GB2098219A - Daunorubicin-protein conjugates - Google Patents

Abstract

Daunorubicin-protein conjugates in which the daunorubicin is bound to the protein through 9-acetyl side-chain of the daunorubicin, rather than through the 3'- primary amino group are provided. They may be prepared by reaction of 14-bromo-daunorubicin with an excess of a protein in aqueous- methanolic solution at ambient temperature for 2 to 4 hours. Neutrality is maintained by addition of dilute sodium hydroxide as necessary.

Description

SPECIFICATION Daunorubicin-protein conjugates DESCRIPTION The invention relates to the preparation of daunorubicin-protein conjugates useful as antitumoural agents.

It is common knowledge that the major problem in cancer chemotherapy still remains the lack of selectivity of the available drugs. For increasing the selective toxicity of antitumour drugs for the tumour cells, and consequently their therapeutic effectiveness, linkage or association of those drugs to a carrier macromolecule has been reported (A. Trouet, Europ. J. Cancer 14: 105-111, 1978; M.Szerkerke and J.S. Driscoll, ibid, 13: 529-537, 1977). Anthracycline antibiotics have been used in the immunochemical approach in the attempt to increase the selectivity of those cytoxic agents, but the use of antibodies as specific carriers has been limited by difficulties to obtaining antitumour antibodies.Moreover all reported procedures for linking daunorubicin covalently to antitumour antibodies or also to less specific proteins, involve the oxidation or modification of the primary amino group of the sugar moiety of the antibiotic. Since the presence and stereochemistry of the unsubstituted amino function in the antibiotic molecule is a critical determinant of both DNA binding and biological activity, the reported methods produce generally less potent drugs as a consequence of loss of basic character and electrostatic binding component in the drug.

To avoid this disadvantage, we have found and developed an alternative procedure for the preparation of daunorubicin-protein conjugates in which 14-bromo-daunorubicin (Belgian Patent No. 731,398) is used to bind the drug, via ita acetyl side-chain, to various proteins.

The invention provides a process for the preparation of a daunorubicin-protein conjugate, the process comprising reacting 1 4-bromo-daunorubicin with an excess of a protein in aqueousmethanolic solution at ambient temperature for a period of from 2 to 4 hours while keeping the solution at neutrality by addition of dilute sodium hydroxide. A pure solution of daunorubicinprotein conjugate may be obtained from the reaction mixture by removing any preciptate, for example by centrifugation, and purifying the supernatant liquid. The purification may be effected by passage over an ion-exchange or adsorption resin, the former when negatively charged proteins are used and the latter when positively charged proteins are used.

The resultant daunorubicin-protein conjugates, in which the utilization of the primary amino group of the antibiotic, essential for its cytoxic activity, is still retained, show a cytotoxic selectivity either in "vitro" by inhibition of the colony forming ability of Hela cells or in "vivo" against Ehrlich ascites tumour.

Since its assumed that the 1 4-bromo derivative of daunorubicin reacts with the free amino groups or with the caaboxylic groups of the involved proteins, a molar excess of protein (with respect to the amino acids residues) has been used for the preparation of the new daunorubicinprotein conjugates as reported in the following Example which illustrates the invention.

EXAMPLE: 20 mg of protein were dissolved in 2 ml of water and the pH of the solution was adjusted to 8.5 by adding a dilute aqeuous solution of sodium hydroxide. 2 mg of 14-bromo-daunorubicin (freshly dissolved in 0.1 ml of methanol) were then added and the mixture was left under stirring, at room temperature, for 120-240 minutes while contemporaneously adjusting the pH of the solution to neutrality by a cautious addition of a dilute aqueous solution of sodium hydroxide to neutralize the hyrobromic acid during the reaction. When the reaction was over (pH = 7), the mixture was centrifuged at 5000 rpm for 1 5 minutes and the clear supernatant, in the case of negatively charged proteins, was passed through an ion-exchange column filled with Dowex 5 OW-X2 resin (Trade Mark) to remove any free antibiotic.When positively charged proteins were used, for avoiding the binding of the protein to the ion-exchange resin, the eventual free drug was removed by adsorption chromatography on Amberlite XAD-2 resin (Trade Mark). The resultant pure solutions of daunorubicin-protein conjugates (no free drug detected by HPLC) were used as such for the evaluation of their biological activity.

BIOLOGICAL ACTIVITY The inhibition of the colony forming ability of HeLa cells was used to quantitate activity of the drug linked to various proteins. In general, the activity of the conjugates was lower than that of the free daunorubicin, since typically four times (or more) higher concentrations of conjugates (expressed as daunorubicin content) than that of free antibiotic are required to produce 50% inhibition. This reduction is expected since the limited cellular uptake of protein macromolecules reduce intracellular drug accumulation.

However the effect of the various carrier proteins on the cytotoxic activity of daunomycin is different. Although the actual mechanism of antitumour activity of the macromolecular derivative of cytotoxic drug remains to be established, it is clear that the use of some proteins offered a consistent advantage in most cases. In the case of daunorubicin-proteins conjugates, if the cytotoxicity is correlated to the drug content, a considerable variation in the inhibitory potency of the conjugates (Table I) suggests some protein specific effects.

Daunorubicin linked to casein, ribonuclease A, asialo fetuin, immunoglobulin and concanavalin A is appreciably more active than drug linked to bovine serum albumin, fetuin, lysozyme, histones. The cytoxic effect cannot be attributed to the protein component of the conjugates since unmodified proteins when tested in the absence of daunorubicin, did not show any cytoxic effect at similar or higher concentration (up to 201lg/ml.

Daunorubicin bound to immunogloblin retained a relevant activity.

Table 1 BIOLOGICAL ACTIVITY OF DAUNORUBICIN-PROTEIN CONOUGATES Daunorubicin-Protein Drug/protein Drug/proein Concentration required for 50% inhibition of Conjugate weight ratio molar ratio colony-forming abiliy of HeLa cells.

Concentrations are expressed as daunorubicin content in the conujugates.

Daunosrubicin (Da) 6.5(4-12.5) Da-CASEIN 1:38 1.21 35 Da-RIBONUCLEASE A 1:26 0.93 37.5(25-50) Da-ASIALO FETUIN 1:17 5.2 42,5(35-50) Da-IMMUNOGLOBULIN 1:28 9.5 50(40-60) Da-CONCANAVALIN A 1:27 1.77 71(40-115) Da-FETUIN 1:26 > 117 Da-BSA 1:28 4.18 100 (70-130) Da-LYSOZYME 1:34 0.75 105(100-110) Da-HISTONE 1:30 0.95 175(150-200) HeLa cells were exposed to drug for 8 h. The drugs were removed and cells were diluted and plated into 50 mm tissue culture dishes. Colonies were countes after 8 days of incubation. Data were deduced from dose-response curves in experiments performed at 4 different concentration leveis (5 replicate dishes per concentration). The dats are presented as mean of value deduced from at least 3 replicate experiments (in brackets, range of individual experiments).

In vivo antitumour studies CDL mice (22-28 g) were inoculated intraperitoneally with Ehrlich ascites tumour cells, 5x106 cells per animal) 24 hours prior to treatment with a single dose. Under these conditions, the tumour is lethal to 100% of the untreated mice in 16-18 days. Each experimental group consisted of at least ten mice. Preliminary results obtained with the daunorubicin-immunoglobulin (bovine, non specific) conjugate indicated that the protein-bound drug retained, at least in part , its activity at higher doses. However, the reduction of potency is associated with an appreciable reduction of toxicity. (Table ill). Daunorubicin linked to the synthetic polypeptide, poly-l-lysine, showed a reduction of antitumour potency, as in the case of the daunorubicinimmunoglobulin conjugate.However, the daunorubicin-poly-l-lysine conjugate was as active (or more active) as the free drug at the maximal dose tested (16 mg/kg). Again the protein-bound drug was less toxic than free drug. The antitumour activity could not be due to the protein component of the conjugate, since the ummodified protein, at the same dose was completely inactive.

ACTIVITY of danorubicin-protein conjugates on Ehrlich ascites tumour Table II Compound Dosea MSTb Daunorubicin (DA) 4 1 88 5 197 6 138 10.5 75 Da-immunoglobulinc 10.5 143 14 153 Immunoglobulind - 100 Da-poly-i-lysineC 1 2 1 66 16 222 Poly-l-lysined 100 aTreatment i.p. on day 1 (mg/kg of body weight) bMedian survival time expressed as percent of untreated control.

CDose is expressed as daunorubicin content in the conjugate dProteins were tested at the same dose as the daunorubicin-protein conjugate.

Claims (5)

1. A daunorubicin-protein conjugate in which the daunorubicin is bound through its 9-acetyl side-chain to the protein.

2. A daunorubicin-protein conjugate according to claim 1 in which the protein is casein, ribonuclease A, asialo fetuin, immunoglubulin, concamavlain A, fetuin, bovine serum albumin, lysoxyme, histone or poly-Iysine.

3. A process for the preparation of a daunorubicin-protein conjugate, the process comprising reacting 14-bromo-daunorubicin with an excess of a protein in aqueous-methanolic solution at ambient temperature for a period of from 2 to 4 hours while keeping the solution at neutrality by addition of dilute sodium hydroxide.

4. A process according to claim 3 further comprising removing any precipitate and purifying the supernatant liquid by passing it over an ion-exchange or adsorption resin, the former when negatively charged proteins are used and the latter when positively charged proteins are used.

5. A process for the preparation of a daunorubicin-protein conjugate, the process being substantially as described herein with reference to the Example.