -itizers, to 25 address the ~1 ini~Al problem of multidrug resistance.
Toward this end Arpli rAnts clQqi~nQd a set of novel llydLu~hobic peptide-based --1QC111Q~:: called Reversins.
Reversins effectively inhibit QYr~ of therapeutic drugs. They are effective at low 30 cullc~ .at.ion, non-toxic, and reversible. Reversins also leave the organism without adverse side effects, and in some cases even enhance the activity of the drug therapy itself. They can be ~-e~aL~d in high purity, in large quantities, and at relatively low cost. Reversins are 35 Q~reriAl ly advantageous for preventing resistance against ~ W095~31474 21 8~978 r~ [~4~

ant;r~n~-Pr drugs, without causing serious side-effects in other tissue6, and without irreversibly disrupting the l natural physiological function of the MDRl transport protein itself.
Nethods of Prer,aration Reversins are peptide derivatives consisting of naturally occurring L amino acids with bulky aromatic or alkyl groups, and carhoYAn~ P or carboxylic acid ester groups. The hydrophobic side-chains of the molecules 10 enhance their interaction with the MDRl Lrellla~lur ~er for which they are a substrate. The overall size of the - lPcllle also influences this interaction. Dipeptides and tripeptides are preferred. A computer-analyzed sP~ nn~lAry structure of a preferred dipeptide, Reversin 15 121, is illustrated in Fig. 2.
In general, Reversins are synthPC; 7Pd by an ~l~ylu~Iiate rL _ ~ rnnrlPncAtion reaction, ~PpPn~;n~ on the ,hPmic~l character of the amino acid moiety in the _ ' being prepared. Reversins are syn~hPsi ~s:l by 20 traditional stepwise rnn~Pnc~tion methods (The Peptides:
Methods of peptide synthesis, Eds. E. Schoder, K.Lubke, Acad. Press, NY, 1966; The Peptides: Analysis, synthesis and biology, Ed. by E. Gross, J. Neienhofer, Acad. Press, I~Y 1979, each hereby inou~uL~ted by reference), or by 25 automated solid-phase peptide synthetic methods, which are relatively l~l~y_ E:cc.le and inPyppncive. Reversins are also ~- e~d~6d by methods known to those of ordinary skill in the art, and as exemplified by r lPq 1-12, below.
Each of the r 1PC 1-12, below, are l.IL ~ed from amino acids that were protected to a desired grade.
The starting materials are dissolved in apolar solvents, e.g., DNF, acetonitril, or DMSû. C'nn~lPncAtion reactions are performed in solution, or by passage of the solution 35 over a solid-phase column. The solution is cooled, for W095~31474 - 14 - r~ 44 --example to 0-10C, and the pH is adjusted to the neutral range with, e.g., triethylamine, N-methylmorpholine, trimethyalmine, or diisopropylethylamine, and the product is allowed to form slowly, e.g. by stirring overnight.
5 Finally, the Reversin ~ _ 1 is purified from the reaction mixture by, e.g., filtration of the precipitate, or by evaporating away the mother liquor, and the product is washed.
The final products can receive additional 10 purification in order to remove any con~Tn;nAntq from the Reversin ' . The f inal purif ication step can include one or a combination of the following ~L~ceduL,as:
ethyl acetate; gel filtration; preparative high ~Las-liquid chromatography (HPLC); medium ~L~::5 UL~ column 15 chromatography; or silica gel column chromatography. The purity of the peptides obtained is ~lotPrm; nPd by thin layer chromatography lTLC) analysis.
In the examples that follow, Rf values were obtained by TLC on Kiesel gel sheets (DC Alufolien Merclc) 20 using the following solvent mixtures (in the ~ les the solvent mixtures will be identified by the numbers listed below):
(1) acetone:toluene, 1:1 (2) chloroform:acetic acid:benzene, 85:10:5 (3) acetic acid:benzene, 1:7 ( 4 ) ethyl acetate: pyridine: acetic ac id: water 240:20:6:11.
In general, Rever5in5 are only slightly soluble in water (maximum solubility is about 10 ,~g/ml), while 30 freely soluble in dimethyl sulfoxide (DMS0), glycerol, or ethanol. When testing a Reversin in an in vitro c~ r screening assay, the presence of serum in the culture media increases the solubility of the Reversin.

~ W095~31474 2~ 8~978 r~ 4~

The following non-limiting examples are provided to illustrate methods of preparing the Reversin - '-of the invention.
r le 1 5 p,~ n of BOC-Agp(OBzl)-Ly:l~Z)-OtBu ~R~versin 121) After dissolving 1 mmol of BOC-Asp(OBzl)-OH amino acid derivative (mole wt. = 323) in 50 ml of DMF, 1.1 mmoles of DCC (mole wt. = 206) and then 1 mmole of N'-carb~,hPn7~rylysine tert.-butyl ester hydrochloride (mole 10 wt. = 371) were added while stirring and cooling with ice water. The pH value of the reaction mixture was adjusted to 7-8 with triethylamine and the mixture was stirred at room t~ _ aLuLe: overnight. The precipitate was filtered off and the mother li~uor was ~:vay~lLated. After taking 15 up the residue in 150 ml of ethyl acetate, the extract was washed three times with 120 ml of 10% citric acid solution each, then three times with saturated sodium hydLuyo~ ;aiL,-,.,ate solution, and finally three times with ~a~uL~Ited saline solution. After drying the ethyl 20 acetate phase over anhydrous sodium sulfate and filtering off the drying agent, the organic phase was ~va~,LaLed and the residue was L~:LYI~ .d from 30~6 alcohol to give the title product. m.p.: 128-129C, R~ (4) = 0.9.
The possible conformation of Reversin 121 is shown in 2 5 Fig . 2 .
The molecular mass of Reversin 121 is 641.5.
r le 2 E~, -- '~on of succinyl--bi~tGlu~OBsl)--oBsl]
To a solution containing 1 mmole of dibenzyl 30 glutamate hydrochloride (mole wt. = 362) in 50 ml of DMF, 0.5 mmole of bis(pentafluorophenyl) succinate was added while stirring and cooling with ice water. The pH value of the reaction mixture was adjusted to between 7 and 8 with triethylamine, and the mixture was stirred at room 35 ~ aLuL~ overnight. Then the precipitate was filtered Wo 9~131474 2 1 8 9 9 7 8 - r~ 14~ --off, the mother liquor was evaporated and the residue was taken up in 150 ml of ethyl acetate. The organic solution was successively washed three times with 120 ml of 10% citric acid solution each, three times with 5 saturated sodium }.ydL~,g~ carbonate solution, and finally with saturated saline solution. After drying the ethyl acetate the phase over anhydrous sodium sulfate and filtering o~f the drying agent, the organic phase was evaporated, and the ~v~,Lcltion reside was recrystA~ 9cl 10 from aqueous alcohol to give the white crystalline title product, m.p.: 106-107C, Rf (4) = 0.9.
ExamPle 3 PL~ ~ ..tion of ~-D-Glu~OBzl)-D-A~p~OBsl)-OBzl To a solution containing 1 mmol of the amino acid 15 derivative Z-D-Glu(OBzl)-OH (mole wt. - 371) in 50 ml Or D~F, 1.1 mmoles of DCC and then 1 mmol of H-D-Asp(OBzl)-OBzl hydrochloride (mole wt. - 349) were added while stirring and cooling with ice water. The pH value of the reaction mixture was adjusted to between 7 and 8 with 20 triethylamine, and the mixture was stirred at room t, Cl~UL'2 overnight. After filtering off the precipitate, the mother liquor wa~ ~v~ ,Lc-ted, and the residue was taken up in 150 ml of ethyl acetate. me organic colntlon was sl~rco~ively washed three times with 25 120 ml of 10% citric acid solution each, three times with saturated sodium 1IYdLOg~ .LL.,.,ate solution, and finally with sal uLc~l~ed sa~ ine solution. After drying the ethyl acetate phase over anhydrous sodium sulfate, the drying agent was filtered off and the organic phase was 30 ~v~,~.,L,-ted to give the title ~ __ ' as a slightly yellow crystalline product. Rf (4) = 0. 9 .
r le 4 PL~ ..tion of DB~rA--~ois ~D-PI~ T ~ ONe) To a solution containing 1 mmole of the dipeptide 35 hydrochloride D-Phe-Trp-OMe (mole wt. = 401.8) in 50 ml Wo ss/3l474 2 1 8 9 9 7 8 P~ 44 of DMF, first 1.1 mmoles of DCC, and then subsequently 0. 5 mmole of DBTA- (OPFP) 2 active ester (mole wt. = 696), were added while stirring and cooling with ice-water.
The pH value of the reaction mixture was adjusted to 5 between 7 and 8 with triethylamine and the mixture was stirred at room t~ al ULa overnight. Then, the precipitate was filtered off, the mother liquor was ~va,.uLated, and the residue was taken up in 150 ml of ethyl acetate. The organic solution was s~-c~cively 10 washed three times with 120 ml of 10% citric acid solution each, 3 times with saturated sodium 11Y SLU~
carbonate solution, and f inally three times with ~ ted saline solution. After drying the ethyl acetate phase over an.~.y.lLuu6 sodium sulfate and filtering 15 off the drying agent, the organic phase was ~vapu aLed to obtain the title _ ' as a foam-like amorphous "uLa~ion residue. m.p.: 79-80C, Rf (4) = 0.9.
r le 5 ion of D~lTA--[GlU~ûBZ1)2]2 To a solution containing 1 mmole of the active ester DBTA- (OPFP) 2 (mole wt. = 696) in 50 ml of DMF, 2 mmoles of Glu(OBz1)2 tosylate (mole wt. - 500) and then 0.28 ml (2 mmoles) triethylamine, were added while stirring and cooling with ice water. The pl~ value of the 25 reaction mixture was adjusted to between 7 and a wit~
triethylamine and the mixture was stirred at room t~ _ a~uLa overnight. Then, the precipitate was filtered off and after evaporating the mother liquor the residue was taken up in 150 ml of ethyl acetate. The 30 organic solution was successively washed three times with 120 ml of 1096 citric acid solution each, three times with saturated sodium IIYdL ZCj-:SI carbonate solution, and finally three times with saturated saline solution. After drying the ethyl acetate phase over csl*lydLuus sodium sulfate, 35 the drying agent was filtered off and the organic phase

4 2 1 8 ~ ~ 7 8 r~ l4~ ~

was evaporated to give the title ~_ ' as an oily residue. Rf (4) = 0.9.
Example 6 Prep~rntion of N~, N'-bis[BOC-Glu~O}~zl) ]-~ys-ONo IRev~r~n

5 205~
Method A: To a solution containing 1 mmol of the amino acid derivative BOC-Glu(OBzl)-OH (mole wt. = 337) in 50 ml of DMF, 1.1 mmole6 of DCC and 1 mmoles of the amino acid derivative Lys-O~e dihydrochloride (mole wt. =
10 228) were added while stirring and cooling in ice water.
After adjusting the pH value to between 7 and 8 with triethylamine, the reaction mixture was stirred at room t~ _ C~tUL~ overnight. Then, the precipitate was filtered off and the mother liquor was t:v-~u~clLed. The 15 residue was taken up in 150 ml of ethyl acetate and ~lcc~ ively washed three times with 120 ml of 10% citric acid each, three times with saturated sodium l-y~
carbonate solt~tion, and finally three times with saturated saline solution. After drying the ethyl 20 acetate phase over d~ly~u~ sodium sulfate, the drying agent was filtered off and the organic phase was y~ ~.ted. After ri _Ly~ i n~ t~te eVO,~UL ~tion residue from the aqueous alcohol, the title ~ ' was r~hts~n~l as a slightly yellow crystalline product. m.p.:
25 79--81C, Rf (1) = 0.7, Rf (4) = 0.95.
Method B: A second method of preparing N3, N~-bistBOC-Glu(OBzl) ]-Lys-OMe is according to the ~ dU'~
of Example 11, except that 5. 03 g of the amino acid active ester BOC-Glu (OBzl) -OPFP was used as the starting 30 material, and the other constituents were used in the amounts of 1.15 g of Lys-OMe dihydrochloride, 20 ml of DMF and 1. 38 ml of triethylamine.
The molecular mass of Reversin 205 is 875 . 5 .

Wo gsl3l474 r ~1/.. . L~ 144 2 1 89q78 r le 7 P~a~ _tion of BOC-Tyr(Bsl)-Tyr~Bzl)-oMe To a solution containing 986 mg of the amino acid derivative BOC-Tyr(Bzl)-ONp (mole wt. = 491) in 50 ml 5 Dl~F, first 682 mg of H-Tyr(Bzl)-O~le. O~e hydrochloride salt (mole wt = 384), and then 0 . 28 ml triethylamine were added while stirring and cooling with ice water. The pH
value of the reaction mixture was adjusted to between 7 nnd 8 with triethylamine, and the mixture was stirred at 10 room ~ LuLa for 24 hours. After e vcLL~uLc~ing the mother liquor the residue was taken up in 50 ml of ethyl acetate. The organic solution was sllrc~qsively washed three times with 2N KHS04 solution, three time6 with saturated sodium llydL~ carbonate solution, and three 15 times with saturated saline solution. After drying the ethyl acetate phase over anhydrous sodium sulfate, the drying agent was filtered off. The organic phase was ~:vapuL~lt~d to give an oil which can be crys~ l 7ed from the mixture of ethanol:water (7:3) to obtain the title0 - _ '. Rf(1) :0.95; (R~(4) :0.9; m.p.=161-163 C.
~ 8 }1._ --..t.lon of BOC-D--~er(Bzl)--I,ys~8)--OtBu To a solution containing 10 mmoles of the amino acid derivative BOC-D-Ser(Bzl)-OH (mole wt. = 294) in 20 25 ml D~F, were first added 11 mmoles of DCC (mole wt. =
206), 3.5 g of H-Lys(Z)-OtBu hydrochloride salt (mole wt.
- 336). Then 1.13 ml of triethylamine were added while stirring and cooling in ice water. The pH value of the reaction mixture was adjusted to ~etween 7 and 8 by using 30 triethylamine, and the mixture was stirred at room t~ clLuLa for 24 hours. After tlv~l~uLclting the mother liguor the residue was taken up in 50 ml of ethyl acetate. The organic solution was successively washed three times with 2N RHSO~ solution, three times with 35 saturated sodium 11YdLUY~II carbonate solution, and finally Wo95J31474 21 8997~ r~ 44 ~¦

three times with saturated saline solution. After drying the ethyl acetate phase over al~.y-lLous sodium sulfate, and filtering off the drying agent the organic phase was t:vapuL ~ ted to obtain the title - ' as an oil which 5 i8 triturated in petroleum ether. Rf(4):0.85; Rf(5): 0.65.
~i~YAml'lle 9 Pr~paration of BoC-Glu~OBsl)-1y~[Z)-OtBu To a solution containing 2.12 g of BûC-Glu(OBzl)-ûFFP amino acid active ester (mole wt. = 503) in 20 ml 10 DMF, first 744 mg H-Lys(Z)-OtBu hydrochloride salt tmole wt. = 371), then 0.28 ml triethylamine were added while stirring and cooling by ice-water. The pH value of the reaction mixture is adjusted between 7 and 8 by using triethylamine and the mixture is stirred at room 15 t~ ~ULe for 24 hours. After evaporating the mother liquor the residue is taken up in 30 ml of ethyl acetate.
The organic solution is c~ cescively washed three times with 10% citric acid, three times with saturated sodium llydLu~ carbonate solution, and finally three times with 20 saturated saline solution. After drying the organic phase over anhydrous sodium sulf ate, the drying agent was filtered off and the organic phase was evc.~uLa~ed to give an oil which was then triturated in petroleum ether and crystAl 1 i ~o~l from ethanol with water to obtain the title '. Rf (1) :0.95; m.p.=79-81 C.
Examnle 10 F ~ tion o~ BOC-GlUlOB~l)--LystZ)-ON~
To a 5011lt~0rl containing 2.1 g of the amino acid active ester BOC-Glu(OBzl)-OPFP (mole wt. = 503) in 10 ml 3 0 DNF, was f irst added 1. 3 g of H-Lys ( Z ) -ONe hydrochloride salt (mole wt. = 329) . Then 0 . 28 ml of triethylamine were added while stirring and cooling with ice water. The pH
value of the reaction mixture was adjusted to between 7 and 8 with triethylamine, and the mixture was stirred at 35 room t~ __L-ltULe for 24 hours. After ~v~puL~ting the ~ Wo 95131474 r~ 4~
2 ~ 89q78 mother liquor the residue was taken up in 50 ml of ether.
The organic solution was successively wa6hed three times with 2N KHS04 solution, three times with saturated sodium hydLo5~ LLu--c-Le solution, and finally three times with 5 saturated saline solution. After drying the organic phase over an~y-lLuus sodium sulfate, the drying agent was filtered off, and the organic phase was t v~ ~c,.~lted to give an oil which was triturated in petroleum ether and crys~l l; 7 .d from ethanol with water to obtain the title 10 _ '. R~(1)=0.80; Rft4) :0.85; R~(5) :0.90; m.p. = 102-105C.
T;~yAmnle 11 Pr~p~rntion of Na,N~-bistBOC-Lys ~BOC) ]-Lys-OMe To a solution containing 5.12 g of the amino acid 15 active ester BOC-Lys(BOC)-OPFP (mole wt. = 512) in 50 ml DMF, was first added 1.15 g of Lys-OMe dihydrochloride salt (mole wt. = 231) . Then 1. 38 ml of triethylamine were 21dded while stirring and cooling with ice water. The pH
value of the reaction mixture was adjusted to between 7 20 and 8 with triethylamine, and the mixture was stirred at room t~ ~LULO for 24 hours. After ~v~u ~lting the mother liquor the residue was taken up in 50 ml of ether.
The organic solution was sll~ coccively washed three times with 2N KElso4 solution, three times with a~lLuLc.ted sodium 25 ~ LUgC.. carbonate solution, and finally three times with saturated saline solution. After drying the organic phase over cll~lydLuus sodium sulfate, the drying agent was filtered off, and the organic phase was e:va~uL~ted to give the title ' as an oil. R~(1) :0.90; Rf (4) :0.90.
r le Prop~rntion o~ Na,N~-bis~Z-Glu~OtBu) ]--Lys-OM~
Na,N~-bistZ-Glu(OtBu) ]-Lys-OMe was prepared according to the ~L UC6:dUL e of example 11, except that 2 . 5 g of the amino acid active ester Z-Glu (OtBu) -OPFP was WO 95131474 2 1 8 9 9 7 8 r~l,~ L'~ 144 u6ed as the starting material, and other constituents were used in the amounts of O . 6 g of Lys-OMe dihydrochloride, 25 ml DMF, and O.1 ml triethylamine.
The title ~ ' i8 a white crystalline material.
5 Rf(l) :0.70; R~t4) :0.75; m.p. = 83-84 C.
Meth~ c for D~ I ~Latin~ the Efficacv of Reversins Any of the various Reversin ~ ul,ds of the invention can be su~ .ed for their ability to reduce the activity of the MDR1 protein according to the following 10 in vitro and in vivo methods. In addition to the instructions and experiments provided below, each method is supported by one or more publications, each of which is hereby in~;UL~UL-ted by reference.
A. In vitro ~ethods Two test systems were developed to specif ically assess the ability of a Reversin ' to interact with the human MDRl protein. The f irst system measures the ATPase activity of MDRl, while the second system ~s the level of a f luorescent indicator extruded by 2 O the MDRl protein .
1. ATPase Assay In the first assay, MDRl-ATPase activation reflects the interaction and relative affinity MDR1 has f or a candidate _ ' . The MDRl-ATPase is stimulated 25 by cytotoxic drugs, e.g., vincristine, while it is insensitive to c-h~;cAl~ that are not LLe..,a~uLLed by the MDRl protein. The MDRl-ATPase is also stimulated by known ~ h~ itizing agents, such as the multidrug-resistance reversing agents verapamil and quinine (Fig.
3 0 3 ), probably ~ i n~ with drug extrusion of the multidrug LL ~u~ uL Ler . Thus, by measuring this MDRl-ATPase a relatively simple in vitro assay system became available for A~ 5ln~ direct drug interactions with the W095/31474 P~l~. '.'~ 14~

NDR1 protein (Sarkadi et al. J. Biol. Chem. 267:4854-4858, 1992).
The assay was developed by expressing the human MDR1 protein in Spodoptera frugiperda insect cells. The 5 cultured cells were infected with a baculovirus into which the cDNA of human NDRl was genetically Pn~i nPP~ed.
The ~ inAnt virus-infected cells produce a large amount of the NDRl protein, properly folded and fl~n~-fil~ns~lly inserted into the membrane. Additional 10 details regarding construction of these recombinant strains are provided by Germann et al. tPiorhPmi.ctry 29:2295-2303, 1988) and Sarkadi et al. (1992 supr~).
Measurements of the effect of Reversins on the MDR1 ATPase activity are performed as follows, and 15 according to the methods provided by Sarkadi et al.
(1992, supra).
MDRI ATP~se mea;.ul~ ts:
SL~t?A~ frugiporda (S~9) cells were infected with a 1 ,_ ; nAnt baculovirus carrying the human NDRl 2 0 gene, and cultured according to the y uceduL c:s described previously (Germann et al. 1990 supra; Sarkadi et al.
1992 supra~. The virus-infected Sf9 cells were harvested, and their membranes isolated and stored as described (Sarkadi et al. J. Biol. Chem. 267:4854-4858, 25 1992). The amount of ATP cu..:,, Lion measured in these membranes ref lects the ATP-~Ppnn~l~nt transport function of the multidrug LL~IJ.~.LLer. ATPase activity of the isolated Sf9 cell membranes was estimated by measuring inorganic phosphate (Pi) liberation. To do this, a 30 membrane F--CpPn~; on (about 10 ,~lg of membrane protein) was incubated at 37C in 0.1 ml of a medium containing 50 mN
Tris-Mes (pH 6.8), 2 mN EGTA, 2 mN DTT, 50 mM KCl, and 5 mM Na-azide. The ATPase reaction was started by the addition of 5 mN NgATP. The reaction was stopped by the 35 addition of 0.1 ml of 5% SDS solution, and the amount of W0 95/31474 2 1 ~ ~ 9 7 8 I~ ~ ..'C 144 Pi ~ntc~rmin~d immediately. ATPase activity was estimated by the difference obtained in PL levels by a sensitive colorimetric assay between zero minutes (reaction stopped immediately with SDS) and 20 minute incubation periods.
5 The data points show the means of triplicate (lPtorm;n:~tions in a re~Last:-lLative experiment. The differences between the ATPase activities meagured in the absence and ~Lasèl-ce of vanadate (100 ~M) are plotted.
Isolated ~ .es of uninfected or B galactosidase 10 infected Sf9 cells had no drug-stimulated ATPase activity (Fig. 3).
When Reversins 121 and 205 were tested in the ATPase assay system, they greatly stimulated the ~5DRl-ATPase, but were effective at significantly (one to two lS orders of magnitude) smaller .c,l~cel~LLations than the known reversing agents verapamil and quinine. The half-maximal activating u ,l~o~ tion (~a) of verapamil was approximately 1 ~M, while the Ka value for Reversin 121 was approximately 60 nM and for Reversin 205 this value 20 was about 30 nM (Fig. 4). Thus, the multidrug LLe.ll~UL Ler seems to interact with Reversins with an exceptionally high affinity. As shown in Fig. 4, a strong inhibition of the MDRl-ATPase was observed at higher .<,l~ce~.LLations (above 1 ~M) of Reversin 205.
2 . FluoLes- e~ce Assay:
The second assay is based on the mea~ uL~ of fluuLe~cellL dye uptake into intact cells. FluuLasc.~
dyes are often used to indicate intraCo~ r calcium or p~ changes. An effective technique for colllll~r dye 30 loading is the application of acetoxy-methylester (AM) derivatives. These hydrophobic dye esters are non-fluoIa~.cellL outside the cell are cleaved by intracelllll;~r esterases into hydrophilic fluuL~:sc~:llL free acids. This intr~o~ r "trapping" of the free dye and the 35 c~t;nl~uC inward gradient of the AM ~ ' results in WO9~131474 2 1 89978 r~l~ L'~144 . 1 the A~ lAtion of large amounts of fluuLasu~..L
indicator inside the cell.
Cell culturing:
NIH 3T3 cells were cultured under standard 5 conditions in D-NEN medium. MDRl-transfected cells (NIH
NDRl G185) were ~rt~a~- d and characterized for their drug-resistant properties as described (A-' ~ ' r et al.
Proc. Natl. Acad. Sci. USA 89:8472-8476, 1992; BL~
et al. J. Biol. Chem. 267:21020-21026, 1992; Sarkadi et 10 al. 1992 supra). 8efore each experiment the cells were tryp~;nl~ed, then washed and stored in D-NEM medium at 37C. K8Vl (NDRlt) and KB3 (~DRl ) human tumor cells were also cultured in D-NEN, which K562 human tumor cells were grown in RPMI medium, supplemented with 10% FCS.
The effects of Reversins 121 and 205 on NDRl function in intact cells was PY;lm;nPd by a flu~,L-~sc~..L
dye extrusion assay (Fig. 5). Nouse NIH 3T3 fibroblasts, transfected with human NDRl cDNA and expressing human NDRl protein, actively extrude the ~ LU~JllObiC AN
20 derivatives of fluuLesctl-L dyes, e.g., Fura2-AN (Homolya et al. J. B~ol. Chem. 29:21493-21496, 1993; Hollo et al.
Bioch. Biophy. Acta. 1191:384-388, 1994). Similar experiments can be performed with NDRl ~ .Lessing human tumor cells. Verapamil, vincristine, and Reversins 121 25 and 205 inhibit this dye extrusion, most probably by competing with the dye on the transporter. In the experiments shown here r-~ir-l ly effective Cu"-~"LLations of Reversins were used. However, Reversins also act in at least one order of magnitude smaller cullc~llLLations 30 than verapamil.
Human NDRl-transfected mouse fibroblasts and NDRl-expressing human tumor cells actively extrude the AN
forms of several fluoL~su~llL indicators, lowering the level of intracellular f luorescence in cells with active 35 multidrug LLa~l-yuLL. Thus the ArCllm~llAtion in such cells WO9~/31474 1~ 14~ 1~
21 8~978 of fluuLa~ce..~ dye is strongly inhibited. This MDR1-specif ic dye-AM extrusion is blocked by competing 6ubstrates and inhibitors of the MDR1 LL ~ uu~ Ler, e.g., by verapamil, vincristine, sodium orthovanadate, and a ~ n;~l anti-MDRl antibody. In contrast, these agents have no effect on dye ~ tion in fibroblasts which do not uveLe~Less MDR1 (Fig. 6). See also Kessel et al.
(Cancer Res. 51:665-670, 1991~; Neyfakh et al. (E~xp.
Cell. Res. 174:168-176, 1988); and Sarkadi et al. (,J.
Biol. Chem. 268:21493-21496, 1993).
Fluorescence studies:
Dye uptake was measured by incubating 2 X 106 cells/ml D-MEM medium at 37C in the ~Lasel~ce of 0.5 ~LM
Fura-2 AM (added in 5 mM stock solution in DMSO), then rapidly spinning the cells (15 sec, 12 , 000 x g), and rinsing the pellet with HPM1 medium (containing 120 mM
NaCl, S mM kcl, 0.4 mM MgC12, 0.04 mM CaC12, 10mM HEPES-Na (pH 7.4), 10 mM NaHCO3, 10 mM glucose, and 5 mM Na2HP04).
The cells were L~ C~ in 2 ml HPM1 and fluu~c~"ce 20 was measured with rapid stirring in a Hitachi F-4000 fluc,~;esce..L ~e.:LLuul~otometer. The excitation wavelength was 340 nm, and P~ i Csinn was measured at 410 nm. Maximum flUULeSCtllCe and dye ou..ce..LL Lion were - .d after the addition of 0 . 596 Triton X-100 and 2 mM CaC12 to the 25 medium. The dye c ullcellLL~ltion was calibrated based on the mea~uL Ls of free acid dye fluolescence in the same in~LL, L under identical conditions.
Flow-cytometric - _ ~ -,tc Fluorescence mea:iuL Ls were done in 60 sec 30 8n:~nninq periods using a Cytoronabsolut in~,LL, L (Ortho Diagnostic Systems, NJ). The excitation was set to 488 nm. Green fluuLes-:e~ was r - _Led with a filter with a range of 515-548 nm, while red fluuLecce1-~.e was measured above 62 0 nm .

~ WO 95131474 2 1 8 9 9 7 8 P.. ,~. ~ 14~

~ With this method drug-interactions with the MDRl protein can be measured by following cc ~ Ar fluuL~ ..ce allowing a flow ;yt LLic or single cell imaging detection of the function of MDRl in tumor cells.
5 As shown in the single cell images of Fig . 7, f ibroblasts expressing the NDR1 protein, in ~UII~L~ to the control cells, are not loaded with a flUULC:s_~lt dye, while verapamil, which inhibits the multidrug ~L~ln~yuLLer, restores dye uptake.
When assaying the effect of Reversin molecules on the f luuL ~sc~ dye uptake and the drug-resistance in various MDRl-transfected and multidrug-resistant tumor cell lines in vitro, these experiments indicated a strong inhibitory action on drug extrusion by low cul.c~ L~Lions 15 of Reversins, again showing a specific interaction of the multidrug ~L~n~l!uLLer with these molecules.
Binding l~xperiments:
The relative ability of Reversins to bind to the MDRl protein, or to be removed from the cell - ' c...e, is 20 --- ed by "wash-out experiments" (Fig. 9). NIH 3T3-NDRl cells were pre-treated with 15 ~lN verapamil, 5 ~N
Reversin 121, or 5 ~LM Reversin 205 for 5 minutes, which ~L U lu~ed a ~ lete inhibition of dye extrusion by NDRl with 1% serum in the media. The cells were washed once 25 with the standard incubation media and a 5 min centrifugation at 800 x g. Dye uptake was then - e:d with or without the addition of 15 ,~IM verapamil during the uptake period. As shown, preincubation with veLc~amil had no effect on dye uptake, while both 30 Reversin 121 and Reversin 205 had a major effect even ~fter washing the cells. Thus verapamil could be eliminated by a single wash, while 121 and 205 r~ in~d effectively bound to MDRl.

WO95/31474 21 8 9 9 7 8 P~ rDl44 ~csPcsing Reversin activity by its ability to enhance the cytotoxicity caused by other agents Another method for IcRaRRin~ the ability of 5 Reversins to inhibit drug resistance is to measure the cytotoxicity of drugs in multidrug-resistant human tumor cell lines. Since MDRl normally lowers the cu..~.:..LL~tion of cytotoxic agents to subtoxic cu--ct:~-LL ~,tions by extruding them from the cell, inhibition of MDR1 would be0 expected to enhance cytotoxicity.
In these studie6, adriamycin, vincristine, and vinblastine, in originally ineffective Co~ LLc.Lions, become effectively cytotoxic in the presence of 1-10 flg/ml of Reversins 121 or 205. Figs. lOA and lOB present 15 6uch an experiment for Reversin 205 using K562 human ~:LyLl ~ ublastoid tumor cells and their adriamycin-selected multidrug-resistant subline. Figs. llA and llB are similar experiments with the intestinal tumor cells KB3 and RBVl, the latter being a multidrug resistant subline.
20 In the experiment shown in Fig. ll (A), Reversin 121 was found to be cytotoxic in the NDRl _,~L~ssing cell6 (but not in the control cells) even without the addition vinblastine. Such a collateral toxic effect (which may be due to the AL1 ~,; R " ;n~ futile functioning of the 25 drug LL~ UL Ler) can be greatly advantageous in treating drug-resistant tumors.

wo 9S/31474 2 1 8 9 9 7 8 1 ~.,~, . 14~

Application of the in vitro fluorescent ~ssay for clinical ~;~r~nn5iç
By using the some flu~.L-:~ce1-L dye extrusion assay described above the in vitro effects of Reversins on the 5 multidrug-resistant leukocytes of a lellkPm; ~ patient were studied. Cells were isolated by withdrawing blood from the patient, and isolating white blood cells by centrifugation. Cells can be isolated from other types of tumors by biopsy . A f low cytometer was used to l0 measure fluu~asc~ dye loading of individual cells, which had been previously shown to express MDRl, in the absence or presence of Reversin 205 (Fig. 8). The uptake of fluu.ascel.L dye under these conditions models the uptake of cytotoxic drugs into the tumor cells.
15 F1U~Le SC~ dye uptake was measured at 37 C for l0 minutes. In Fig. 8, the MDRl containing cells in the control experiment had a low fluuLas~ dye uptake tA), while the addition of 5 ~lM Reversin 205 blocked dye extrusion by MDRl (8). Reversin 205 thereby 20 significantly increased dye uptake and yielded a uniformly high fluorescence in the leukocytes. Further details for the method are provided by Hollo et al., supra .
Addition in vitro methods:
Additional in vitro methods of screening the ability of a Reversin ' to act as a rh iti~ing agent for the reversal of multidrug resistance are provided by Ford et al. supra, at Tables 1--6 .
30 B. In vlvo Methn-l~
I~ vivo animal studies for the effectiveness of - Reversins can be conducted using any suitable animal model system known to those skilled in the art. One example of an appropriate system is the mouse P388 35 l ~llk~ model system (Tsurouo et al., supra) . This Wo 95/3147 21 ~ 9 9 ~ 8 r~ l44 animal model is widely accepted for testing the effect of cytotoxic antileukemic agents or response - '1fier .
Inbred (DBA x black F1) mice received 106 P388 5 lollkomi~ cells by illL~ eLltoneal in~ection. The survival of the ~ice was followed. The mice were injected with control P388 cells, as well as with P388 cells that had been solectecl under drug e~O~ULe. Drug ~L} O~uLe induced uvel-~k~Lession of the NDRl protein 10 (P388-MDR cells~.
In one trial, the mice had a mean survival time of 14-16 days (they die in a generalized leukemia caused by the P388 cells). The mice injected with the P388 cells were treated with doxorobicin (adriamycin) in a dose of 15 1 mg/kg/day for 6 days after the injection of the P388 cells. The results showed that adriamycin prolonged the survival time in the case of mice injected with control P388 cells, ~Y~eo~n~ the period of 30 days. In cc,..L.c.:.L, adriamycin therapy did not significantly 20 prolong the lifetime of mice injected with P388-NDR
cells. Reversin can be administered to the animals in a dose of 2 mg/kg, a level which has no toxic effect on the control animals, as ~ll RCIlCCP~ above.
Another test system is to conduct similar trials 25 using human xenografts in nude mice. Human erythroleukemia (R562) cells are injected into tolerant mice. The developing ~ koml ~ is treated with cytotoxic _ '- with or without a Reversin candidate '.
Another animal model system for testing the 30 ability of a Reversin ~_ ' to reverse multidrug resistance is to use a tr~ncg~niC~ mouse which e~.esses the human MDR1 gene, e.g., in its bone marrow (Pastan et al. FASEB Jour. 5:2523-2528, 1991).
Human cl inic~l trials can be performed according 35 to methods known to those skilled in the art. For ~ WO9~131474 2 ~ 89'~78 r~., . 1~ 144 example, Dalton et al. provide methods of testing the - --,citiger verapamil for its ability to modify resistance to a chemotherapeutic tJo1lr. Clin. Oncol.
7:415-424, 1989). Additional methods for cnn~ t;n~
5 human cl ;n;r~l trials of Reversins include, but are not limited to, those provided by Berenbaum et al.
(phn 7. ~ev. 41:93-141, 1989); Benson et al. (Cnncer TreAt . R~p. 69 : 795-799 , 1985); Cairo et al . (Cancer Res .
49:1063-1066, 1989); Fine et al. (J. Clin. Oncol. 5:489-10 495, 1987); Frishman et-al. (J. Cardiol. 50:1180-1184, 1982); r~iller et al. (J. Clin. Oncol. 6:880-888, 1988);
ozOls et al. (J. Clin. Oncol. 5:641-647, 1987); and Presant et al. (Am. J. Clin. Oncol. 9:355-357,1986).
~laLa,U~U~iC Use of Rever8;nc The Reversins of the invention can be used therapeutically to inhibit the in vivo activity of the ~lDR1 protein in a patient experiencing drug resistance or poor drug absorption. An effective amount of the Reversin can be administered il~LL~v~ u81y to t_e 20 patient, or administered orally according to conventional methods, in the form of a capsule, liquid, tablet, powder, or pill. Reversin can also be incuL~ Lc-ted into an ; _ 1 ~nted or orally administered slow release device.
Reversin can be EJL~Led for therapeutic use by 25 mixing the _ ' with rh~rr-ce~ltical carriers and/or additives that aid solubility, absorption, flavor, or texture to the vehicle or its contents , e . g ., physiological saline, oil, e.g., refined soy bean oil, gelatin, glycerin, or purified water.
An appropriate dosage is between 50 ,ùg/kg and 100 mg/kg. An effective and safe dosage can be det~rm;n~d by conventional methods or by the methods taught herein, or by calibration to a given patient on an individual basis.

Wo95/31474 2 1 89978 P~ l44 It has been found that _ - of the formula (I) according to our invention are capable of stimulating, in a concentration of 0. 03 ,~M, or of inhibiting, respectively, in Cul~- ell~Lc~Lions of 1 to 5 ~M, 5 the activity of the MDR1 protein. The stimulating c-- L- ations are by 1 to 3 orders, the inhibitory c ~ ations are by 1 to 2 orders lower than the C~L~ n~ CullC~ LaLiO118 of 6ubstances known from the literature, e.g., verapamil.
The safety of Reversins for human administration can be confirmed in CI~L~r iate ani~al models. For example, the in vivo acute and subacute toxicity of Reversins was DY~minD~ in laboratory rats. Reversins 121 and 205 solutions were ~re~a~ed and administered as 15 follows. Type A solutions contained 50 ~g/ml Reversin 121 or 205 in 20% ethanol in physiological saline. Type B solutions contained 1 mg/ml in glycerol, contA~nin~ 10%
ethanol. Typê A solutions (0.5 ml) were administered ~ Lave~uusly to three laboratory rat6 weighing 200-250 g 20 each (about 100-125 ,ILg/kg) twice dally for 3 days. Type B ~ol~ltinnf: (0.5 ml) were given to similar rats (2 - 2.5 mg/kg) by i~ eLitoneal injection, twice daily for three days. Control rats received the same solutions without the Reversins. During the three days of 25 injections and in a ~ ]c follow-up period, no acute or subacute toxicity of the _ '~ was observed.
Additional toY~city testing can be performed by the methods of Pastan et al. (Proc. Natl. Ac~d. Sci. rJSA
85: 4486-4490, 1988) and Tsuruo et al. (Cancer ~es.
30 43:2905-2910, 1983).
other ~ 8 are within the claims set forth below .
What is claimed is:

Claims (23)

1. A composition of the formula (I):
X1n-X2-X3(X4)n-X5 (I) wherein n is 0 or 1, and each n is the same or different;
X1 is selected from the group consisting of tert.-butyloxycarbonyl, BOC-Asu, Z-Asu, benzyloxycarbonyl, Glu(OBzl)-OBzl, Trp-OMe, Trp-Phe-OMe, Phe-Trp-OMe, Phe-Phe-OtBu, Trp-Trp-OtBu, indoleacetyl, benzoyl, alkylamine of 1-4 carbons, dibenzylamide, tryptamide, 1-amino-adamantine, aminomethylcyclohexane, indoline, phenylethylamide and dicyclohexylamide;
X2 is selected from the group consisting of Glu(OBzl), Asp(OBzl), succinyl, O,O-dibenzoyltartaroyl, diphenoyl, muconyl, Thx, Cap, Asu, Nal, Pen, Phg, Dbt, Lys(BOC), Lys(Z), Cys(Bzl), Thr(Bzl) Glu(OtBu), tert.-Leu, Leu, Nle, Pro, Phe, Tyr(Bzl), and Ser(Bzl);
X3 is selected from the group consisting of Asp, Asu, Lys, Glu, Trp, Thx, Cpa, Nal, Pen, Phg, Dbt, Glu(OtBu), tert.-Leu, Leu, Nle, Pro, Tyr, Phe, and Tyr (Bzl);
X4 is selected from the group consisting of BOC-Glu(OBzl), Glu(OBzl), OBzl, Bzl, BOC, BOC-Lys(BOC), Asu, Z-Glu(OtBu), Asp(OBzl), Asp(OBz)-OBzl, benzyloxycarbonyl, 0-(cyclo-hexyl), fluorenylmethyl ester, Glu(OtBu), Glu(OtBu)-OBzl, 1-amino-adamantine, aminomethylcyclohexane, indoline, phenylethylamide, and dicyclohexylamide;
and X5 is selected from the group consisting of OMe, OBzl, OtBu, Phe-OMe, Trp-OMe, -0-(cyclohexyl), (chlorophenyl)-isobutylamide, fluorenylmethyl ester, ONp, 1-aminoadamantine, aminomethylcyclohexane, indoline, phenylethylamide, and dicyclohexylamide, with the proviso that said formula (I) is not BOC-Leu-Tyr-OMe.

2. The composition of claim 1, wherein said formula (I) is BOC-Asp(OBzl)-Lys(Z)-OtBu.

3. The composition of claim 1, wherein said formula (I) is succinyl-bis[ Glu(OBzl)-OBzl].

4. The composition of claim 1, wherein said formula (I) is Z-D-Glu(OBzl)-D-Asp(OBzl)-OBzl.

5. The composition of claim 1, wherein said formula (I) is DBTA-bis(D-Phe-Trp-OMe).

6. The composition of claim 1, wherein said formula (I) is DBTA-[ Glu(OBzl)2]2.

7. The composition of claim 1, wherein said formula (I) is N.alpha., N.epsilon.-bis[ BOC-Glu(OBzl)]-Lys-OMe.

8. The composition of claim 1, wherein said formula (I) is BOC-Tyr(Bzl)-Tyr(Bzl)-OMe.

9. The composition of claim 1, wherein said formula (I) is BOC-D-Ser(Bzl)-Lys(Z)-OtBu.

10. The composition of claim 1, wherein said formula (I) is BOC-Glu(OBzl)-Lys(Z)-OtBu.

11. The composition of claim 1, wherein said formula (I) is BOC-Glu(OBzl)-Lys(Z)-OMe.

12. The composition of claim 1, wherein said formula (I) is N.alpha.,N.epsilon.-bis[ BOC-Lys(BOC)]Lys-OMe.

13. The composition of claim 1, wherein said formula (I) is N.alpha.,N.epsilon.-bis[Z-Glu(OtBu)]Lys-OMe.

14. The composition of claim 1, wherein said formula (I) is combined with a pharmaceutically acceptable carrier.

15. The composition of claim 1, wherein said formula (I) is combined with a drug.

16. A method for preparing the composition of claim 1, said method comprising the steps of:
(a) providing a combination of one or more of said X1n, X2, X3, X4n, X5, or X1nX2 in a solution;
(b) cooling said solution;
(c) adjusting the pH of said solution to the neutral range; and (d) purifying said formula (I) from said solution.

17. The method of claim 16, wherein said method comprises active ester coupling.

18. The method of claim 16, wherein said method comprises a dicyclohexylcarbodiimide condensation.

19. A method of reducing the activity of the multidrug transporter protein in a mammal, said method comprising administering to said mammal an amount of the composition of claim 1 that is effective for reducing said activity.

20. The method of claim 19, wherein said method is used to lower resistance to a drug.

21. The method of claim 20, wherein said drug is selected from the group consisting of a chemotherapeutic drug, an antiparasitic drug, and an antibiotic drug.

22. The method of claim 19, wherein said method is used to facilitate administration of a drug through the blood-brain barrier or through the blood-testis barrier.

23. The method of claim 19, wherein said composition is co-administered with a drug.