WO1987000530A1

WO1987000530A1 - Protein conjugates of bis-indole alkaloids, bis-indole alkaloids, their preparation and application

Info

Publication number: WO1987000530A1
Application number: PCT/FI1986/000074
Authority: WO
Inventors: Aarre Huhtikangas; Seppo Lapinjoki; Jarmo Niemi
Original assignee: Huhtamäki Oy
Priority date: 1985-07-16
Filing date: 1986-07-04
Publication date: 1987-01-29
Also published as: DK132787D0; FI860456A0; DK132787A; AU6135586A; EP0264376A1; FI860456A

Abstract

A protein conjugate of bis-indole alkaloids having the formula (I) Ar-N=N-bisindole alkaloid, wherein Ar is a benzoe- or arylalkylcarboxylic acid group which contains a protein group. The invention also concerns a process for the preparation of a protein conjugate of bis-indole alkaloids having the formula (I), in which process is reacted with a diazonium acid derivative having the formula Ar-N=N(+), wherein Ar represents the same as above with the exception, however, that Ar may not contain a protein group in which case a protein group is attached to an aromatic acid group. The invention further concerns a bis-indole alkaloid derivative, having the formula Ar1-N=N-bis-indole alkaloid, wherein Ar1 represents a benzoe-or arylalkylcarboxylic acid group. Said derivative is useful compound in preparation of the conjugates of formula (I). Finally conjugates of formula (I) in immunometric methods and in the treatment of diseases, especially cancer.

Description

Protein conjugates of bis-indole alkaloids, bis-indole alkaloids, their preparation and application

The present invention relates to protein conjugates of bis-indole alkaloids and their preparation as well as application. The invention also relates to bis-indole alkaloids which are particularly useful when producing the above protein conjugates.

So-called immunotoxines are conjugates wherein a specific carrier protein, which is generally but not necessarily an antibody, is provided with a toxic component, such as a vegetable- or microbe-based proteintoxin, anti- neoplastic drug or radioactive agent (Hofstaetter, Gronski and Seiler, "Immunotoxines - Theoretical and Practical Aspects", Behring Inst. Mitt. No. 74 (1984) 113 - 121).

As indicated in the above article, the general immunotoxin principle is already known but inventive improvements are suggested to this basic idea, regarding the carrier, toxic segment and the manner for linking these two to each other.

Various immunotoxines are useful especially in the treatment of cancer diseases but also in other treatments which necessitate the destruction of a certain cell population without damaging other cells, if possible. Thus, the carrier segment must be a protein that is capable of binding itself specifically to such surface structures of an animal cell which are characteristic of a target cell, i.e. the cells of a cell population to be destroyed. Such a protein is especially an antibody molecule produced against the above type of surface structures. Said antibody can in principle be prepared with prior known methods by immunizing an animal with a purified antigen, the above type of surface structure thus acting as such antigen.

Most preferably, however, the above type of antibody can be^produced by applying conventional hybridoma technique. This technique, which has been described e.g. in "Monoclonal Hybridoma Antibodies:- Techniques and Applications", editor Murrell, J.G.R., publisher CRC Press. Inc., Boca Raton, Florida, 1982, makes antibody production possible also against unpurified surface structures of target cells (e.g. pages 151 - 168 of this work). It is also known that, instead of a whole antibody molecule, it is possible to use such fragments thereof that have the molecule segment binding to an antigen, such as proteolytically producible fragments Fab and (Fab)₂. Instead of an antibody, the carrier can also be some other protein having a property of binding itself selectively to target cells. The Patent application GB 2 116 979 describes the conjugates of transferrine or ceruloplasmine with antituτnor drugs, said conjugates being useful in the treatment of cancer diseases since transferrine receptors are abundant in cancer cells.

The target cells may be cancer cells but, as pointed out in the article "Ex-vivo treatment of donor bone marrow with anti-T-cell immunotoxins for prevention of graftversus-host disease", Filipovich et al. The Lancet, March 3, 1984, pages 469 - 471, also an immunotoxin directed at a normal cell system is useful whenever the treatment of a disease (or in this particular case: contraception) requires selective destruction of a cell population in question.

The Patent application GB 2 137 210 discloses immunoglobuline conjugates of vinca alkaloids wherein, however, a vinca- or bis-indole alkaloid is bound to a protein, in this case to an immunoglobulin, with an ester bond at the point of a vindoline unit, designated hereinafter with R₂. This is just the area where bis- indole alkaloids of various properties differ from each otffer and this area can be expected to have a great significance in the medical activity of a molecule. Thus, the method of conjugation described in the Patent application GB 2 137 210 must be considered unfavourable since a large-sized protein segment prevents sterically the medical activity of an alkaloid.

What is described in this invention is a method of conjugating bis-indole alkaloids to a protein in a manner that leaves free the fragments which have the greatest effect on the activity of an alkaloid molecule.

Bis-indole alkaloids, some of which are vegetable-based natural molecules and some synthetically derived therefrom, have proved to be so-called cytostats suitable for medical destruction of cancer cells.

Such bis-indole alkaloids or their synthetic derivatives can be illustrated with the following general formula:

catharanthine unit

D R₁ R₂ R₃ R₄

1 vinblastine -CH3 -OCOCH₃ -OH -OCH₃

2 vincristine -"- -CHO -OCOCH₃ -OH -OCH₃

3 vindesine -"- -CH₃ -OH -OH -NH₂

4 vinzolidine -"- -CH₃ -OCOCH₃

A compound of the invention is characterized in that formula (I) of the conjugate is

Ar-N=N-bisindole alkaloid (I) wherein Ar is a benzoe- or arylalkylcarboxylic acid group which contains a protein group.

Preferably in formula (I), the general formula (II) of indole alkaloids is

(II)

wherein A represents a lower C₁ -C₅ alkylene group and B represents a lower C₁-C₅ alkylene or alkylenyl group which can be substituted with a hydroxy group and/or a lower alkyl group and Ar represents a group having a formula

T-NH-CO-(CH₂)_n

wherein n is 0 - 5 , which alkylene chain can be substituted and T represents a protein group, such as gluco- protein, immunoglucoprotein or enzyme.

Obviously, there is no commercially available sensitive and specific sequence-analytical method for the above- type of compounds, vincristine, vinblastine and vinde- sine, presently in clinical use. An essential difference compared with the prior published immunoanalytical methods (Ria, Irma, Elisa etc.) for these compounds is the high specificity of the antibodies produced on the basis of this presently developed method. This is based on the fact that the process for preparing a protein conjugate described herein leaves one part, presently understood as the therapeutically active part of bis-indolealkaloid free and hence active in interaction processes on which the analysis procedures are based.

The available high specificity is useful in the analysis e.g. in a manner that possible metabolic products in medicines or sideproducts in production do not disturb the assay.

Bis-indole alkaloids consist of vindoline and catharanthine units, wherein the indolic part of a catharanthine unit offers a possibility of conjugating a protein group to an aromatic six-member ring in question by using an electrophile aromatic substitution reaction. The protein group relates generally to protein, glycoprotein, e.g. enzyme, albumin, human or animal serum albumin and particularly to immunoglobulinor the like. Since the reactions of protein chemistry occur in aqueous solution, it will be appropriate here to use a diazo coupling with a diazonium salt to which a protein in the formation of a final immunogen can be covalencely bonded as a reaction step performed either prior to or after the diazo coupling.

The present vindoline and catharanthine units are both provided with an aromatic six-member ring for diazo coupling. In the vindoline section, the most active part of the ring is not free for this purpose as that is exactly where the catharanthine unit is attached with a covalence link. Moreover, in vindoline, the second most reactive carbon atom of said ring is in view of a diazo coupling in a sterically cramped area. In the aromatic six-member ring of the catharanthine unit there are in principle 4 carbon atoms available for diazo coupling. By performing the coupling on the diazo coupling principle of aromatic amines, in other words, by allowing an indolic nitrogen atom to activate an aromatic ring, it is quite justified to expect the coupling to occur in p-position relative to said nitrogen atom, this being the most reactive point of the ring in.view of coupling and, furthermore, this is sterically one of the best available positions of the ring. Thus, it is possible to obtain a quite uniform reaction product:

A difficulty in the reaction is a tendency of the diazonium ion to react with water molecules. If diazo coupling is slow due to unfavourable reaction conditions, the last-mentioned side reaction, phenol formation, will become the main reaction. Thus, a phenolic compound in question may also be diazo coupled whereby, in addition to a desired diazo compound, there is formed a contamination compound produced by said side reaction.

Side reactions can be avoided most effectively by adjusting the acidity (pH) of the reaction solution to comply as accurately as possible with the optimum value of this particular sysnthesis reaction.

Reaction conditions in which the above alkaloid structure is diazo coupled as quickly as possible and, at the same time, the side reactions remain as few as possible require adjustment of the acidity of the reaction solution. Also, the solution must not be so alkaline that the diazonium ion concentration goes down too much but neither should the solution be so acidic that the concentration of non-protonated amine (indolic part of the catharanthine unit) remains too low. In the present diazo coupling raction, the pH- value of the reaction solution must be adjusted within the range of pH 6,60 to pH 6,90 for producing an immunogen that is sufficiently uniform and able to measure specifically and separately bis-indole alkaloids for satisfying the requirements of ELISA assay procedure. Preparation of an antibody is effected in a prior known manner, e.g.: L. Hudson & F.C. Hay, Practical Immunology, Blackwell Scientific Publications, Oxford, 1976, and R.H. Kennett, K.B. Bechtol & T.J. McKearn (edit.). Monoclonal Antibodies and Practical Cell Lines, Plenum, New York, 1984.

Antibody can be produced by means of the ixnmunosystem of a test animal immunizing the animal with the above conjugate as immunogen and by collecting the antibody from blood circulation. Alternatively, it is possible to produce so-called monoclonal antibodies either in vivo or in vitro principle by isolating antibody-producing cells from the system of the animal either after or before the immunization treatment and by further developing production cell systems. The anibody can be purified, if necessary, by using conventional and appropriate techniques.

The analysis is based on a binding reaction between the above antibody and a bis-indole alkaloid to be analysed. The amount of a binding analysate is determined by means of a competing binding tracer. The tracer can be a corresponding commercial compound labelled with a radioactive isotope or a corresponding compound conjugated to a measurable enzyme. Radioactivity and enzymatic activity can be measured by conventional equipment marketed for these purposes. This principle is described in publication Voller et al. Bull. Wild. Hlth. Org. (1979) 53, 55-56.

The workability of this method has been practically, tested in all respects for vincristine by using both radάoisotope and enzyme as a tracer. A radioimmuno- metric method (RIA) works with a trithionated tracer within a concentration range of 0,1 to 50 ng, is highly -specific on a target material and fulfils the criteria generally accepted for the reproducibility of corresponding methods. The method has been found applicable in the assays of the vincristine concentration of plasma. An enzyme-immunoanalytical method (EIA) corresponds to the above in other characteristics but it is more sensitive (measuring range circa 10 - 500 pg) and, in addition to plasma, it is also suitable for vincristine assays of whole blood and cellular material in blood.

The performance of a RIA analysis sequence takes appr. two days during which one person can perform appr. 200 analyses. Respectively, the duration of an EIA sequence is appr. eight hours, including appr. 500 samples. The speed and capacity of both assays can be improved significantly if they are developed into a so-called laboratorium kit in which the final laboratorium work phases are minimized and conditions accurately optimized.

In therapeutical application, the protein conjugates of formula (I) according to the invention can as a protein group contain a protein binding specifically to the surface structures of an animal or human cell, an antibody produced against individual structures or an anti- body which is monoclonal and prepared by using hybridoma technique. As pointed out in the introduction to this specification, the antibody can be typical or specific to cancer cells.

The following examples illustrate the invention without limiting it.

Example 1

Preparation of an albumin conjugate of bis-indole alkaloid

50 mg of aminophenylalanine is dissolved in 5 ml of water. To this solution is added 50 mg of bovine serum albumin and 50 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbbdeimide (EDC) and the mixture is incubated overnight at room temperature.

Reaction product is separated from reagents by dialysing (ultrafiltration) at +4 against water. The solution pH is adjusted to 1,5 with hydrochloric acid in ice bath. 100 mg of NaNO₂ is dissolved in 1 ml of water and added dropwise to the hydrochloric acid solution to which is further added 50 mg of ammoniumamidosulphonate dissolved in 1 ml of water. 15 mg of bis-indole alkaloid (vincristine) is dissolved in the minimum volume (1 - 2 ml) of a borate buffer, pH 6 (0,1 M Na-borate solution whose pH adjusted with 1 M HCl). Added to the thus obtained solution is 1,8 ml of the above-produced acidic solution and the mixture is left overnight at room temperature in a shaker. The end product is separated from reagents by dialysing against water (gel filtration Sephadex G - 25 M). Dialysate is cold dried. The formation of a final conjugate can be determined by analysing the product solution spectrophotometrically on a wavelength suitable for the chromophors of bis- indole alkaloids. A typical yield is approx. 70 %.

In the example, the bis-indole alkaloid was vincristine but the method can be effected also with other alkaloids mentioned in the specification, such as vinblastine, vindesine or vinzolidine.

Example 2

Conjugation of bis-indole alkaloid to protein

1 mg of aminobenzoate is dissolved in 0,5 ml of 0,2 M HCl to which is dropwise added 1 mg of sodium nitrite dissolved in 0,5 ml of water. The obtained solution is kept in ice bath for 45 minutes with occasional slow stirring. Added dropwise to this solution is 1 mg of sulphamine acid dissolved in 0,5 ml of water and 3 mg of vincristine is dissolved in the minimum volume of the mixture of N-N-dimethylformamide and a borate buffer (1 : 1) (borate buffer: pH of 0,1 M Na-borate is adjusted in the range of 6,5 - 7,5 with 0,1 M boric acid solution).

The above prepared acid solution is added dropwise to a vincristine solution. During the addition, pH is monitored and kept at approx. 7 by means of 0,1 M Na-borate (forming yellow colour). Reaction is allowed to proceed for 3 to 4 hours in darkness followed by adding 3 mg of protein to the solution (solution pH approx. 6). 1-ethyl- 3-(3-dimethylaminopropyl)-carbodeimide (4 mg) dissolved in the minimum volume of water is added to an enzyme solution and reaction is allowed to proceed overnight at +4°C. The end product is separated from reagents by ultrafiltration at +4ºC against a suitable buffer (e.g. a phosphate-buffered physiological salt solution). The fact that just one single point is subjected to diazo coupling can be determined by running a HPLC analysis on the product of vincristine diazo coupling, A typical yield from this stage is approx. 90 %.

Claims

1. A protein conjugate of bis-indole alkaloids, c h a r a c t e r i z e d in that formula (I) of the conjugate is

Ar-N=N-bisindole alkaloid (I)

wherein Ar is a benzoe- or arylalkylcarboxylic acid group which contains a protein group.

2. A protein conjugate of bis-indole alkaloids as set forth in claim 1, c h a r a c t e r i z e d in that in formula (I) the indole alkaloids have a general formula (II)

(II)

wherein

A represents a lower C₁-C₅ alkylene group and

B represents a lower C₁-C₅ alkylene or alkylenyl group which can be substituted with a hydroxy group and/or with a lower alkyl group and

Ar represents a group having a formula

T-NH-CO-(CH₂)_n wherein n is 0 - 5, which alkylene chain can be substituted and T represents a protein group, such as glucoprotein, immunoglucoprotein or enzyme.

3. A protein conjugate of bis-indole alkaloids as set forth in claim 1 or 2, c h a r a c t e r i z e d in that formula (III) of the bis-indole is

(III)

in which formula

D is - or -

R₁ is -CH₃ or -CHO,

R₂ is -OCOCH₃ or -OH,

R₃ is -OH,

R₄ is -OCH₃, -NH₂ or and R₄ form together a chain

R₃ l

- O - C = N - CH₂CH₂Cl ''

4. A protein conjugate of bis-indole alkaloids as set forth in any of the preceding claims, c h a r a c t e r i z e d in that the protein.group containing acid group is in 5-position of the indole ring of a second alkaloid group.

5. A process for the preparation of a protein conjugate of bis-indole alkaloids whose formula (I) is

Ar-N=N-bisindole alkaloid

wherein Ar is a protein group-containing benzoe- or arylalkylcarboxylic acid group, c h a r a c t e r i z e d in that a bis-indole, whose formula (III) is

(III)

in which formula 2

D is - C - CH₂ - or -

R₁ is -CH₃ or -CHO, R₂ is -OCOCH₃ or -OH,

R₃ is -OH, R₄ is -OCH₃, -NH₂ or

R₃ and R₄ form together a chain - O - C = N - CH₂CH₂Cl

is reacted with a diazonium acid derivative having a formula

Ar-N=N⁽⁺⁾,

wherein Ar represents the same as above with the exception, however, that Ar may not contain a protein group in which case a protein group is attached to an aromatic acid group.

6. A process as set forth in claim 5, c h a r a c t e r i zre d in that bis-indole is reacted with a diazo acid whose formula IV is

-N=N⁺-AR

wherein Ar represents the same as above but does not contain a protein group, whereafter a protein group is attached to the resulting acid by means of an electrophile substitution reaction, or an aromatic amino acid,

NH₂-Ar-COOH

wherein Ar is the same as above, is diazotized and reacted with bis-indole.

7. A process as set forth in claim 5 or 6, c h a r a c t e r i z e d in that the process is performed in acidic conditions, preferably in slightly acidic conditions, most preferably within the pH range of 6 to 6, 9.

8. A bis-indole alkaloid derivative, c h a r a c t e r i z e d in that its formula (V) is

Ar₁-N=N-bis-indole alkaloid (V)

wherein Ar₁ represents a benzoe- or arylalkylcarboxylic acid group.

9. The application of a bis-indole alkaloid derivative, whose formula (V) is

Ar₁-N=N-bis-indole alkaloid (V)

wherein Ar₁ represents a benzoe- or arylalkylcarboxylic acid group, in the preparation of a protein conjugate of bis-indole alkaloids set forth in claim 1.

10. The application of a bis-indole alkaloid derivative, whose formula (I) is

Ar-N=N-bis-indole alkaloid

wherein Ar represents a protein group containing benzoe- or arylalkylcarboxylic acid group, in an immunometric assay method.

11. A protein conjugate as set forth in claim 1 , wherein the protein group is a protein specifically binding to the surface structures of an animal or human cell.

12. A conjugate as set forth in claim 11, wherein the protein group is an antibody produced against individual surface structures of an animal or human cell.

13. A conjugate as set forth in claim 12, wherein the antibody is monoclonal and produced by hybridoma technique.

14. A conjugate as set forth in claim 12 and 13, wherein the surface structure, against which the antibody is produced, is typical or specific to cancer cells.

15. A conjugate as set forth in claim 11, wherein the protein group is transferrine.

16. A method for the preparation of conjugates as set fortfh in claims 11 - 15.

17. The application of conjugates as set forth in claims 11 - 15 in the treatment of a disease.

18. The application of conjugates as set forth in claims 11 - 15 in the treatment of cancer.