CA1323303C - Advanced anticancer therapy and cytotoxic medicaments for its implementation - Google Patents
Advanced anticancer therapy and cytotoxic medicaments for its implementationInfo
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- CA1323303C CA1323303C CA000543912A CA543912A CA1323303C CA 1323303 C CA1323303 C CA 1323303C CA 000543912 A CA000543912 A CA 000543912A CA 543912 A CA543912 A CA 543912A CA 1323303 C CA1323303 C CA 1323303C
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- egf
- cytotoxic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
ABSTRACT
Cytotoxic targeted drug polymers comprising a backbone of polyglutamic or polyaspartic acid or copolymers threreof having part of the .gamma. -carboxy-late groups bound to daunomycin or other antitumor agents and conjugated with epidermal growth factor and/or other antibodies and internalizing factors as a targetting device.
Cytotoxic targeted drug polymers comprising a backbone of polyglutamic or polyaspartic acid or copolymers threreof having part of the .gamma. -carboxy-late groups bound to daunomycin or other antitumor agents and conjugated with epidermal growth factor and/or other antibodies and internalizing factors as a targetting device.
Description
1 3 2 ~ ~, IJ v ADv~NrEn ANTICANOER THERAPY AND CYTOTOXIC MEDICAMENTS
FOR ITS IMPLEMENTATION
r~
R~U~ N
The present invention deals with anti-cancer therap~ and, more parti-cularly, concerns a new metnod of combat.ing ~alignar.t di_eases by adminis-tering a medicamen~ ~hich ~will preferentially ~ill cancer cell and have oniy a limited, or insignificant detrimental effect on ;~ealthy cells.
The invention also concerns new selective cytotoxic compounds for impiementing the method, i.e. conjugates consisting of three main covalen-tly bound components: a seiective tumor targetting vector, a biodegradable poly~er backbone acting as an endocellular drug release carrier and leavir.g no toxic residue after resorption and a cytotoxic drug labei especially designed to inhibit cellular growth or to kill malignant cells.
The invention also concerns a method for manufacturlng the nelA medica-ment compounds.
T~E PRIOR ~T
It is ~ell ~nown ~hat current cancer therapy involves the use or ant;mitotic drugs such as adriamycin, vinchristine, bombesin, daunomycin and metothrexate which all have strong undesirable side-effects on the normal cells of the patient. It is therefore important that the activity of antitumor drugs be specifically directed to the malignant cells and have little toxic effect on the normal cells.
Many targeted cytotoxic agents have already been reported based on anti~odies recognizing speciric cell surface antigens coupled to radio-active isotopes with cytotoxic properties, or to toxins such as auromycin, hematoporphyrin, abrin, ricin, diphteria toxin, pseudomonas, exotoxin, gelonin or to the above-mentioned antimitotic drugs. For reviews see P.E.
THORPE et al., (1985), Monoclonal Antibodies 84; Biological & Clinical Applications; Ed. A. RIMCHER~ e; al., Complexes o~ antibodies and antiviral agents, such as interferon have also been tried ~New Scientist ref. April 17, lg86). Further US-A-4,455,985 discloses the binding of Pseudomonas exotoxin to antibodies, for instance antibodies to s?ecific human cell receptors such as the transferrin receptor.
1 3233~
Slowing the release of anticancer drugs by firs, link ng to d sui'able polymer carrier which will then graduall/ rree th* drug and kee? its con-centration effect ve for a longer time at ~.he ~ar~et site const-~ates an improved approach to the problem. For instance W.A.R. VAN HEESE'~lJK et al., (19~5) J. of Control Release 1, 310-15 discloses the binding of anthracy-clin to a polymer which slowly releases the drug and thus maintains a constant concentration thereof in the blood of treated patlents. In this connection, endocellular drug-releasing systems are particularly interes-ting, for instance antimitotic drugs coupled to the side-,-hain of poly-glutamic acid. Indeed, these side-chains are slowly degraded in the presen-ce of y -glut~myl-transferase, an endocellular enzyme ~hich is particularly abundent in tumor cells as compared to normal cells and, consequently, releasing the anti-mitotic drug within the targeted cells. Thus Y. KATO et al., (1984) in J. Medicinal Che. 27. 1602-7, reports bir.ding Daunomycin (~M) to polyglutamic acid (PLGA) and coupling the resulting c~/totoxl-polymer with rat ~ -fetoprotein ( ~-~FP) antibody. Upon trial, the cytotoxic activity of the resulting ~ -AFP-PLGA-DM conjugate was shown to be more effective than nIg (a control antibody), ~ -A~P, unconjugated DM, PLGA-DM or nIg-PLGA-DM.
EP-~ ,720 (TEIJIN) discloses a conjugate comprising an i.~moglobulin capable of binding selectively to a particular antigen possessed by a cell to be killed, a polymer carrier and a cytotoxic substance lin~ed thereto, for instance p-(N,N-bis(2-chloroethyl))-phenylenediamine, Melphalan, 1-( ~-D-arabino-furanosyl)-cytosine and its phosphate, methotrexate, actinomycin D, mitomycin C and the li~e.
US-A-4,485,093 discloses an immunotoxin conjugate for treating mali-gnant deseases, which consists of arsanilic acid and tumor specific anti-odies covalently bound to a poiyslutamic acid bac~-bone.
In the aforesaid approach~ selection of an effective antigen or rece-ptor to target the drug is of prime importance. However, even more impor-tant is the possibility of causing the medicament to penetrate into tne target cell, i.e. to be internalized therein at suitables sites so that, by fast degradation of the back-bone drug carrier, the killing or inhibiting effect of the cytocoxic substance be e~phasized or amplif ed.
Thus, the therapy achieved by the method of claim 1 is an important step toward this objective.
132330, According to one aspect of the invention there is pro~ided the use in the treatment of malignant diseases a covalent conjugate medicament consisting essentially of the following components bound covalently together, a) a cytotoxic substance, b) a poly~.er carrier having an endocytic biodegradable, nontoxic, polyaminoacid backbone, c) a cell homing vector with the properties of first selectively targetting the conjugate toward malignant cells to be fought and second, providing for the internalization of the conjugate into said cells, wherein fast biodegradation of the carrier will occur with consecutive release of the cytotoxic drug which will selectively combat said malignant cells.
According to another aspect of the invention there is provided a cytotoxic targetted conjugate for the application of the method of Claim 1, this conjugate having the formula Z
- ~G~ Cv -F- ~ - H-~
(lc~;2~p ~rly ~0 ~_ CO-.~. ~-DIY CO-.~. ' y~`_~!' in which p and o are 1 or 2;A and A' represent chain extending amino-acid intermediate links of formula -~NH CHY-C0) and x and y, which define the number of these links per molecule, can be zero or any integer from 1 to 20; y is an amino-acid rest; DM and DM' represent one or more cytotoxic substances covalently bound to the aminoacid carboxyl group through an _,.
1 3 2 3 3 0 ~
amide or ester lin~ but DM can also represent an OH of a free carboxyl; EGF defines a homing vector for promoting malignant cell recognition and internalization therein; R is a group of formula.
-C~ a- ( G~ iH wi.h ~ ~einq ~2- ~r~ e~
~rom -(C~ with r .r ~ d; or ~ ; o~ ~ ; ~
In some embodiments, the factor promoting cell internalization of the homing vector (c) also acts as the "arrow-head", i.e. it recognizes and binds to the cells at suitable receptors thereof. Examples of such vectors are the epidermal growth factor tEGF) or other EGF analogues of growth factors such as urogastrone, -TGF, or synthetic and natural derivatives thereof, transferin, low density lipoprotein (LDL),Nerve Growth Factor (NGF), Platelet Derived Growth Factor (PDGF), and some viral receptors, all providing cell internalization.
~O Otherwise, in some other embodiments, the homing vector can comprise separate binding and internalizing factors; for instance, the binding element can be non-internalizing antibody (such as FAB) to a specific antigen of the cells and the internalizing element can be any known internalizing factor specific or unspecific.
The use of EGF factors to specifically direct medicaments toward EGF receptors on malignant cells is not unobvious per se since several investigations have demonstrated the -3a-. ~ ~
1323'~'~
presence of these receptors in high concentration on many squamous cell carninomas as well as sarcomas. Such results include breast, lung, brain and skin tumors (see the following references; J. Hunts et al., (1985) JPn. J. Cancer Res. 76, 663-66; B. GUSTERSON et al., (1985), The Lancet, 364-68; F.J. Hendler et al., (1984), Cancer Res. 443, 753-60;
T. BAUKNECHT et al., (1985), Dermatologica 171, 16-20.
It is known also that these receptors are particularly efficient targets for EGF-linked cytotoxic drugs because of internalization into the -3b-13233~ J
cells upon interaction with the EGF-receptors (see for instance T.H. HAIGLER et al., (1979), The Journal of Cell Biology 81, 382-394). Exemplifying the targeting of a drug with EGF is provided by document W0-85/03357 (MD. WATERFIELD) which discloses antibodies recognizing EGF receptors which effectively inhibit the activity of these growth factor receptors in tumor cells.
Another example is provided by an article of N. SHIMIZU
et al., in FEBS letters 118 (1980), 274-278, who reports the preparation of a conjugate between EGF and diphtheria toxin.
However, as far as the present inventors are aware, no therapeutic method using a conjugate of EGF (or analogue) with an endocellular drug-release polyaminoacid back-bone carrying a cytotoxic su~stance in high mole-ratio and with a so impredicatably potent effect (as will be seen hereafter) has been reported.
The new therapeutic compounds of the present invention using EGF or analogues as a homing vector can be illustrated by the formula below:
~--~
EGF-R-(C0-~H-NH)-~..- Cv-CH-NH)~n~~
(I 2)P (I 2)o (I) - C0-A~-DM ~A~y~~M~
in which EGF designates the homing vector ~c) as previously defined.
In the above formula, p and 0 are the integers 1 or 2; A
and A' represent chain extending amino-acid links of general formula -(NH-CHY-C0)- and x and y define the number of these links. Actually x and y can be zero or an integer from 1 to about 20. It should be made clear that A and A' can define a sequence of identical or different amino-acids, i.e. in each X
~3233& ~, link Y can be the same or can be different. Y defines the rest of the amino-acids in question; for instance Y can be H
(glycine), CH3 (alanine); iso-Bu (leucine), benzyl (phenylalanine); p-hydroxy-benzyl (Tyrosine), -CH3-COOH
(aspartic acid); -(CH2)-COOH (glutamic acid), and other rests of other aminoacids. The nature and the length of the A and A' extenders allows to 4a ;F
13233Q, control the biodegradation rate of comp.und I ar.d '~enre the re'ea~o ~f the la~el ~M. In genæral the strucrllrP of the pol~mer ~ack-Done and the chain extending segmen.s is adapted for qul~-k biov~ograrion b-~ the endsce -lular enzyma.ic system. For instance a polyglutamic structure i3 readil-~deyraded by endocellular a-glutamylt.ansferase ~i~h fast release of the cytotoxic label. hhen re~uired the aminoacid st ucture of the polymer bac.~-bone and segments ~ and A' can be adap~e-~ for preferential attack by ot.~er endocellular en~ymes.
Ir. formula I .'1~ and DM' represer.t the sami or different cyt5~xic substances selected from Dauno~ycin Ac~iam~cin me ~otre-.ate ar.d any of the cytotoxic substances recited in the -ntroductior. and t.'e ~rior ar .
Ncrmaliy the cytotoxic substance is Do~nc (by m.e~ns o~ a ?r~ary a~i?e function thereof) through an amide linc to the terminal carbo~y group.
Since the e~;ten- or bir.dir.g of the cytotoxic substances i3 general'y belcw lOQ~ scme of the carboxy'ic end-group in rormula ~ are free anc p2rr of the DM or DM' groups represent ar. -0~ Jroup. A .on~enien.t wa-~ of ?ict~ring this situation wlth formula I is to cefine ~M as the c-ytotoxic substances DM' as an -OH and the ratio m/n as the deg~ee of amidation (or esteri ica-tion! of the carboxylic end-grou? 3y the cyto-oxic subst~nces. .hus the carbcxy'vte group in compound I is there for er.sur ng -~a'er so'u~ y. The ind ces m arc n rerpresent numbers 3uf.-'c.en; -o ?ro-~iide mole.1'ar ~eights !~r about 50CO ~o 100~00 ~a ar.d n/r~ is in ~ e ance 2 to 'O.
In ror~.ula I ~ is a group of fcrmula IVI) i.e -CO-X-N ~ -S ~ ~H- where ~ ~s a bridg-ng o hyZrocarbon chaln selected from alky_ene (~ith 1 tO 4 CH2!, 1, 3-cyclohe-gylene m-phenylene p-toluylene p-trime.hyler.e-phenylene e'c.
In for~ula VI the acyclic carbonyl can aiso be a thiocarbonyl.
As said before svmbols A and A' represent si?.gie aminoacid units or oligo?ep.-dês _~ntaining ~rom 2 to 70 a.~~noa- d U?i's uch o'igo?e?tides are acapred tv ?ro~tide control'ed h~drol~r'~ (en~-~ar'-) re'ea_e o~ the drug dependins on the selected target s te. mhe am-no aclds selected for m2king the A ard A bridges can be any usual am~no-aclv li~e alanine glycine leucine serine pheny;alarine arglnine et.... a"d the-y vare sele_~ed in n~mbver anc kind depencilng cn .he needs ~nà .h- a??:i-ation.
132~ .~l` i This technique of making the release of the drug controllable by linker structure has been detailed in applications EP-A-150,184; EP-A-130,935 and WO-PCT-CH86/00177.
Compound I can be prepared by coupling a 4-(N-maleimido) derivative II of EGF (or other EGF activated compound) with the thiol-copolymer III according to the following scheme (in this scheme Z designates the polyglutamic moiety of compound I) Q,_ EGF-NHC0~ H~ N-Z ---~ I
Compound II, can be prepared by the activation of EGF
with N-succinimidyl-4-(N-maleinimido)-butyrate, but other heterobifunctional bridging compounds (see below) can also be used to activate EGF and can be prepared according to the aforementioned KATO reference. Said activation consists in reacting one terminal -NH2 of the antibody on the butyrate carbonyl, thus providing the -NHCO-link in formula II.
Other agents suitable for activating EGF, thus providing compounds usable in place of II in the above scheme, are those having at one end a reactive NHS-ester functional group and a sulfhydryl reactive group at the other end; exampIes of such compounds are the m-maleimidobenzoyl-N-hydroxy-succinimide ester, m-maleimidobenzoylsulfosuccinimide ester, succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate, succinimidyl-4-carboxylate, succinimidyl-4-~p-maleimidophenyl) butyrate, sulfosuccinimidyl-4-~N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate, etc. A
good description of the use of these bridging agents can be found in the PIERCE 1985-1986 Handbook ~ General catalog, page 326 onward; other known suitable bifunctional compounds could also be used.
~r~
i.3233Q .~, Labelling the intermediate polyaminoacid backbone of the intermediate polyaminoacid-thiol (i.e. formula III in which DM and DM' are still OH) with Daunomycin, or any other cytocide, can be done also according to KATO, J. Medical Chem. (1984), 1602-7 by first protecting the thiol function by a 2-pyridyl-disulfide group and reacting the protected intermediate V in the presence of 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide hydrochloride (EDCI) according to the following scheme, then removing the protective thiol-pyridine with dithiothreitol (DTT).
ITI ~ ~ -SS~ S~ ~!iH-Z
(DM in Z is 0~ M in Z is OH) V
~ -S~ ~ ~H-Z + DM ~ IlI
~ 2. DT. (r~ = Qbuno~ycin) EGF, a single 53 aminoacids polypeptide, can be extracted from submaxillary`glands of mice (see J. SAVAGE et al., ~1972) J. Biol. Chem. 247, 7609) or it can be synthesised by genetic engineering methods, for example using a cloned gene. EGF can also be of human origin.
To introduce the chain extension segments A and A', usual techniques prevalent in polypeptide chemistry can be used. Such techniques are detailed for instance in the foregoing references EP-A-130,935 and WO/PCT-CH86/00177.
Compound I has other advantages in addition to being t internalized into malignant cells and controllably releasing therein its bound cytotoxic drug. Quite unexpectedly, its selectivity toward malignant cells over that of previously known cytotoxic drugs is considerable as will be seen hereafter. Further, the polymer construction should prevent 132~3Q ~
the polymer drug complex from penetrating into heart tissue, thus removing one of the major shortcomings of free daunomycin and other known antitumoral drugs.
Reference is now made to the accompanying drawings in which:
Fig. 1a is a photomicrograph of a culture of A-431 human squamous carcinoma cell line used to test the effect of the compound of the invention. The presence of large amounts of EGF receptors thereon is indicated by indirect immunoperoxidase staining (dark color).
Fig. lb is a photomicrograph similar to that of fig. la but of a culture of normal WI 38 embryonic fibroblast cells used as a control. The low level of EGF receptors is indicated by the quasi absence of staining 7a ~ i _ 13233G~
(light color).
Fig 2a is a photomicrograph of an A-431 cell line culture after a 4a hrs incubation with 1 yg/ml of free daunomycin and testing for dead cells with Trypan Blue exclusion dye.
Fig 2b is a photomicrograph similar to that of fig. 2a but after 48 hrs incubation Nith, instead, 1 ~g/ml of Dbunomycin in the form of compound I. The dark areas represent the killed cells.
Eig. 3a is a photomicrograph at time zero of a mixed culture of A-431 and r~I ~8 cell lines. The round cells are the tumor cells.
Eig. 3b is a photomicrograph si~ilar to that of fig. 3a showing the situation after 24hrs incubation with 1 ~g/ml of compounZ I. the round tumor ceils have greatly diminished.
~ ompound I demonstrates very useful properties in cancer therapy. A
first valuable property relates to iis strons affinity toward malignant cells over normal cells. Eor demonstrating this characteristic, a human squamous carcinoma cell line was selected, for instance A-431 cell line shown in fig. la (see M.~. WATERFIEL~ 8,), J. Cell. Biochem. 20. 14~-161), although other tumor cells can be used as well. The membrane of these cells contains a very large concentration of EGF receptors which makes them highly suitable for test wi'h compound I. The presence of these receptors is shown by indirec' im.munoperoxidase staining and appears as dark color areas of fig la. Control cell lines, for instance WI 38 embryonic fibro-blast cells with low amount of EGF receptors ~see fig. lb), Nere treated identically for comparison and appear as lighteer color areas on the photo-micrograph.
- Thus, when a known amount of compound I (p = o = 2; x = y = O, DM =
daunomycin; DM' = OH; n/m = 6) radio-labelled with I125 ~iodine is part of the EGF moiety) was incubated with A-431 cells using WI 38 cells 2S con-trol, 31% of total radioactive iodine was retained in the intra-cellular compartment and 2% in the membrane of the tumor cells, ~hile in the cGntrol cells, the corresponding values were 2% and 0~, respectively. Using free EGF in place of I in these experiments gave the corresponding 14%/0% and 7%/0% values instead. The reason of the greater accumulation of compound I
as compared to free EGF is possibly due to retention of EGF by the polymer backbone of compound I by endocytic vesicles, whereas EGF is recycled freely back to the medium.
A second valuable and unexpected ?roperty of compound I relates to its enhanced toxicity as compared with free daunomycln toward malignant cells.
Thi~ is illustr3ted by tigs 2a and 2b.
In flg ~a, d cui~ure of A-431 cells is shoNn after 4~ hr_ incubation with a med~u.~ contal`nlng `~ ug/ml or free daunomycin. ~/iability ~as scored by ~eans of the Trypan ~lue exclusion dye ~ethod. E~clusion of the dye from the cells demonstrates the viability thereof. In contrast, when an equiva-lent amount of daunonycin in the form of compound I was used in a s~ilar experinent ~see fig. ~b), a very large number of ce'13 were :~-lled as shown by the shrivelied up cells and the dar~ areas where the dye 'na~ accumula-ted.
The seiectivity of compound I to~ard tumor celis was further tested.
For instance, fig. 3a and 3b show the effect of compound I in the case of a mi~ed culture of A-431 andd WI 38 cells. The round celis repre~ent the A-431 tumor celis ,~nd the elonsated cells are the WI 3~ control cells. The culture medium was Dhot3sraphed at time -ero t'ig. 3a). Eig 3~ illustrates this situa-ion after ~4hrs in a medium conta-'nir.g i ~g/.~.l or compound I. It can be seen tha~ t~e round darker malignant cells have stron~ly re~ressed.
In conclusion, it has been demonstrated that at equiv~ient molar concentrations (of daunomycin) com?ound I rAas much more lethal to squamous carcinoma ce'ls than free daunomycin itself. Also compound I has a selec-~ive mortal ac~ivity on tuncr cells ~ut leaves normal cells allve, ~hereas, under the same cond '._ons, free daunomycir will kil' norma' cells.
Further advan-ages of the compour.ds of the inver.t1cn ror treatlr.g-tu~or diseases relate to the follo~inc characteristlcs:
Molecular size: The compounds or tne invention are smaller than most usual targetted drugs using monoclonal antibodies, wherefrQm improved pene-tration into tumor bodies is assured ~ith consequent better cytoto~ic efficiency. Yet, the compounds Ot the invention are bigger than the free cytotoxlc drugs ~hich ?revents penerr3tion lnto nornal cells. Ir contrast, malignant cells being susceptible to endocytocls they will allow penetra-tion of compounds of moderate size like that of the invention.
Effect of EGF and related factors: EGF has an an~iogenic effect and develops mali~nant cell vascularization wi.~ consequent cell mitosis and increased suscepti~ility to antimitotic drugs. In otner words, t~e com-?ounds of the irventicn tenporarily incroase the ~umor cel' metabolism ar.d render it more sensitive to d.ug killing actior.
The following Exam?les lllustrate the invention in more detail.
1~2~30' EXPERIMENTAL SECTION
Example l Synthesis of compound I, a conjugate of EGF and daunomycin-grafted polyglutamic acid.
A polymeric compound ~V) with a poly~lutamate ba-.'ibone structure (p o = 2; 3 = y = O, in Nhich some ~ -carboxylic groups are condensed with daunomycin and having a 2-pyridyl-dithio-ethylamido heaZing group was pre-pared according to Y. KATO et al., (lg84~, J. Med. Chem. 27, 1602-1607 (compound 5 in scheme II of KATO).
The identity of the compound was chec.~ed analytically:
Mw = 29000 Da by quantitatively determining the 2-pyridyldithio group;
ratio; ratio of daunomycin to carboxylate = 1i6 as determined by spectrome-trlc quantitation of daunomycin at 480 nm.
To twenty five mg of the polymer dissolved in 2 ml of lOmM sodium phosphate buffer at pH 7 were added O,l ml of a 0,3 M solution of dithio-threitol (~TT). After l hr at 40C the solution was dialyzed overnight against a 0,1 M sodium phosphate buffer at pH o,O (SPECTRAPOR membrane, MW
cutoff 3500); this regenerated 'he thioethylamido group of the molecule (compound 6 in scheme II of ~ATO); yield ~3 mg of a red compound after free2e-drying (poly-(DM)-Glu-SH). The poly-(~M)-Glu-SH polymer was reacted with an excess of thiopropyl-SEPHAROSE~in the pyridylsulfide form (4C; 12 hrs; phosphate buffer, pH~) and the gel was rinsed with an excess of the same uffer in order to eliminate the polymer lackin the SH extremity. The polymer was conserved ur.der this form in the cold. It was then regenerated by treating the gel with an excess of mercaptoethanol (12 hrs), dialysed against water (overnight, 4C) and freeze-dried before reacting with EGF.
One hundred ~g of EGF (SIGMA) ~as dissolved in 500 ~1 of lO mM phos-phate buffer, pH 7,0 containing 0,14 M NaCl. Then a quantity of l25I-EOE
sufficient to provide an activity of lO,OOO cpm/~g EGF was added followed by 50 ~1 of a 32 ~ solution of N-succinimidyl-4-~-maleimido)-butyrate (SMBU) (origin: Sigma), in dimethylformamide. The mixture ~as allo~ed to stand for l hr at 25~C, then it was dialyzed aga~nst a O,l M sodium phos-phate buffer-O,l M NaCl,, pH 6,0, to eliminate the excess of SMBU.
The desired product (II) resulting from t~e cor.densation of S~BU and EGF was not isolated ~u to tne 500 al of the d aly-ed ~GF solution ~ere 132330~
added 1 mg ot poly-(DM)-Glu-SH and the mixture ~as slowly agitated over-night at 4C. ~ive ml of thiopropylsepharose (pyridylsulfide form) were then added and the reaction was continued at 4C for 12 hrs. The gel was washed with successively 3 portions of l ml of sodium phosphate buffer, the eluent was concentrated under reduced pressure and subjected to gel filtra-tion on SEPHADEX G75~ column 40 x 0,8 cm), using 0,l M ammonium carbonate solution pH 7,0. The fraction containing the EGF-poly-(~M)-Glu conjugate ;I), absorption 490 nm, was collected and freeze dried. Yield: 80 ~g of solid.
Example 2 Selection of A-431 test cells lines from evidence of receptor concentration on the cell membrane by indirect immunoperoxydase stain-ing Indirect immunoperoxydase staining on cell lines were performed on trypsinised cells in 35 mm P~C plates. The surface was pre-treated with phosphate buffered saline (PBS) ph 7,~, the excess was then removed and the PBS washed cells (105/well in 50 ~l ?BS) were added to the plated and centrifuged for 5 minutes at `2000 rpm. 50 ~l/well of 0,5% glutaraidehyde in cold PBS were then added to the dish and incubated for 15 minutes at room temperature. After two round of washes with PBS, the wells were filled with 100 mM glycine in a 0,1% BSA solution and allowed to rest for 30 minutes at room temperature to block gutaraldehyde activity. After two PBS washes, indirect immunoperoxydase was done by first denaturing the cells with an ice cold mixture of 9~:l ethanol-acetic acid for 30 minutes at 4C. During this period, 3 ~1 of antibody were diluted in 1 ml PBS + 1% foetal calf serum (FCS). The wells were then washed twice with PBS and incubated for 5 minutes with a solution of 20~o FCS in PBS. This was then replaced by 200 ~l/well of the antibody solution and incubated for 30 minutes at room temperature. The wells were then washed twice with PBS, once with PBS +
Tween 0~l~o and once again with PBS. 200 ul/well of a 11400 dilution of swine anti-mouse perodydase ~POD) conjugated ant.body (DAK0) in PBS l~ FCS
was then washed twice with PBS, once with PBS + 0,1% tween and twice with dlstilled water. The in situ coloring was achieved by incubating the cells at room temperature with a solution of lO ml 0,Ol M phosphate buffer pH
6,0, 5 ~l 35~O oxygenated wa~er and lQu ~l of l~, ortho-àianisidi.. (MERCC) in methanol. 132330~
Example 3 Internalisation of compound I in A-431 squamous carcinoma cells compared to in HI-38 fibroblasts The entry of compound I into the cells is essential for .t3 action.
Therefore, internalisation Ot the molecule was demonstrated by showing its presence in the membrane and inside the cell compartment. This has been done using A-431 and WI-38 cells. t Compound I, radiolabeiled with 125I on the EGF, was incubated with confluent cell cultures for 6 hrs at 37C in solution A (4 parts ~MEM and 1 part of 50 mM Tris, 1~0 ~M NaCl and 0,1% BSA adjusted a. ph 7,4). The cells were then washed ~our times ~ith ice-cold PB~T (lmM Cd Cl2and 1 .~M
MgC12). Fifty % trichloroacetic acid ~as added in a proportion of 1!5 to the pooled solution a and PBS+ and counted on a y counter. The cell mem-brane was destabilized by a treatment on ice with 200 mM acetic acid and 150 mM NaC1 (solution B) for 6 minutes. The solution B was then removed and the cells wa~hed tNice with solutlon B. These pooled solutions were assayed for 125I. This treatment releases the EGF rece?tors bound to the cell surface. The cells were then complete1y dissolved in 0,7 N NaOH. The radioactivity found then represented the internali,~ed compound I. Ln con-trol experiments, using free EGF 125I, a proportlon of the EGF is recycled to the medium therefore lowering the intracellular EGF. The results on the internalization of 125I labelled EGF and compound I in ~1-38 and ~-431 cell cultures are given in the following Table in terms of counts per min (background 30 cpm) for successively: EGF not integrated in the cell, EGF
bound to the membrane, and EGF in the intra-cellular compartment. The percent of total cpm is given in brackets.
132330 ~
TABLE
EGF (%) Compound I (%) WI-38 530 (93) 1169 (98) " 29 (O) 28 (O) " 67 (7) 52 (21 A-431 536 (86) 831 (67) " 31 (O) 57 (2) " 111 (14) 390 (31) These results show that compound I is more efficiently internalized in malignant cells (31%) than in normal cells (2%~.
Example 4 Comparative cytotoxic effects of free daunomycin and compound (I) on a A-431 squamous carcinoma cells All cells were maintained in Dubelcco's Modified Medium (DMEM), 10%
foetal calf serum (FSC) (Gibco), 2% penicillin-streptomycin (Gibco) and 1%
fungizon (Gibco~ in 5% C02. They were plated at 50 to 60% confluency 24 hours before the addition of the toxin. 1 ~g/ml of daunomycin, or equiva-lent in compound I, was added in DMEM 10% FCS and the ceil death rate visualized by trypan blue exclusion. 4 volumes of 0,2% (w/v) Trypan Blue in water was freshly mixed with 1 volume of a saline solution 4,25% (w~v) of NaCl in water. 1 volume of this solution was mixed with 1 volume of PBS on the cell monolayer or to 1 volume of cell suspension in PBS. Observation and scoring took place 48 hours after addition of the toxin (see fig. 2a and Zb). These results show that daunomycin is much more effective against malignant cells when in the form of compound I than when in the free state.
14 13233~
Example 5 Effect of compound I on mixed cultures of tumor (A-431) and normal (~I-38) cells.
Cells were maintained as described in e~ample 4. The ~I-38 fibroblasts were first plated and the A-431 squamous carcinoma cells were plated the next day. The mixed culture was left growing for 24 hours and then 1 ,ug/ml of compound I was added to DMEM 10% FCS. Nearly all the A-431 cells were selectively killed after 24 to 48 hours whereas the ~I-38 fibroblast were left allve. Hence, the mixed culture became free from the tumor cells demonstrating that compound I can be used to separate the normal cells from cancer cells. The experiment was repeated but using only 0,1 ~g/ml of compound I (for the controls, equivalents of daunomycin were used in free form). Observation of the cultures after 15 days showed that the test samples contained no more of A-431 cells, which situation was confirmed by continuing culturing for 6 weeks under normal conditions, this resulting in no reformation of tumor cells. In contrast, in the controls all cells, malignant and normal, had died after 15 days.
Example 6 A compound of general formula V (see description) was prepared accor-ding to KATO et al., G. Med. Chem. 27 (1984), 160Z-1607 (compound defined as 4 in scheme I of KATO). This compound has the formula VA below in this paticular embodiment.
SS ~ NH~(CO~CH~NH)q~~H VA
( C;~2 ) 2-COoH
in which q may be fro~ about 150 to 250 depending on the polymeri7ation conditions.
This compound was subjected to a procedure reported by HESS-~IJK e~
al., J. of Controlled 2elease 1 (4) (1985), 312 for e~tending the side chain with an H-Gly-Gly-Leu-OH peptide (segment A in formula I), as fol-lows: 40 mg of VA and 68 mg of saccharin (0.31 mmole) were dissolved in 1 ml of DMF and the solution was allowed to rest far a few hours (solution A).
On the other hand, 0.32 mmole (40 ~1) of N,.~,N'~'-tetr~methylganidine (TMG) ware slowly added to a stirred suspension of 0.3Z mmole (8C mg) of H-Gly-~ly-Leu-OH in DWF Stirring was continued until all sollds had dissolved ~solution B).
Then 0,43 mmole (70,3 mg) of ~,N'-carbonyl-diimidazole were added to soiution ~ and, after stlrring for 30 min, solut~on 3 waa added. The mi~ture was further stirred for 3 days at room-tempera~ure. The mi~.ure was added into 15 ml of 0.1 M phospha~e buffer (pH 7.0) ar.d the resulting solution was dialyzed into water (17 hrs) filtered on a millipore membrane (0.45 ~m) and the filtrate was freeze-dried. The polymer ~yield 80% mg) was analy~ed by hydroly~ing an aliquot in ~ N HCl for lZ nrs at 180C. Deter i-nation of the aminoacids in the hydrolyzate was carr_ed out ~y High Perfor-mance ~iquid Chromatosraphy of the amlnoacld-~Atalaldeh~fde derivatives (detection by fluorescence). The followlng ratlo of glutam~c~lycine/'eucine was measured: 0.95/2/i. These results indicate that the grafting level was aroung 95%r i.e. the compound can be represented conventionally by the formula:
PYR-SS ~ ~lH ~ CO-jH-NH)- 05........... -(C0-jn-NH) ~5 ~ . H
(CH2)2 COO C~.2(CH2CONH)3-CH(iso'Bu)COO
as the tetramethylguanidlne salt. ('~B) The pyrldine-S group was removed ~ith dithlothreitol according to KATO
et 21., and analysis Nas performed by measuring the absorbance at 343 nm of the ll~erated pyridine-2-thione. Neqlecting the presence of the TMG+ ion, a molecular weight of 25.500 was found meaning that in the product t (the degree of polymerizat on) i3 about 75-90.
Labellinq compound ~B with Daunomycin waa ac_om?liahed ~s follows:
33.7 mg (74.7 ~mole) of '~B ~ere disolved in 15 ml of ~ a(iueous NaCl and 20 mg (35,5 ~mole) of 2aunomycin hydrochloride were added. The pH ~as brought to 5.5 Nith 0.1 N ~aOH arter which ~8 mg ~0.1 .~r~cle! of 1-ethyl-3-C3-(dimethylamino)-carbodiimide hydrochlor~de ~EDC) ~ere added under s;irring.
16 1323"\J~
After agitation for 18 hrs, the mixture was dilute~ with 15 ml of 1 M NaCl and dlaly7ed in ~ater~ The residue was free7e-dried wh ch provided 31.5 mg of polymer. Splitting t;r~e dlsulfide with DTT as before and analy7ing spec-troscopically the pyridine-thione indicated the prosence of about 10 DM per molecule, i.e. a ratio of labelled side-chains to unlabelled side-chains of about 1:7.
Conversion of an aliquot o~ the above disulfide to the deslred cor-res?Gndi.rig thiol I -~B with DTT was done as Ec~ ows: 2 mg of pol-~me- were dissolved in 2 ml or 0.1 ~ phosphat- buEtar (?H 7.0) and 150 ul of 0.3 M
DTT were added. After allowins tG stand for one hr at 42, the solution was dialy~ed for 4 hrs agaln~t freshly degassed phosphate buffe. ~pu 6.0); A
SPECTRAPOR bag was used (MN cut-off = lOOC).
Simultaneously, EGE was ai-t~ated by tak ng 0,2 ~g of _GE (Sigma) and dissolving in 3.?5 ~1 oE 10 .~M ?hosphate bu~ter in 0.14 M NaCl, pH 7.0;
the-, adding 0.5 ml of 2 10 ~g/ml S~BU solur on in DI~F. After 2 hrs at '0C, the mi~ture was dialy7i?d at d~C ~gainst a 0. ~ Phosphate ~uffer contalning 0.1 M NaCl at pH 5.0 'membrane Mw cut-oEf = 1000).
The overall wlume of both the dlalyzed polymer and dialy~ed EGF
solution Nere reduced to 1 ml by absorption with C`~C powder, then they were mixed together and allowed to stand for Z4 hrs n one dialysis Jas. The m :~ture was c;lrcmatGsraphed on S~P~ADE~ G75 ~eluent O.lM N~o~ and the fract ons absorbing a~ 4~0 nm were collected and cleaned from unreacted ?olymer by treating with thiopropy sepharose overnight at 4C. The gel -~as washed with O.lM NH4Co3 and 22.5 ml (O.D. of 0.176) of solution was collected. Yield about 80% of compound IA. The respective weight contri-butions of DM and EGF in the product are about equal.
If, in the above pre~aration, Daunomicin is replaced by Adriamycin, a similar product, incorporatins Adriamycin is obta ned.
The presence and bir.dlng efficiency of the EGF factor in .ompour.d IA
was checked by immunoprecipitation with EGF antibody andd attachment of the immunocomple~ to a protein A - Sepnarose gel (C~4B, Pharmacia). The proce-dure was as follows:
Protein A - Sepharose gel was rahydrated to ~rovide a 50~, (VtV) solution in NET-NP40 buf-er (100 .~M NaCl, 1 .~ A, 10 ml`~ Tri,, pY 7.5, 0,5% NP40). Bovine serum albumin (BSA) was added to 20 ~1 or the buf ered sepharose solution to provide a 0,3% (by weight~ ~SA solution ~S!. On the o~her har.d, BSA was added to 12 ul of a sc uticn of antiserum aga-nst EGF
(Collaborative ~esearch) so as to ?rc~; d- ~ 0,_~ by weiqn. BSA so u _on in 17 13233G, antiserum (Ab). ~oth (S) and (Ab) were incubated overnight at 4~C, then a quantity of compound IA corresponding to 120 ~g of daunomycin was added to sample Ab and incubated for 7 hrs at ~C under agitation. Then solution (S) was added and the mixture was agitated per 12 hrs at 4C. A control (C) was prepared by adding the same quantity of compound IA to another identical sample of solution (S). Both the above mixture (M) and the control were centrifugated for 5 min at 2000/rpm and the optical density (OD) of the supernatant liquid measured at 480 ~m. The results are 0.41 for M and 0.955 for C which shows that compound IA c~rries EGF, the conformation of which is recognized by the ~ntibody on the gel.
Example 7 The toxicity oE compound IA and of free daunomycin toward A431 malig-nant and W~3~ (control) cells was compared. The effect of these drugs was evaluated by the degree of inhibition of cellular protein synthesis. For this, we measured the level of incorporation of 35S methionine (35S-met NEN) into newly synthesized proteins after a 48 hrs exposure to different concentrations of the drugs.
Cells were plated on 1,5 cm Petri dishes at 50% to 60% of confluency before the addition of the toxin. 0,1, 0,5 or 1 ~g/ml doses of daunomycin (controls) or its equivalents in the form of compound IA were added in DMEM
10% FC~. Cell death consecutive to this addition was measured as follows:
The cells were exposed for 1 hour at 37C in 500 ~1 DMEM low methionine medium (Gibco) containing radioactive 35S met. The medium was then removed and the dishes were washed with PBS (137 mM NaC1, 2,7 mM KU , 8,1 mM
Na2HpC4, 1,5 mM KH2P04, 0,9 mM CaC12, 0,5 ~M MgC12 pH 7.2). These solutions were stored for ~ counting. The cells were lyzed with 1 ml 0,1 N NaOH and placed in a tube Nith 500 ~1 Trichloroacetic acid (TCA) 10% to precipitate the proteins. This mixture was filtered on GF/A filters (Whatman) to eliminate unreacted 35S-met. The filters were washed twice with 1 ml of 10%
TCA and once with 100% Ethanol. The filters were dried at 80% for two hours and sub~ected to ~ counting in Econofluor (NE~) sc-ntillation medium. The results reported in the following table show that compound IA is very cytospecific but less cytotoxic than daunomycin alone.
18 ~233aj drug concentration % Oe Protein synthesis inh~bition (~g/ml A431 WI38 0,1 30% 10%
FREE DAUNOMYCIN O,5 69% 46%
1,0 97% 80%
=========================================================
0,1 4% 0%
CONPOUND IA O,5 17% 6%
1,0 47% 4%
Example 8 IN VIVO TEST OF COMPOUND I
We induced A431 xenografts in nude mice and when the tumors reached 3 to 5 mm, we in~ected into two sets of nude mice 0,1 mg/kg of daunomycin either in the form of compound I (see example 1) or in free form. In the prior art, 10 mg/kg doses have been used in mice (Schwarz S. et al., 1975, Cancer Chem. Rep., 6, no 2, 107-114). The drug was injected either directly in the tumor or in the caudal vein 4 times at 3 days intervals. The measu-res were taken a week after the last injection.
By visual inspection of the animals, it was noted that the growth of the tumors treated with compound I was significantly reduced, as compared to control mice or to treatments with free daunomycin. These results were similar whether the injection of the drug was directly in the tumor or in the caudal vein which shows that biodegradation and release of the cytotoxic substance occurs substantiallly only in the target cells. Results are shown in the follo~ing Table (I.V. = intra-veinous; I.T. = intra-tumor) 19 1 3 2 3 c~ Q
injection initial surface final surface growth of the tumor ~A) of the tumor (8) coefficient (B/A
Daunomycin I.V. 30.5 500 16 Compound I I.~. 25 54 2 Daunomycin I.T. 9 170 18.7 Compound I I.T. 9 50 5.6 Control* 9 320 35.6 * Placebo injection At the end of the experiment, the tumors were dissected and weighed.
Results are given below. If corrections for slight differences of tumor si~e at the beginning of the treatment are taken into account, it can be seen that the results correlate well with the surface estimate glven in the previous Table.
~aunomycin I.V. : 1.178 g.
Compound I I.V. : 0.359 g.
Daunomycin I.T. : 6.789 g.
Compound I I.T. : 0.816 g.
in conclusion, the compounds of the invention show better performances in selectively killing squamous carcinoma cells than daunomycin, both in vitro and in vivo. In the animal tests, we noted that very low amounts of com-pound I have a remarkable effect on tumor growth.
E~ample g The in-vivo tests reported in Example 8 were repeated using compound IA instead of compound I. Thus, 2 mg/kg of daunomycin, free or in the form of compound IA were injected every 3 days over 9 days in the caudal vein of nude .mice bearing A431 tumors. The tumor growth inhibitlon by compound IA
132330.~
was significantly greater than by free daunomy.cin (D), free EGF or DM
labelled but untargetted polymer (DMA). These results are shown in the Table below where the values correspond to tumor diameter (in mm) measured with a scalliper after a number of days. The lethal dose of compound I and compound IA have not been measured but are presumably less toxic than free daunomycin which enters freely into most normal cells.
T A B L E
Dayg 0 4 7 11 14 Control 10 14 17 2Z 24 Free EGF 10 13 14 17 19 DMA 10 11 12 16 i7 IA 10 11 12 14 i5
FOR ITS IMPLEMENTATION
r~
R~U~ N
The present invention deals with anti-cancer therap~ and, more parti-cularly, concerns a new metnod of combat.ing ~alignar.t di_eases by adminis-tering a medicamen~ ~hich ~will preferentially ~ill cancer cell and have oniy a limited, or insignificant detrimental effect on ;~ealthy cells.
The invention also concerns new selective cytotoxic compounds for impiementing the method, i.e. conjugates consisting of three main covalen-tly bound components: a seiective tumor targetting vector, a biodegradable poly~er backbone acting as an endocellular drug release carrier and leavir.g no toxic residue after resorption and a cytotoxic drug labei especially designed to inhibit cellular growth or to kill malignant cells.
The invention also concerns a method for manufacturlng the nelA medica-ment compounds.
T~E PRIOR ~T
It is ~ell ~nown ~hat current cancer therapy involves the use or ant;mitotic drugs such as adriamycin, vinchristine, bombesin, daunomycin and metothrexate which all have strong undesirable side-effects on the normal cells of the patient. It is therefore important that the activity of antitumor drugs be specifically directed to the malignant cells and have little toxic effect on the normal cells.
Many targeted cytotoxic agents have already been reported based on anti~odies recognizing speciric cell surface antigens coupled to radio-active isotopes with cytotoxic properties, or to toxins such as auromycin, hematoporphyrin, abrin, ricin, diphteria toxin, pseudomonas, exotoxin, gelonin or to the above-mentioned antimitotic drugs. For reviews see P.E.
THORPE et al., (1985), Monoclonal Antibodies 84; Biological & Clinical Applications; Ed. A. RIMCHER~ e; al., Complexes o~ antibodies and antiviral agents, such as interferon have also been tried ~New Scientist ref. April 17, lg86). Further US-A-4,455,985 discloses the binding of Pseudomonas exotoxin to antibodies, for instance antibodies to s?ecific human cell receptors such as the transferrin receptor.
1 3233~
Slowing the release of anticancer drugs by firs, link ng to d sui'able polymer carrier which will then graduall/ rree th* drug and kee? its con-centration effect ve for a longer time at ~.he ~ar~et site const-~ates an improved approach to the problem. For instance W.A.R. VAN HEESE'~lJK et al., (19~5) J. of Control Release 1, 310-15 discloses the binding of anthracy-clin to a polymer which slowly releases the drug and thus maintains a constant concentration thereof in the blood of treated patlents. In this connection, endocellular drug-releasing systems are particularly interes-ting, for instance antimitotic drugs coupled to the side-,-hain of poly-glutamic acid. Indeed, these side-chains are slowly degraded in the presen-ce of y -glut~myl-transferase, an endocellular enzyme ~hich is particularly abundent in tumor cells as compared to normal cells and, consequently, releasing the anti-mitotic drug within the targeted cells. Thus Y. KATO et al., (1984) in J. Medicinal Che. 27. 1602-7, reports bir.ding Daunomycin (~M) to polyglutamic acid (PLGA) and coupling the resulting c~/totoxl-polymer with rat ~ -fetoprotein ( ~-~FP) antibody. Upon trial, the cytotoxic activity of the resulting ~ -AFP-PLGA-DM conjugate was shown to be more effective than nIg (a control antibody), ~ -A~P, unconjugated DM, PLGA-DM or nIg-PLGA-DM.
EP-~ ,720 (TEIJIN) discloses a conjugate comprising an i.~moglobulin capable of binding selectively to a particular antigen possessed by a cell to be killed, a polymer carrier and a cytotoxic substance lin~ed thereto, for instance p-(N,N-bis(2-chloroethyl))-phenylenediamine, Melphalan, 1-( ~-D-arabino-furanosyl)-cytosine and its phosphate, methotrexate, actinomycin D, mitomycin C and the li~e.
US-A-4,485,093 discloses an immunotoxin conjugate for treating mali-gnant deseases, which consists of arsanilic acid and tumor specific anti-odies covalently bound to a poiyslutamic acid bac~-bone.
In the aforesaid approach~ selection of an effective antigen or rece-ptor to target the drug is of prime importance. However, even more impor-tant is the possibility of causing the medicament to penetrate into tne target cell, i.e. to be internalized therein at suitables sites so that, by fast degradation of the back-bone drug carrier, the killing or inhibiting effect of the cytocoxic substance be e~phasized or amplif ed.
Thus, the therapy achieved by the method of claim 1 is an important step toward this objective.
132330, According to one aspect of the invention there is pro~ided the use in the treatment of malignant diseases a covalent conjugate medicament consisting essentially of the following components bound covalently together, a) a cytotoxic substance, b) a poly~.er carrier having an endocytic biodegradable, nontoxic, polyaminoacid backbone, c) a cell homing vector with the properties of first selectively targetting the conjugate toward malignant cells to be fought and second, providing for the internalization of the conjugate into said cells, wherein fast biodegradation of the carrier will occur with consecutive release of the cytotoxic drug which will selectively combat said malignant cells.
According to another aspect of the invention there is provided a cytotoxic targetted conjugate for the application of the method of Claim 1, this conjugate having the formula Z
- ~G~ Cv -F- ~ - H-~
(lc~;2~p ~rly ~0 ~_ CO-.~. ~-DIY CO-.~. ' y~`_~!' in which p and o are 1 or 2;A and A' represent chain extending amino-acid intermediate links of formula -~NH CHY-C0) and x and y, which define the number of these links per molecule, can be zero or any integer from 1 to 20; y is an amino-acid rest; DM and DM' represent one or more cytotoxic substances covalently bound to the aminoacid carboxyl group through an _,.
1 3 2 3 3 0 ~
amide or ester lin~ but DM can also represent an OH of a free carboxyl; EGF defines a homing vector for promoting malignant cell recognition and internalization therein; R is a group of formula.
-C~ a- ( G~ iH wi.h ~ ~einq ~2- ~r~ e~
~rom -(C~ with r .r ~ d; or ~ ; o~ ~ ; ~
In some embodiments, the factor promoting cell internalization of the homing vector (c) also acts as the "arrow-head", i.e. it recognizes and binds to the cells at suitable receptors thereof. Examples of such vectors are the epidermal growth factor tEGF) or other EGF analogues of growth factors such as urogastrone, -TGF, or synthetic and natural derivatives thereof, transferin, low density lipoprotein (LDL),Nerve Growth Factor (NGF), Platelet Derived Growth Factor (PDGF), and some viral receptors, all providing cell internalization.
~O Otherwise, in some other embodiments, the homing vector can comprise separate binding and internalizing factors; for instance, the binding element can be non-internalizing antibody (such as FAB) to a specific antigen of the cells and the internalizing element can be any known internalizing factor specific or unspecific.
The use of EGF factors to specifically direct medicaments toward EGF receptors on malignant cells is not unobvious per se since several investigations have demonstrated the -3a-. ~ ~
1323'~'~
presence of these receptors in high concentration on many squamous cell carninomas as well as sarcomas. Such results include breast, lung, brain and skin tumors (see the following references; J. Hunts et al., (1985) JPn. J. Cancer Res. 76, 663-66; B. GUSTERSON et al., (1985), The Lancet, 364-68; F.J. Hendler et al., (1984), Cancer Res. 443, 753-60;
T. BAUKNECHT et al., (1985), Dermatologica 171, 16-20.
It is known also that these receptors are particularly efficient targets for EGF-linked cytotoxic drugs because of internalization into the -3b-13233~ J
cells upon interaction with the EGF-receptors (see for instance T.H. HAIGLER et al., (1979), The Journal of Cell Biology 81, 382-394). Exemplifying the targeting of a drug with EGF is provided by document W0-85/03357 (MD. WATERFIELD) which discloses antibodies recognizing EGF receptors which effectively inhibit the activity of these growth factor receptors in tumor cells.
Another example is provided by an article of N. SHIMIZU
et al., in FEBS letters 118 (1980), 274-278, who reports the preparation of a conjugate between EGF and diphtheria toxin.
However, as far as the present inventors are aware, no therapeutic method using a conjugate of EGF (or analogue) with an endocellular drug-release polyaminoacid back-bone carrying a cytotoxic su~stance in high mole-ratio and with a so impredicatably potent effect (as will be seen hereafter) has been reported.
The new therapeutic compounds of the present invention using EGF or analogues as a homing vector can be illustrated by the formula below:
~--~
EGF-R-(C0-~H-NH)-~..- Cv-CH-NH)~n~~
(I 2)P (I 2)o (I) - C0-A~-DM ~A~y~~M~
in which EGF designates the homing vector ~c) as previously defined.
In the above formula, p and 0 are the integers 1 or 2; A
and A' represent chain extending amino-acid links of general formula -(NH-CHY-C0)- and x and y define the number of these links. Actually x and y can be zero or an integer from 1 to about 20. It should be made clear that A and A' can define a sequence of identical or different amino-acids, i.e. in each X
~3233& ~, link Y can be the same or can be different. Y defines the rest of the amino-acids in question; for instance Y can be H
(glycine), CH3 (alanine); iso-Bu (leucine), benzyl (phenylalanine); p-hydroxy-benzyl (Tyrosine), -CH3-COOH
(aspartic acid); -(CH2)-COOH (glutamic acid), and other rests of other aminoacids. The nature and the length of the A and A' extenders allows to 4a ;F
13233Q, control the biodegradation rate of comp.und I ar.d '~enre the re'ea~o ~f the la~el ~M. In genæral the strucrllrP of the pol~mer ~ack-Done and the chain extending segmen.s is adapted for qul~-k biov~ograrion b-~ the endsce -lular enzyma.ic system. For instance a polyglutamic structure i3 readil-~deyraded by endocellular a-glutamylt.ansferase ~i~h fast release of the cytotoxic label. hhen re~uired the aminoacid st ucture of the polymer bac.~-bone and segments ~ and A' can be adap~e-~ for preferential attack by ot.~er endocellular en~ymes.
Ir. formula I .'1~ and DM' represer.t the sami or different cyt5~xic substances selected from Dauno~ycin Ac~iam~cin me ~otre-.ate ar.d any of the cytotoxic substances recited in the -ntroductior. and t.'e ~rior ar .
Ncrmaliy the cytotoxic substance is Do~nc (by m.e~ns o~ a ?r~ary a~i?e function thereof) through an amide linc to the terminal carbo~y group.
Since the e~;ten- or bir.dir.g of the cytotoxic substances i3 general'y belcw lOQ~ scme of the carboxy'ic end-group in rormula ~ are free anc p2rr of the DM or DM' groups represent ar. -0~ Jroup. A .on~enien.t wa-~ of ?ict~ring this situation wlth formula I is to cefine ~M as the c-ytotoxic substances DM' as an -OH and the ratio m/n as the deg~ee of amidation (or esteri ica-tion! of the carboxylic end-grou? 3y the cyto-oxic subst~nces. .hus the carbcxy'vte group in compound I is there for er.sur ng -~a'er so'u~ y. The ind ces m arc n rerpresent numbers 3uf.-'c.en; -o ?ro-~iide mole.1'ar ~eights !~r about 50CO ~o 100~00 ~a ar.d n/r~ is in ~ e ance 2 to 'O.
In ror~.ula I ~ is a group of fcrmula IVI) i.e -CO-X-N ~ -S ~ ~H- where ~ ~s a bridg-ng o hyZrocarbon chaln selected from alky_ene (~ith 1 tO 4 CH2!, 1, 3-cyclohe-gylene m-phenylene p-toluylene p-trime.hyler.e-phenylene e'c.
In for~ula VI the acyclic carbonyl can aiso be a thiocarbonyl.
As said before svmbols A and A' represent si?.gie aminoacid units or oligo?ep.-dês _~ntaining ~rom 2 to 70 a.~~noa- d U?i's uch o'igo?e?tides are acapred tv ?ro~tide control'ed h~drol~r'~ (en~-~ar'-) re'ea_e o~ the drug dependins on the selected target s te. mhe am-no aclds selected for m2king the A ard A bridges can be any usual am~no-aclv li~e alanine glycine leucine serine pheny;alarine arglnine et.... a"d the-y vare sele_~ed in n~mbver anc kind depencilng cn .he needs ~nà .h- a??:i-ation.
132~ .~l` i This technique of making the release of the drug controllable by linker structure has been detailed in applications EP-A-150,184; EP-A-130,935 and WO-PCT-CH86/00177.
Compound I can be prepared by coupling a 4-(N-maleimido) derivative II of EGF (or other EGF activated compound) with the thiol-copolymer III according to the following scheme (in this scheme Z designates the polyglutamic moiety of compound I) Q,_ EGF-NHC0~ H~ N-Z ---~ I
Compound II, can be prepared by the activation of EGF
with N-succinimidyl-4-(N-maleinimido)-butyrate, but other heterobifunctional bridging compounds (see below) can also be used to activate EGF and can be prepared according to the aforementioned KATO reference. Said activation consists in reacting one terminal -NH2 of the antibody on the butyrate carbonyl, thus providing the -NHCO-link in formula II.
Other agents suitable for activating EGF, thus providing compounds usable in place of II in the above scheme, are those having at one end a reactive NHS-ester functional group and a sulfhydryl reactive group at the other end; exampIes of such compounds are the m-maleimidobenzoyl-N-hydroxy-succinimide ester, m-maleimidobenzoylsulfosuccinimide ester, succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate, succinimidyl-4-carboxylate, succinimidyl-4-~p-maleimidophenyl) butyrate, sulfosuccinimidyl-4-~N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate, etc. A
good description of the use of these bridging agents can be found in the PIERCE 1985-1986 Handbook ~ General catalog, page 326 onward; other known suitable bifunctional compounds could also be used.
~r~
i.3233Q .~, Labelling the intermediate polyaminoacid backbone of the intermediate polyaminoacid-thiol (i.e. formula III in which DM and DM' are still OH) with Daunomycin, or any other cytocide, can be done also according to KATO, J. Medical Chem. (1984), 1602-7 by first protecting the thiol function by a 2-pyridyl-disulfide group and reacting the protected intermediate V in the presence of 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide hydrochloride (EDCI) according to the following scheme, then removing the protective thiol-pyridine with dithiothreitol (DTT).
ITI ~ ~ -SS~ S~ ~!iH-Z
(DM in Z is 0~ M in Z is OH) V
~ -S~ ~ ~H-Z + DM ~ IlI
~ 2. DT. (r~ = Qbuno~ycin) EGF, a single 53 aminoacids polypeptide, can be extracted from submaxillary`glands of mice (see J. SAVAGE et al., ~1972) J. Biol. Chem. 247, 7609) or it can be synthesised by genetic engineering methods, for example using a cloned gene. EGF can also be of human origin.
To introduce the chain extension segments A and A', usual techniques prevalent in polypeptide chemistry can be used. Such techniques are detailed for instance in the foregoing references EP-A-130,935 and WO/PCT-CH86/00177.
Compound I has other advantages in addition to being t internalized into malignant cells and controllably releasing therein its bound cytotoxic drug. Quite unexpectedly, its selectivity toward malignant cells over that of previously known cytotoxic drugs is considerable as will be seen hereafter. Further, the polymer construction should prevent 132~3Q ~
the polymer drug complex from penetrating into heart tissue, thus removing one of the major shortcomings of free daunomycin and other known antitumoral drugs.
Reference is now made to the accompanying drawings in which:
Fig. 1a is a photomicrograph of a culture of A-431 human squamous carcinoma cell line used to test the effect of the compound of the invention. The presence of large amounts of EGF receptors thereon is indicated by indirect immunoperoxidase staining (dark color).
Fig. lb is a photomicrograph similar to that of fig. la but of a culture of normal WI 38 embryonic fibroblast cells used as a control. The low level of EGF receptors is indicated by the quasi absence of staining 7a ~ i _ 13233G~
(light color).
Fig 2a is a photomicrograph of an A-431 cell line culture after a 4a hrs incubation with 1 yg/ml of free daunomycin and testing for dead cells with Trypan Blue exclusion dye.
Fig 2b is a photomicrograph similar to that of fig. 2a but after 48 hrs incubation Nith, instead, 1 ~g/ml of Dbunomycin in the form of compound I. The dark areas represent the killed cells.
Eig. 3a is a photomicrograph at time zero of a mixed culture of A-431 and r~I ~8 cell lines. The round cells are the tumor cells.
Eig. 3b is a photomicrograph si~ilar to that of fig. 3a showing the situation after 24hrs incubation with 1 ~g/ml of compounZ I. the round tumor ceils have greatly diminished.
~ ompound I demonstrates very useful properties in cancer therapy. A
first valuable property relates to iis strons affinity toward malignant cells over normal cells. Eor demonstrating this characteristic, a human squamous carcinoma cell line was selected, for instance A-431 cell line shown in fig. la (see M.~. WATERFIEL~ 8,), J. Cell. Biochem. 20. 14~-161), although other tumor cells can be used as well. The membrane of these cells contains a very large concentration of EGF receptors which makes them highly suitable for test wi'h compound I. The presence of these receptors is shown by indirec' im.munoperoxidase staining and appears as dark color areas of fig la. Control cell lines, for instance WI 38 embryonic fibro-blast cells with low amount of EGF receptors ~see fig. lb), Nere treated identically for comparison and appear as lighteer color areas on the photo-micrograph.
- Thus, when a known amount of compound I (p = o = 2; x = y = O, DM =
daunomycin; DM' = OH; n/m = 6) radio-labelled with I125 ~iodine is part of the EGF moiety) was incubated with A-431 cells using WI 38 cells 2S con-trol, 31% of total radioactive iodine was retained in the intra-cellular compartment and 2% in the membrane of the tumor cells, ~hile in the cGntrol cells, the corresponding values were 2% and 0~, respectively. Using free EGF in place of I in these experiments gave the corresponding 14%/0% and 7%/0% values instead. The reason of the greater accumulation of compound I
as compared to free EGF is possibly due to retention of EGF by the polymer backbone of compound I by endocytic vesicles, whereas EGF is recycled freely back to the medium.
A second valuable and unexpected ?roperty of compound I relates to its enhanced toxicity as compared with free daunomycln toward malignant cells.
Thi~ is illustr3ted by tigs 2a and 2b.
In flg ~a, d cui~ure of A-431 cells is shoNn after 4~ hr_ incubation with a med~u.~ contal`nlng `~ ug/ml or free daunomycin. ~/iability ~as scored by ~eans of the Trypan ~lue exclusion dye ~ethod. E~clusion of the dye from the cells demonstrates the viability thereof. In contrast, when an equiva-lent amount of daunonycin in the form of compound I was used in a s~ilar experinent ~see fig. ~b), a very large number of ce'13 were :~-lled as shown by the shrivelied up cells and the dar~ areas where the dye 'na~ accumula-ted.
The seiectivity of compound I to~ard tumor celis was further tested.
For instance, fig. 3a and 3b show the effect of compound I in the case of a mi~ed culture of A-431 andd WI 38 cells. The round celis repre~ent the A-431 tumor celis ,~nd the elonsated cells are the WI 3~ control cells. The culture medium was Dhot3sraphed at time -ero t'ig. 3a). Eig 3~ illustrates this situa-ion after ~4hrs in a medium conta-'nir.g i ~g/.~.l or compound I. It can be seen tha~ t~e round darker malignant cells have stron~ly re~ressed.
In conclusion, it has been demonstrated that at equiv~ient molar concentrations (of daunomycin) com?ound I rAas much more lethal to squamous carcinoma ce'ls than free daunomycin itself. Also compound I has a selec-~ive mortal ac~ivity on tuncr cells ~ut leaves normal cells allve, ~hereas, under the same cond '._ons, free daunomycir will kil' norma' cells.
Further advan-ages of the compour.ds of the inver.t1cn ror treatlr.g-tu~or diseases relate to the follo~inc characteristlcs:
Molecular size: The compounds or tne invention are smaller than most usual targetted drugs using monoclonal antibodies, wherefrQm improved pene-tration into tumor bodies is assured ~ith consequent better cytoto~ic efficiency. Yet, the compounds Ot the invention are bigger than the free cytotoxlc drugs ~hich ?revents penerr3tion lnto nornal cells. Ir contrast, malignant cells being susceptible to endocytocls they will allow penetra-tion of compounds of moderate size like that of the invention.
Effect of EGF and related factors: EGF has an an~iogenic effect and develops mali~nant cell vascularization wi.~ consequent cell mitosis and increased suscepti~ility to antimitotic drugs. In otner words, t~e com-?ounds of the irventicn tenporarily incroase the ~umor cel' metabolism ar.d render it more sensitive to d.ug killing actior.
The following Exam?les lllustrate the invention in more detail.
1~2~30' EXPERIMENTAL SECTION
Example l Synthesis of compound I, a conjugate of EGF and daunomycin-grafted polyglutamic acid.
A polymeric compound ~V) with a poly~lutamate ba-.'ibone structure (p o = 2; 3 = y = O, in Nhich some ~ -carboxylic groups are condensed with daunomycin and having a 2-pyridyl-dithio-ethylamido heaZing group was pre-pared according to Y. KATO et al., (lg84~, J. Med. Chem. 27, 1602-1607 (compound 5 in scheme II of KATO).
The identity of the compound was chec.~ed analytically:
Mw = 29000 Da by quantitatively determining the 2-pyridyldithio group;
ratio; ratio of daunomycin to carboxylate = 1i6 as determined by spectrome-trlc quantitation of daunomycin at 480 nm.
To twenty five mg of the polymer dissolved in 2 ml of lOmM sodium phosphate buffer at pH 7 were added O,l ml of a 0,3 M solution of dithio-threitol (~TT). After l hr at 40C the solution was dialyzed overnight against a 0,1 M sodium phosphate buffer at pH o,O (SPECTRAPOR membrane, MW
cutoff 3500); this regenerated 'he thioethylamido group of the molecule (compound 6 in scheme II of ~ATO); yield ~3 mg of a red compound after free2e-drying (poly-(DM)-Glu-SH). The poly-(~M)-Glu-SH polymer was reacted with an excess of thiopropyl-SEPHAROSE~in the pyridylsulfide form (4C; 12 hrs; phosphate buffer, pH~) and the gel was rinsed with an excess of the same uffer in order to eliminate the polymer lackin the SH extremity. The polymer was conserved ur.der this form in the cold. It was then regenerated by treating the gel with an excess of mercaptoethanol (12 hrs), dialysed against water (overnight, 4C) and freeze-dried before reacting with EGF.
One hundred ~g of EGF (SIGMA) ~as dissolved in 500 ~1 of lO mM phos-phate buffer, pH 7,0 containing 0,14 M NaCl. Then a quantity of l25I-EOE
sufficient to provide an activity of lO,OOO cpm/~g EGF was added followed by 50 ~1 of a 32 ~ solution of N-succinimidyl-4-~-maleimido)-butyrate (SMBU) (origin: Sigma), in dimethylformamide. The mixture ~as allo~ed to stand for l hr at 25~C, then it was dialyzed aga~nst a O,l M sodium phos-phate buffer-O,l M NaCl,, pH 6,0, to eliminate the excess of SMBU.
The desired product (II) resulting from t~e cor.densation of S~BU and EGF was not isolated ~u to tne 500 al of the d aly-ed ~GF solution ~ere 132330~
added 1 mg ot poly-(DM)-Glu-SH and the mixture ~as slowly agitated over-night at 4C. ~ive ml of thiopropylsepharose (pyridylsulfide form) were then added and the reaction was continued at 4C for 12 hrs. The gel was washed with successively 3 portions of l ml of sodium phosphate buffer, the eluent was concentrated under reduced pressure and subjected to gel filtra-tion on SEPHADEX G75~ column 40 x 0,8 cm), using 0,l M ammonium carbonate solution pH 7,0. The fraction containing the EGF-poly-(~M)-Glu conjugate ;I), absorption 490 nm, was collected and freeze dried. Yield: 80 ~g of solid.
Example 2 Selection of A-431 test cells lines from evidence of receptor concentration on the cell membrane by indirect immunoperoxydase stain-ing Indirect immunoperoxydase staining on cell lines were performed on trypsinised cells in 35 mm P~C plates. The surface was pre-treated with phosphate buffered saline (PBS) ph 7,~, the excess was then removed and the PBS washed cells (105/well in 50 ~l ?BS) were added to the plated and centrifuged for 5 minutes at `2000 rpm. 50 ~l/well of 0,5% glutaraidehyde in cold PBS were then added to the dish and incubated for 15 minutes at room temperature. After two round of washes with PBS, the wells were filled with 100 mM glycine in a 0,1% BSA solution and allowed to rest for 30 minutes at room temperature to block gutaraldehyde activity. After two PBS washes, indirect immunoperoxydase was done by first denaturing the cells with an ice cold mixture of 9~:l ethanol-acetic acid for 30 minutes at 4C. During this period, 3 ~1 of antibody were diluted in 1 ml PBS + 1% foetal calf serum (FCS). The wells were then washed twice with PBS and incubated for 5 minutes with a solution of 20~o FCS in PBS. This was then replaced by 200 ~l/well of the antibody solution and incubated for 30 minutes at room temperature. The wells were then washed twice with PBS, once with PBS +
Tween 0~l~o and once again with PBS. 200 ul/well of a 11400 dilution of swine anti-mouse perodydase ~POD) conjugated ant.body (DAK0) in PBS l~ FCS
was then washed twice with PBS, once with PBS + 0,1% tween and twice with dlstilled water. The in situ coloring was achieved by incubating the cells at room temperature with a solution of lO ml 0,Ol M phosphate buffer pH
6,0, 5 ~l 35~O oxygenated wa~er and lQu ~l of l~, ortho-àianisidi.. (MERCC) in methanol. 132330~
Example 3 Internalisation of compound I in A-431 squamous carcinoma cells compared to in HI-38 fibroblasts The entry of compound I into the cells is essential for .t3 action.
Therefore, internalisation Ot the molecule was demonstrated by showing its presence in the membrane and inside the cell compartment. This has been done using A-431 and WI-38 cells. t Compound I, radiolabeiled with 125I on the EGF, was incubated with confluent cell cultures for 6 hrs at 37C in solution A (4 parts ~MEM and 1 part of 50 mM Tris, 1~0 ~M NaCl and 0,1% BSA adjusted a. ph 7,4). The cells were then washed ~our times ~ith ice-cold PB~T (lmM Cd Cl2and 1 .~M
MgC12). Fifty % trichloroacetic acid ~as added in a proportion of 1!5 to the pooled solution a and PBS+ and counted on a y counter. The cell mem-brane was destabilized by a treatment on ice with 200 mM acetic acid and 150 mM NaC1 (solution B) for 6 minutes. The solution B was then removed and the cells wa~hed tNice with solutlon B. These pooled solutions were assayed for 125I. This treatment releases the EGF rece?tors bound to the cell surface. The cells were then complete1y dissolved in 0,7 N NaOH. The radioactivity found then represented the internali,~ed compound I. Ln con-trol experiments, using free EGF 125I, a proportlon of the EGF is recycled to the medium therefore lowering the intracellular EGF. The results on the internalization of 125I labelled EGF and compound I in ~1-38 and ~-431 cell cultures are given in the following Table in terms of counts per min (background 30 cpm) for successively: EGF not integrated in the cell, EGF
bound to the membrane, and EGF in the intra-cellular compartment. The percent of total cpm is given in brackets.
132330 ~
TABLE
EGF (%) Compound I (%) WI-38 530 (93) 1169 (98) " 29 (O) 28 (O) " 67 (7) 52 (21 A-431 536 (86) 831 (67) " 31 (O) 57 (2) " 111 (14) 390 (31) These results show that compound I is more efficiently internalized in malignant cells (31%) than in normal cells (2%~.
Example 4 Comparative cytotoxic effects of free daunomycin and compound (I) on a A-431 squamous carcinoma cells All cells were maintained in Dubelcco's Modified Medium (DMEM), 10%
foetal calf serum (FSC) (Gibco), 2% penicillin-streptomycin (Gibco) and 1%
fungizon (Gibco~ in 5% C02. They were plated at 50 to 60% confluency 24 hours before the addition of the toxin. 1 ~g/ml of daunomycin, or equiva-lent in compound I, was added in DMEM 10% FCS and the ceil death rate visualized by trypan blue exclusion. 4 volumes of 0,2% (w/v) Trypan Blue in water was freshly mixed with 1 volume of a saline solution 4,25% (w~v) of NaCl in water. 1 volume of this solution was mixed with 1 volume of PBS on the cell monolayer or to 1 volume of cell suspension in PBS. Observation and scoring took place 48 hours after addition of the toxin (see fig. 2a and Zb). These results show that daunomycin is much more effective against malignant cells when in the form of compound I than when in the free state.
14 13233~
Example 5 Effect of compound I on mixed cultures of tumor (A-431) and normal (~I-38) cells.
Cells were maintained as described in e~ample 4. The ~I-38 fibroblasts were first plated and the A-431 squamous carcinoma cells were plated the next day. The mixed culture was left growing for 24 hours and then 1 ,ug/ml of compound I was added to DMEM 10% FCS. Nearly all the A-431 cells were selectively killed after 24 to 48 hours whereas the ~I-38 fibroblast were left allve. Hence, the mixed culture became free from the tumor cells demonstrating that compound I can be used to separate the normal cells from cancer cells. The experiment was repeated but using only 0,1 ~g/ml of compound I (for the controls, equivalents of daunomycin were used in free form). Observation of the cultures after 15 days showed that the test samples contained no more of A-431 cells, which situation was confirmed by continuing culturing for 6 weeks under normal conditions, this resulting in no reformation of tumor cells. In contrast, in the controls all cells, malignant and normal, had died after 15 days.
Example 6 A compound of general formula V (see description) was prepared accor-ding to KATO et al., G. Med. Chem. 27 (1984), 160Z-1607 (compound defined as 4 in scheme I of KATO). This compound has the formula VA below in this paticular embodiment.
SS ~ NH~(CO~CH~NH)q~~H VA
( C;~2 ) 2-COoH
in which q may be fro~ about 150 to 250 depending on the polymeri7ation conditions.
This compound was subjected to a procedure reported by HESS-~IJK e~
al., J. of Controlled 2elease 1 (4) (1985), 312 for e~tending the side chain with an H-Gly-Gly-Leu-OH peptide (segment A in formula I), as fol-lows: 40 mg of VA and 68 mg of saccharin (0.31 mmole) were dissolved in 1 ml of DMF and the solution was allowed to rest far a few hours (solution A).
On the other hand, 0.32 mmole (40 ~1) of N,.~,N'~'-tetr~methylganidine (TMG) ware slowly added to a stirred suspension of 0.3Z mmole (8C mg) of H-Gly-~ly-Leu-OH in DWF Stirring was continued until all sollds had dissolved ~solution B).
Then 0,43 mmole (70,3 mg) of ~,N'-carbonyl-diimidazole were added to soiution ~ and, after stlrring for 30 min, solut~on 3 waa added. The mi~ture was further stirred for 3 days at room-tempera~ure. The mi~.ure was added into 15 ml of 0.1 M phospha~e buffer (pH 7.0) ar.d the resulting solution was dialyzed into water (17 hrs) filtered on a millipore membrane (0.45 ~m) and the filtrate was freeze-dried. The polymer ~yield 80% mg) was analy~ed by hydroly~ing an aliquot in ~ N HCl for lZ nrs at 180C. Deter i-nation of the aminoacids in the hydrolyzate was carr_ed out ~y High Perfor-mance ~iquid Chromatosraphy of the amlnoacld-~Atalaldeh~fde derivatives (detection by fluorescence). The followlng ratlo of glutam~c~lycine/'eucine was measured: 0.95/2/i. These results indicate that the grafting level was aroung 95%r i.e. the compound can be represented conventionally by the formula:
PYR-SS ~ ~lH ~ CO-jH-NH)- 05........... -(C0-jn-NH) ~5 ~ . H
(CH2)2 COO C~.2(CH2CONH)3-CH(iso'Bu)COO
as the tetramethylguanidlne salt. ('~B) The pyrldine-S group was removed ~ith dithlothreitol according to KATO
et 21., and analysis Nas performed by measuring the absorbance at 343 nm of the ll~erated pyridine-2-thione. Neqlecting the presence of the TMG+ ion, a molecular weight of 25.500 was found meaning that in the product t (the degree of polymerizat on) i3 about 75-90.
Labellinq compound ~B with Daunomycin waa ac_om?liahed ~s follows:
33.7 mg (74.7 ~mole) of '~B ~ere disolved in 15 ml of ~ a(iueous NaCl and 20 mg (35,5 ~mole) of 2aunomycin hydrochloride were added. The pH ~as brought to 5.5 Nith 0.1 N ~aOH arter which ~8 mg ~0.1 .~r~cle! of 1-ethyl-3-C3-(dimethylamino)-carbodiimide hydrochlor~de ~EDC) ~ere added under s;irring.
16 1323"\J~
After agitation for 18 hrs, the mixture was dilute~ with 15 ml of 1 M NaCl and dlaly7ed in ~ater~ The residue was free7e-dried wh ch provided 31.5 mg of polymer. Splitting t;r~e dlsulfide with DTT as before and analy7ing spec-troscopically the pyridine-thione indicated the prosence of about 10 DM per molecule, i.e. a ratio of labelled side-chains to unlabelled side-chains of about 1:7.
Conversion of an aliquot o~ the above disulfide to the deslred cor-res?Gndi.rig thiol I -~B with DTT was done as Ec~ ows: 2 mg of pol-~me- were dissolved in 2 ml or 0.1 ~ phosphat- buEtar (?H 7.0) and 150 ul of 0.3 M
DTT were added. After allowins tG stand for one hr at 42, the solution was dialy~ed for 4 hrs agaln~t freshly degassed phosphate buffe. ~pu 6.0); A
SPECTRAPOR bag was used (MN cut-off = lOOC).
Simultaneously, EGE was ai-t~ated by tak ng 0,2 ~g of _GE (Sigma) and dissolving in 3.?5 ~1 oE 10 .~M ?hosphate bu~ter in 0.14 M NaCl, pH 7.0;
the-, adding 0.5 ml of 2 10 ~g/ml S~BU solur on in DI~F. After 2 hrs at '0C, the mi~ture was dialy7i?d at d~C ~gainst a 0. ~ Phosphate ~uffer contalning 0.1 M NaCl at pH 5.0 'membrane Mw cut-oEf = 1000).
The overall wlume of both the dlalyzed polymer and dialy~ed EGF
solution Nere reduced to 1 ml by absorption with C`~C powder, then they were mixed together and allowed to stand for Z4 hrs n one dialysis Jas. The m :~ture was c;lrcmatGsraphed on S~P~ADE~ G75 ~eluent O.lM N~o~ and the fract ons absorbing a~ 4~0 nm were collected and cleaned from unreacted ?olymer by treating with thiopropy sepharose overnight at 4C. The gel -~as washed with O.lM NH4Co3 and 22.5 ml (O.D. of 0.176) of solution was collected. Yield about 80% of compound IA. The respective weight contri-butions of DM and EGF in the product are about equal.
If, in the above pre~aration, Daunomicin is replaced by Adriamycin, a similar product, incorporatins Adriamycin is obta ned.
The presence and bir.dlng efficiency of the EGF factor in .ompour.d IA
was checked by immunoprecipitation with EGF antibody andd attachment of the immunocomple~ to a protein A - Sepnarose gel (C~4B, Pharmacia). The proce-dure was as follows:
Protein A - Sepharose gel was rahydrated to ~rovide a 50~, (VtV) solution in NET-NP40 buf-er (100 .~M NaCl, 1 .~ A, 10 ml`~ Tri,, pY 7.5, 0,5% NP40). Bovine serum albumin (BSA) was added to 20 ~1 or the buf ered sepharose solution to provide a 0,3% (by weight~ ~SA solution ~S!. On the o~her har.d, BSA was added to 12 ul of a sc uticn of antiserum aga-nst EGF
(Collaborative ~esearch) so as to ?rc~; d- ~ 0,_~ by weiqn. BSA so u _on in 17 13233G, antiserum (Ab). ~oth (S) and (Ab) were incubated overnight at 4~C, then a quantity of compound IA corresponding to 120 ~g of daunomycin was added to sample Ab and incubated for 7 hrs at ~C under agitation. Then solution (S) was added and the mixture was agitated per 12 hrs at 4C. A control (C) was prepared by adding the same quantity of compound IA to another identical sample of solution (S). Both the above mixture (M) and the control were centrifugated for 5 min at 2000/rpm and the optical density (OD) of the supernatant liquid measured at 480 ~m. The results are 0.41 for M and 0.955 for C which shows that compound IA c~rries EGF, the conformation of which is recognized by the ~ntibody on the gel.
Example 7 The toxicity oE compound IA and of free daunomycin toward A431 malig-nant and W~3~ (control) cells was compared. The effect of these drugs was evaluated by the degree of inhibition of cellular protein synthesis. For this, we measured the level of incorporation of 35S methionine (35S-met NEN) into newly synthesized proteins after a 48 hrs exposure to different concentrations of the drugs.
Cells were plated on 1,5 cm Petri dishes at 50% to 60% of confluency before the addition of the toxin. 0,1, 0,5 or 1 ~g/ml doses of daunomycin (controls) or its equivalents in the form of compound IA were added in DMEM
10% FC~. Cell death consecutive to this addition was measured as follows:
The cells were exposed for 1 hour at 37C in 500 ~1 DMEM low methionine medium (Gibco) containing radioactive 35S met. The medium was then removed and the dishes were washed with PBS (137 mM NaC1, 2,7 mM KU , 8,1 mM
Na2HpC4, 1,5 mM KH2P04, 0,9 mM CaC12, 0,5 ~M MgC12 pH 7.2). These solutions were stored for ~ counting. The cells were lyzed with 1 ml 0,1 N NaOH and placed in a tube Nith 500 ~1 Trichloroacetic acid (TCA) 10% to precipitate the proteins. This mixture was filtered on GF/A filters (Whatman) to eliminate unreacted 35S-met. The filters were washed twice with 1 ml of 10%
TCA and once with 100% Ethanol. The filters were dried at 80% for two hours and sub~ected to ~ counting in Econofluor (NE~) sc-ntillation medium. The results reported in the following table show that compound IA is very cytospecific but less cytotoxic than daunomycin alone.
18 ~233aj drug concentration % Oe Protein synthesis inh~bition (~g/ml A431 WI38 0,1 30% 10%
FREE DAUNOMYCIN O,5 69% 46%
1,0 97% 80%
=========================================================
0,1 4% 0%
CONPOUND IA O,5 17% 6%
1,0 47% 4%
Example 8 IN VIVO TEST OF COMPOUND I
We induced A431 xenografts in nude mice and when the tumors reached 3 to 5 mm, we in~ected into two sets of nude mice 0,1 mg/kg of daunomycin either in the form of compound I (see example 1) or in free form. In the prior art, 10 mg/kg doses have been used in mice (Schwarz S. et al., 1975, Cancer Chem. Rep., 6, no 2, 107-114). The drug was injected either directly in the tumor or in the caudal vein 4 times at 3 days intervals. The measu-res were taken a week after the last injection.
By visual inspection of the animals, it was noted that the growth of the tumors treated with compound I was significantly reduced, as compared to control mice or to treatments with free daunomycin. These results were similar whether the injection of the drug was directly in the tumor or in the caudal vein which shows that biodegradation and release of the cytotoxic substance occurs substantiallly only in the target cells. Results are shown in the follo~ing Table (I.V. = intra-veinous; I.T. = intra-tumor) 19 1 3 2 3 c~ Q
injection initial surface final surface growth of the tumor ~A) of the tumor (8) coefficient (B/A
Daunomycin I.V. 30.5 500 16 Compound I I.~. 25 54 2 Daunomycin I.T. 9 170 18.7 Compound I I.T. 9 50 5.6 Control* 9 320 35.6 * Placebo injection At the end of the experiment, the tumors were dissected and weighed.
Results are given below. If corrections for slight differences of tumor si~e at the beginning of the treatment are taken into account, it can be seen that the results correlate well with the surface estimate glven in the previous Table.
~aunomycin I.V. : 1.178 g.
Compound I I.V. : 0.359 g.
Daunomycin I.T. : 6.789 g.
Compound I I.T. : 0.816 g.
in conclusion, the compounds of the invention show better performances in selectively killing squamous carcinoma cells than daunomycin, both in vitro and in vivo. In the animal tests, we noted that very low amounts of com-pound I have a remarkable effect on tumor growth.
E~ample g The in-vivo tests reported in Example 8 were repeated using compound IA instead of compound I. Thus, 2 mg/kg of daunomycin, free or in the form of compound IA were injected every 3 days over 9 days in the caudal vein of nude .mice bearing A431 tumors. The tumor growth inhibitlon by compound IA
132330.~
was significantly greater than by free daunomy.cin (D), free EGF or DM
labelled but untargetted polymer (DMA). These results are shown in the Table below where the values correspond to tumor diameter (in mm) measured with a scalliper after a number of days. The lethal dose of compound I and compound IA have not been measured but are presumably less toxic than free daunomycin which enters freely into most normal cells.
T A B L E
Dayg 0 4 7 11 14 Control 10 14 17 2Z 24 Free EGF 10 13 14 17 19 DMA 10 11 12 16 i7 IA 10 11 12 14 i5
Claims (16)
1. The use in the treatment of malignant diseases, of a covalent conjugate medicament consisting essencially of the following components bound covalently together, a) a cytotoxic substance, b) a polymer carrier having an endocytic biodegradable, non-toxic, polyaminoacid backbone, c) a cell homing vector with the properties of first selectively targetting the conjugate toward malignant cells to be fought and second, providing for the internalization of the conjugate into said celis, wherein fast biodegradation of the carrier will occur with consecutive release of the cytotoxic drug which will selectively combat said malignant cells.
2. The use according to Claim 1, wherein the homing vector also comprises one or more EGF, EGF analogues such as urogastrone, ? -TGF, and synthetic or natural derivatives of growth factors providing cell internalization as well as tumor cells specific markers.
3. The use according to Claim 2, wherein the homing vector also comprises one or more antibodies capable of selectively bind to a particular antigen possessed by a malignant cell.
4. The use according to Claim 1, wherein the polymer carrier is polyglutamic or polyaspartic acid.
5. The use according to Claim 3, wherein the polymer carrier is a copolymer of aspartic and/or glutamic acid with other aminoacids such as glycine, leucine, valine, isoleucine, alanine, phenylalanine.
6. A cytotoxic targetted conjugate for the application of the method of Claim 1, this conjugate having the formula (I) in which p and o are 1 or 2; A and A' represent chain extending amino-acid intermediate links of formula -(NH-CHY-CO) and x and y, which define the number of these links per molecule, can be zero or any integer from 1 to 20; Y is an amino-acid rest; DM and DM' represent one or more cytotoxic substances covalently bound to the aminoacid carboxyl group through an amide or ester link but DM can also represent an OH of a free carboxyl; EGF defines a homing vector for promoting malignant cell recognition and internalization therein; R is a group of formula.
with x being selected from -(CH2)r with r from 1 to 4; or ; or ; or , or ; m and n are integers of value sufficient to provide a molecular weight of 10,000 to 500,000 Da, the value of m/n being 1/10 -1/2.
with x being selected from -(CH2)r with r from 1 to 4; or ; or ; or , or ; m and n are integers of value sufficient to provide a molecular weight of 10,000 to 500,000 Da, the value of m/n being 1/10 -1/2.
7. The cytotoxic conjugate of Claim 6, in which said homing vector comprises the epidermal growth factor (EGF), EGF
analogues such as urogastrone, .alpha.-TGF, and synthetic or natural derivatives of growth factors providing cell internalization as well as tumor cells specific markers.
analogues such as urogastrone, .alpha.-TGF, and synthetic or natural derivatives of growth factors providing cell internalization as well as tumor cells specific markers.
8. The cytotoxic conjugate of Claim 6, in which the homing vector further comprises one or more antibodies capable of selectively bind to one or more particularly antigens of malignant cells.
9. The cytotoxic drug of Claim 6, wherein DM and DM' are selected from auromycin, hematoporphyrin, platinum complexes, abrin, ricin, toxins of diphtheria and pseudomonas, bleomycin, gelonin, adriamycin, vinchristine, daunomycin and metothrexate.
10. The cytotoxic drug of Claim 6, wherein A and A' are selected from single aminoacids or polypeptides having from 2 to 20 aminoacids.
11. The cytotoxic drug of Claim 10, wherein said aminoacids are selected from glycine, alanine, serine, leucine, phenylalanine and arginine.
12. The cytotoxic drug of Claim 6, wherein p and o are equal to 1, the backbone polymer being polyasparate.
13. The cytotoxic drug of Claim 6, wherein p=1 and o=2 or p=2 and o=1, the backbone polymer being a copolymer of aspartic and glutamic acids.
14. The cytotoxic drug of claim 6, in which p=o=2, x=0, DM
is OH, DM' is daunomycin or adriamycic and Ay is a Gly-Gly-Leu segment.
is OH, DM' is daunomycin or adriamycic and Ay is a Gly-Gly-Leu segment.
15. A method for manufacturing compound I defined in Claim 6, by the reaction of a thiol-copolymer III of formula (III) with a 4-(N-malaiminido)-butyrate derivative II of formula (II) in which formula Z designates the backbone polymer or copolymer indicated in formula I and the symbols EGF, X, m, n and DM are defined as in Claim 6.
16. The use of compounds of formula I as defined in Claim 6 as cyotoxic agents in cancer therapy.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP86810347 | 1986-08-07 | ||
| EP86810347.4 | 1986-08-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1323303C true CA1323303C (en) | 1993-10-19 |
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ID=8196463
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000543912A Expired - Fee Related CA1323303C (en) | 1986-08-07 | 1987-08-06 | Advanced anticancer therapy and cytotoxic medicaments for its implementation |
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| Country | Link |
|---|---|
| US (1) | US5087616A (en) |
| EP (1) | EP0259904B1 (en) |
| JP (1) | JPH01500435A (en) |
| AU (1) | AU608531B2 (en) |
| CA (1) | CA1323303C (en) |
| DE (1) | DE3770730D1 (en) |
| DK (1) | DK184788A (en) |
| FI (1) | FI881579A0 (en) |
| IN (1) | IN165717B (en) |
| WO (1) | WO1988000837A2 (en) |
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| WO2024006960A1 (en) | 2022-06-29 | 2024-01-04 | Juno Therapeutics, Inc. | Lipid nanoparticles for delivery of nucleic acids |
| WO2024031091A2 (en) | 2022-08-05 | 2024-02-08 | Juno Therapeutics, Inc. | Chimeric antigen receptors specific for gprc5d and bcma |
Family Cites Families (16)
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|---|---|---|---|---|
| IE38892B1 (en) * | 1973-03-28 | 1978-06-21 | Ici Ltd | Pharmaceutical compositions |
| DE3175151D1 (en) * | 1980-05-21 | 1986-09-25 | Teijin Ltd | Reactive polymer and process for the preparation thereof |
| IE53166B1 (en) * | 1980-08-05 | 1988-08-03 | Searle & Co | Synthetic urogastrone gene,corresponding plasmid recombinants,transformed cells,production thereof and urogastrone expression |
| EP0108132A1 (en) * | 1982-05-06 | 1984-05-16 | Applied Molecular Genetics Inc. | The manufacture and expression of genes for urogastrone and polypeptide analogs thereof |
| US4587046A (en) * | 1982-05-18 | 1986-05-06 | The Regents Of The University Of California | Drug-carrier conjugates |
| JPS58219124A (en) * | 1982-06-15 | 1983-12-20 | Nippon Chem Res Kk | Preparation of multiplicative factor of human epithelial cell |
| US4485093A (en) * | 1982-08-13 | 1984-11-27 | Runge Richard G | Immunotoxin conjugate which comprises arsanilic acid, useful for treating malignant tumors, particularly pancreatic cancer |
| JPS59116232A (en) * | 1982-12-24 | 1984-07-05 | Teijin Ltd | Cell toxicity complex and its preparation |
| IL71991A (en) * | 1983-06-06 | 1994-05-30 | Genentech Inc | Preparation of mature human IGF and EGF via prokaryotic recombinant DNA technology |
| EP0148922A4 (en) * | 1983-07-05 | 1987-06-15 | Chiron Corp | Hybrid dna synthesis of epidermal growth factor. |
| JPS6028994A (en) * | 1983-07-08 | 1985-02-14 | Wakunaga Seiyaku Kk | (21-leucine) human urogastrone, corresponding gene, corresponding recombinant plasmid, transformed cell and their preparation |
| IL69719A0 (en) * | 1983-09-14 | 1983-12-30 | Yeda Res & Dev | Synthetic peptides with egf like activity |
| US4545985A (en) * | 1984-01-26 | 1985-10-08 | The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services | Pseudomonas exotoxin conjugate immunotoxins |
| US4522750A (en) * | 1984-02-21 | 1985-06-11 | Eli Lilly And Company | Cytotoxic compositions of transferrin coupled to vinca alkaloids |
| CA1283661C (en) * | 1984-06-20 | 1991-04-30 | Franz Jansen | Imidazolides, process for their preparation and application as intermediates for the synthesis of cytotoxic conjugates |
| IN165717B (en) * | 1986-08-07 | 1989-12-23 | Battelle Memorial Institute |
-
1987
- 1987-07-29 IN IN543/MAS/87A patent/IN165717B/en unknown
- 1987-08-05 JP JP62504770A patent/JPH01500435A/en active Pending
- 1987-08-05 EP EP87201490A patent/EP0259904B1/en not_active Expired - Lifetime
- 1987-08-05 AU AU77887/87A patent/AU608531B2/en not_active Expired - Fee Related
- 1987-08-05 WO PCT/EP1987/000435 patent/WO1988000837A2/en active Application Filing
- 1987-08-05 DE DE8787201490T patent/DE3770730D1/en not_active Expired - Fee Related
- 1987-08-06 CA CA000543912A patent/CA1323303C/en not_active Expired - Fee Related
- 1987-08-06 US US07/082,244 patent/US5087616A/en not_active Expired - Fee Related
-
1988
- 1988-04-06 FI FI881579A patent/FI881579A0/en not_active IP Right Cessation
- 1988-04-06 DK DK184788A patent/DK184788A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01500435A (en) | 1989-02-16 |
| AU608531B2 (en) | 1991-04-11 |
| FI881579A (en) | 1988-04-06 |
| DE3770730D1 (en) | 1991-07-18 |
| DK184788A (en) | 1988-06-06 |
| DK184788D0 (en) | 1988-04-06 |
| WO1988000837A3 (en) | 1988-03-10 |
| US5087616A (en) | 1992-02-11 |
| WO1988000837A2 (en) | 1988-02-11 |
| AU7788787A (en) | 1988-02-24 |
| IN165717B (en) | 1989-12-23 |
| FI881579A0 (en) | 1988-04-06 |
| EP0259904A1 (en) | 1988-03-16 |
| EP0259904B1 (en) | 1991-06-12 |
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