FI80703B - Protein conjugates of bis-indole alkaloids, process for the preparation and use thereof - Google Patents

Description

1 80703

Protein conjugates of bis-indole alkaloids, their preparation and use - Bis-indole alkaloids protein conjugates, deram framställning och använding

The invention relates to conjugates of bis-indole alkaloids, their preparation and their use in immunometric assay methods.

Certain bis-indole alkaloids, some of which are natural molecules derived from plants, some of which are synthetically derived, have been shown to be suitable for the medical destruction of cancer cells. cytostatics. Concentrations of such substances are important both for their development work and, in particular, for the optimization of treatment. Sensitive analytical methods are also useful in the production processes of these compounds.

Such bis-indole alkaloids or synthetic derivatives thereof can be described by the following general formula; catharanthine unit jfy-S V ·· '' —f— Il I J · 'vindolun unit * (- / R1 K * C0R4 2 80703 D R ^ R2 R3 R4

OH

\ / 1 vinblastine C -CH3 -OCOCH3 -OH -OCH3 I Et CHo / * 2 vincristine -CHO -OCOCH3 -OH -OCH3 3 vindesine - "- -CH3 -OH -OH -NH2 4 vincolidine -" - -CH3 -OCOCH3 | j ° Y "n 0 ch2ch2ci

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

Ar-N = N-bisindole alkaloid (I) wherein the general formula (II) of the indole alkaloids is r--: ': 3 R1 S * COR4 3 80703 wherein OH CH2CH3 CH2CH3 D is -C - CH2 - or - C = CH-,

R 1 is -CH 3 or -CHO, R 2 is -OCOCH 3 or -OH, R 3 is -OH, R 4 is -OCH 3, -NH 2 or R 3 and R 4 together form a chain -O-C-N-CH 2 CH 2 Cl

O

Ar represents a group of the formula jSS-T-NH-CO- (CH2) n ^ Y ^ / wherein n is 0 to 5, which alkylene chain may be substituted and T represents a protein group such as a glycoprotein, an immunoglucoprotein or an enzyme.

For compounds of the above type, vincristine, vinblastine and vindesine, currently in clinical use, a sensitive and specific serial analytical method is not known to be commercially available. A key difference from previously published immunoanalytical methods for these compounds (Ria, Irma, Elisa, etc.) is the high specificity of the antibodies produced on the basis of the method now developed. This is based on the fact that the method of preparing the protein conjugate described here leaves the second, currently understood, therapeutically active part of the subject bis-indole alkaloid free and thus functional in the interaction process underlying the method of analysis.

The high specificity achieved is useful in the analysis, for example, so that any drug metabolites or by-products in production do not interfere with the assay.

. 80703 4

Bis-indole alkaloids consist of vindoline and catharanthine units, of which the indole part of the catharanthine unit allows the attachment of a protein group. to an aromatic spruce ring using an electrophilic aromatic substitution reaction. By protein group is generally meant a protein, a glycoprotein, e.g. an enzyme, albumin, human or animal serum albumin, immunoglobulin or the like. Because protein chemistry reactions occur in aqueous solution, it is appropriate herein to use diazoconversion with a diazonium salt to which the protein can be covalently attached in the formation of the final immunogen as either a pre- or post-diazoconversion reaction step.

The vindoline and catharanthine units under consideration each have an aromatic spruce ring for diazo coupling. In the vindoline moiety, the most active site of the ring is not free for this purpose because the catharanthine unit is attached to it precisely by a covalent bond. In addition, in vindoline, the second most reactive carbon atom of the ring is in a sterically narrow region for diazo coupling. The aromatic spruce ring of the catharanthine unit has in principle 4 carbon atoms available for diazo coupling. By performing the coupling on the principle of diazo coupling of aromatic amines, i.e. in such a way that the indole nitrogen atom is allowed to activate the aromatic ring, it can be reasonably expected that the coupling will take place. to the p-position relative to the nitrogen atom, which is the most reactive point on the ring for coupling, and is also one of the sterically best available positions on the ring. Thus, it is possible to obtain a fairly uniform reaction product: 5 80703

R R

ΐ I I

NH NH NH

(^)) + ArNp -> (j ^) -> [Qj + H © H N = N-Ar N = N-Ar

A difficulty in the reaction is the tendency of the diazonine ion to react with water molecules. If diazo coupling is slow due to unfavorable reaction conditions, the latter side reaction, phenol formation, becomes the main reaction. In this case, the the phenolic compound may be diazo-coupled to form, in addition to the desired diazo compound, the contamination compound produced by that side reaction.

Side reactions can be most effectively avoided by adjusting the acidity (pH) of the reaction solution as closely as possible to the optimum value of the synthetic reaction in question.

The reaction conditions in which the alkaloid structure described above is diazo-coupled as quickly as possible while keeping the proportion of side reactions as small as possible require adjustment of the acidity of the reaction solution. The solution must also not be so basic as to reduce the concentration of diazonium ion too much, but the solution must also not be so acidic that the content of the non-protonated amine (indole part of the catharanthine unit) remains too low. In the present diazo coupling reaction, the pH of the reaction solution must be adjusted to a range of pH 6.60 to pH 6.90 to form a sufficiently uniform immunogen suitable for the ELISA assay method that specifically measures bis-indole alkaloids.

The antibody is prepared in a known manner, e.g., 6 80703 L. Hudson & F.C. Hay, Practical Immunology, Blackwell Scientific Publications, Oxford 1976, and R.H. Kennett, K.B. Bechtol & T.J. McKearn (ed.), Monoclonal Antibodies and Functional Cell Lines, Plenum, New York 1984.

The antibody can be produced by the immune system of the test animal by immunizing the animal using the above conjugate as an immunogen and collecting the antibody from the circulation. Alternatively, a so-called monoclonal antibodies, either in vivo or in vitro, by isolating antibody-producing cells from the body of the experimental animal either after or before the immunization treatment and by further developing production cells. If necessary, the antibody can be purified using conventional methods suitable for the purpose.

The analysis is based on the binding reaction between the above antibody and the bis-indole alkaloid to be analyzed. The amount of analyte to be bound is determined to compete with the binding tracer. The label may be a radiolabeled corresponding commercial compound or a corresponding compound conjugated to a measurable enzyme. Radioactivity and enzyme activity are measurable by conventional means, e.g. equipment to be marked for these purposes. The principle is described in Voller et al. Bull. Wild. Hlth. Org. (1979) 53, 55-56.

The functionality of the method has been tested in virtually all respects for vincristine using both a radioisotope and an enzyme as a marker. The radioimmunometric method (RIA) works with a tritiated tracer in a concentration range of 0.1 to 50 ng, is highly specific for the target substance and meets generally accepted criteria for the reproducibility of similar methods. The method has been found to be useful in the determination of plasma 7 80703 vincristine levels. The enzyme-linked immunosorbent assay (EIA) has similar properties to the previous one, but is more sensitive (measuring range approx. 10 to 500 pg) and is suitable not only for plasma but also for whole blood and blood cell material assay in humans.

Implementation of the RIA analysis series takes about 2 days in the summer, during which one person can perform about 200 analyzes. Correspondingly, the duration of the EIA series is about 8 hours, including about 500 samples. The speed and capacity of both assays can be significantly improved if they are developed into so-called an analysis kit in which the final laboratory work steps are minimized and conditions are carefully optimized.

The following examples illustrate the invention without limiting it.

Example 1

Preparation of bis-indole alkaloid albumin conjugate 50 mg of DL-β-aminophenylalanine are dissolved in 5 ml of water. To this solution are added 50 mg of bovine serum albumin and 50 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDO) and the mixture is incubated overnight at room temperature.

The reaction product is separated from the reagents by dialysis (ultrafiltration) at + 4 ° C against water. The pH of the solution is adjusted to 1.5 with hydrochloric acid in an ice bath. 100 mg of NaNO2 are dissolved in 1 ml of water and added dropwise to a hydrochloric acid solution, to which is further added 50 mg of ammonium amidosulfonate dissolved in 1 ml of water. 15 mg of bis-indole alkaloid (vincristine) are dissolved in a minimum volume (1-2 ml) of borate buffer, pH 6 (0.1 M Na-borate solution, pH adjusted with 1 M HCl). To the solution thus obtained is added 1.8 ml of the 880703 acidic solution prepared above, and the mixture is allowed to stand at room temperature on a shaker overnight. The final product is separated from the reagents by dialysis against water (gel filtration Spehadex 6-25 M). The dialysate is lyophilized for use in immunizations.

The formation of the final conjugate can be detected by spectrophotometric analysis of the product solution at the appropriate wavelength for the chromophores of the bis-indole alkaloids. The typical yield is about 70%.

In the example, vincristine has been used as the bis-indole alkaloid, but the process can also be carried out with other alkaloids mentioned in the specification, such as vinbalstin, vindesine or vincolidine.

Example 2

Conjugation of vincristine to alkaline phosphatase 1 mg of β-aminobenzoate is dissolved in 0.5 ml of 0.2 M HCl, to which 1 mg of sodium nitrite dissolved in 0.5 ml of water is added dropwise. The resulting mixture is kept in an ice bath for 45 minutes, stirring gently from time to time. To this mixture is added dropwise 1 mg of sulfamic acid dissolved in 0.5 ml of water and 3 mg of vincristine is dissolved in a minimum volume of a 1: 1 mixture of N, N-dimethylformamide and borate buffer (borate buffer: 0.1 M Na-borate The pH is adjusted to 6 .5 to 7.5 with 0.1 M boric acid solution)

The acid solution prepared above is added dropwise to the vincristine solution. During the addition, the pH is monitored, which is maintained at about 7 by means of 0.1 M Na borate (a yellow color forms). The reaction is allowed to proceed for 3-4 hours at + 4 ° C in the dark, after which 3 mg of alkaline phosphatase 9 80703 (pH of the solution of about 6) are added to the solution. 1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide (4 mg) dissolved in a minimum volume of water is added to the enzyme mixture and the reaction is allowed to proceed overnight at + 4 ° C. The final product is separated from the reagents by ultrafiltration at + 4 ° C against a suitable buffer (e.g. phosphate-buffered saline).

The targeting of the diazo coupling to a single site can be determined by running an HPLC analysis of the diazo coupling product of vincristine. The typical yield from this step is about 90%. By the above method, immunogens can also be prepared from at least vincristine, vinblastine and vindesine using, for example, albumin instead of alkaline phosphatase.

Example 3

Antibody preparation

Lyophilized immunogens were emulsified in Freunds adjuvant and injected into four New Zealand albino rabbits. The VCR-albumin ratio in the immunogens was: immunogen 1 = 245: 1; immunogen 2 = 22: 1 and immunogen 3 = 7.5: 1. Rabbits 1, 2 and 3 were given immunogen 1, 2 and 3 and rabbit 4 immunogen 2. Table 1 shows the immunization schedule and amounts. One week after the last injection, blood samples were taken and antibody production was detected by measuring [3 H] VCR binding in serum samples as described in Lehtola, T .; Huhtikangas, A. and Virtainen, R.,

Application of radioimmunoassay to the quantitation of hyos-cyamine in plant material. Planta Medica, 1982; 45: 237-239. Inununogolbulin G (IgG) was purified from antiserum using DEAE-cellulose. Hudson, L and Hay, F.C., Practical Immunology, London: Blackwell Scientific Publications, 1976.

10 80703

Table I

Immunization of rabbits for antibody production

Time of injection ___ Amount of antigens_ (weeks) _Rabbit 1_Rabbit 2_Rabbit 3_Rabbit 4 0 0.75 1.00 0.40 0.60 4 0.75 - 5 - 0.80 0.50 0.45 9 0.75 - - - 12 - 0 .25 0.25 0.30 13 0.15 - 18 0.30 - 19 - 0.30 0.30 0.20 22 0.15 0.20 0.20 0.10 27 - 0.10 31 - 0 .20 - 0.27 32 - 0.50 -

Figure 1 depicts the generation of VCR antiserum during immunization. A comparison of the results in Figure 1 and the values in Table 1 shows that higher doses of immunogen increase the antibody titer. Figure 1 shows the antibody titer in rabbits 1-4 during immunization according to Table I. The reported serum dilution corresponds to 50% binding of the [3 H] VCR marker.

Claims (8)

1. Protein conjugate of bis-indole alkaloids, characterized in that the formula (I) of Mr Ar-N = N-bisindole alkaloid (I) wherein the general formula of (II) Mr I - νΛ C-0, 11, 0CH3 LHI 1 Protein conjugate of bis-indole alkaloid according to claim 1, characterized in that the protein group containing the acid group is in 5 position in the indole ring of the alkaloid group. 3 is the formula OH CH 2 CH 3 CH 2 CH 3 D is -NC - CH 2 - or - C = CH-, R 2 is -CH 3 or -CHO, R 2 is -COCOCH 3 or -OH, R 3 is -OH, R 4 is -OCH 3 , -NH 2 to R 3 and R 4 together form the chain -O-CN-CH 2 CH 2 Cl 0 21 80703 Ar 2 represents an acid group of the formula & NH 2 -CO- (CH 2) n dir n = 0 - 5, which alkylene chain may be substituted for the preparation of protein conjugates of bis-indole alkaloids according to claim 1. Process for the preparation of bis-indole alkaloid protein conjugates of formula I, characterized in that bis-indole is reacted with a diazonium acid derivative of formula Ar-N = N (+), where Ar refers to the above-defined, as well as Ar, may be without containing a protein group, in which case the kprotein group binds to the aromatic acid group. 3 in which formula OH CH2CH3 CH2CH3 D Mr - C - CH2 - or - C = CH-, R1 Mr -CH3 or -CHO, R2 Mr -OCOCH3 or -OH, R3 Mr -OH, R4 Mr - OCH 3, -NH 2 or R 3 and R 4 together form the chain -O-CN-CH 2 CH 2 Cl 0 Ar refers to a group of the formula JOV T-NH-CO- (CH 2) ie 80703 where n Mr 0-5, which alkylene chain may be substituted and T refers to a protein group, such as glucoprotein, immunoglucoprotein or enzyme. 4. A process according to claim 3, characterized in that bis-indole is reacted with diazoic acid of formula IV , or an aromatic amino acid, NH 2 -Ar-COOH where Ar is defined above, is diazotized and reacted with bisindole. i9 80703 5. A process according to claim 3 or 4, characterized in that the process is carried out under acidic conditions, preferably under mildly acidic conditions, most preferably in the pH range 6-6.9. 6. The bis-indole alkaloid derivative, characterized in that its formula (VI is Ar 2 - N = N - bisindole alkaloid (V) dies the general formula (II) of the indole alkaloid is 0O3 3 * 1 * f ^ R4 in which formula OH CH 2 CH 3 CH 2 CH D is - C - CH 2 - or - C = CH-, R 3 is -CH 3 or -CHO, R 2 is -OCOCH 3 or -OH, R 3 is -OH, R 4 is -OCH 3, -NH 2 or R 3 and R 4 together form the chain - O-CN-CH 2 CH 2 Cl 0 80703 Ar 2 represents an acid group of the formula NH 2 -CO- (CH 2) n where n = 0 - 5, which alkylene chain may be substituted. Use of a bis-indole alkaloid derivative of formula (I) Ar 2 - N = N - bis-indole alkaloid (V) wherein the general formula (II) of the indole alkaloid is r - Use of a bis-indole alkaloid derivative of formula (I) Ar-N = N-bis-indole alkaloid (I) wherein the general formula of the indole alkaloid II Mr Od? Γΰ. in which the formula OH CH2CH3 CH2CH3 D Mr - C - CH2 - or - C = CH-, Mr -CH3 or -CHO, R2 Mr -OCOCH3 or -OH, R3 Mr -OH, R4 Mr -OCH3, -NH2 or 22 80703 R 3 and R 4 together form the chain -OCN-CH 2 CH 2 Cl 0 Ar represents a group of the formula T-NH-CO- (CH 2) n where n = 0 - 5, which alkylene chain may be substituted and T represents a protein group such as glucoprotein, immunoglucoprotein or enzyme, in the preparation of antibody for use in an immunometric assay method.