CONJUGATE OF 9-(2-HYDROXYETHOXYMETHYL)-GUANINE WITH LACTOSAMINATED HUMAN ALBUMIN, PROCESS FOR THE PEPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING IT

Abstract: The conjugate of 9-(2-hydroxyethoxymethyl)-guanine with lactosaminated human albumin is therapeutically more efficacious than the free drug in the treatment of chronical hepatitis induced from Virus B. For the preparation of the conjugate an aqueous solution of 9-(2-hydroxyethoxyme­ thyl)-guanine in form of a derivative, particularly mono­ phosphate, and an aqueous solution of lactosaminated hu­ man albumin are reacted preferably in the presence of 1-­ ethyl-3-(dimethylaminopropyl)-carbodiimide, by adjusting the pH to the value of 7.5 and by maintaining the reaction mixture for 24 hours under stirring and in the dark.

Description: The present invention relates to a novel compound, namely the conjugation product of 9- (2-hydroxyethoxymethyl)-guanine with lactosaminated human albumin, useful in the therapeutical treatment of chronic hepatitis B. 9-(2-hydroxyethoxymethyl)-guanine, also known as acyclovir (ACV), having the formula: is an anti-viral drug of widespread use in the treatment of herpetic infections (Dolin L., Science 227, 1296-1303 (1985). Recently it has also been used in the treatment of chronic hepatitis B with good results (Weller I.V.D. et al., Lancet i, 273, (1982), and Weller I.V.D. et al, J. Antimicr. Chem. 11, 223-231, (1983)), mainly in association with interferon (Schalm S.W. et al, Lancet ii, 358-360, (1985)). However ACV, at the dosages at which the reproduction of the virus of hepatitis B (HBV) is inhibited, (5-15 mg/kg), may cause renal lesions (Weller I.V.D. et al, J. Antimicr. Chem. 11, 223-231, (1983); Balfour H.H., Ann. Rev. Med. 35, 279-291, 1984) and alterations of the central nervous system (Balfour, ibidem.). It has now been found and is the object of the present invention that these problems and drawbacks are substantially and advantageously solved by means of the conjugation product of acyclovir with lacrosaminated human albumin (L-SA) with a bridging unit selected from one of monophosphate, glutarate and succinate. Lactosaminated human albumin is a neoglycoprotein which selectively penetrates the hepatocytes after interaction with a receptor specific for the proteins ending with galactose present only on the parenchimal cells of liver (Wilson G., J. Biol. Chem. 253, 2070, (1978)). It has been sucessfully used in the mouse as the hepatotropic vehicle (carrier) of adenine arabinoside monophosphate (ARA-AMP) (Fiume L. et al, FBS Lett. 129, 261-264 (1981); Fiume L. et al., Gut 25, 1392-1398, (1984)). On the basis of the tests carried out it has been in fact found that the dose of conjugated acyclovir to be administered in order to inhibit the synthesis of viral DNA in the liver is about 6% of that of free acyclovir capable of achieving the same inhibition degree. In other words, the conjugate according to the present invention seems to be capable of increasing the chemotherapeutical index of ACV in the treatment of chronic hepatitis B with a substantial reduction or elimination of the aforesaid side-effects, intolerable or highly disturbing for the patient. Also an object of the present invention is the process for the preparation of the conjugate, comprising the use of a derivative of acyclovir, preferably selected from one of monophosphate succinate and glutarate, and the bonding of the derivative to the lactosaminated human albumin by means of carbodiimides or, as an alternative (in the case of succinate and glutarate), by means of their hydroxysuccinimidic ester, or by the mixed anhydride method. According to a preferred embodiment of the process of the invention, the monosphosphate derivative of acyclovir is bonded to the lactosaminated albumin by means of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (ECDI), the reaction being carried out in aqueous solution at a pH higher than 6.5. Regarding the lactosaminated albumin and the formation of conjugates therefrom further details can be seen from e.g. EP-A-184838. By the term "lactosaminated human albumin" a human albumin is meant those in which at least 20, more preferably 30, of the 59 lysine residues existing in that albumin are linked to galactose molecules through the glucose moiety. More particularly it has been found that the desired results are obtained by operating at pH 7.5 at 25°C and by maintaining the reaction mixture in the dark for 24 hours under stirring. Instead of ECDI other carbodiimides can be used, such as 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)-carbodiimide. ACV monophosphate (ACV-MP) was obtained by adding phosphorous oxychloride to a cooled suspension (-2°C) of ACV in triethylphospate (Yoshikawa M. et al, Tetrahedon Letters 50, 5065-5068, (1967) and Le Page G.A. et al, Cancer Res. 35, 3036-3040, 1975)). It was purified by chromatography through a column of DOWEX®-1 formiate eluted with a continuous gradient of formic acid (O to 4M). Lactosaminated human albumin with 30 sugar residues (L₃₀-HSA) was obtained by reductive amination (Schwartz B-A. et al.,Arch. Biol. Chem. Biophys. 181, 542-549, (1977)). ACV-MP was conjugated with L₃₀-HSA by the following process: 1 g (3.27 mmoles) of ACV-MP and 1 g (12.6 µmoles) of L₃₀-HSA were dissolved in 20 ml H₂O. The pH was adjusted to 7.5 with 10N NaOH and 1 g (5.21 mmoles) ECDI was added. After 24 hours' incubation at 25°C under stirring and in the dark, the reaction mixture was dialyzed against 0.9% NaCl to remove the unbonded ACV-MP, the ECDI and its ureic derivative. When the dialysate no longer showed an absorption at 255 nm the conjugate was collected and concentrated to 500 mg/10ml in minicon B-15 (Amicon) cells. 1 ml fractions (containing 50 mg conjugate and 9 mg NaCl) are lyophilized. At the time of use, a fraction is dissolved in 1 ml H₂O and, if necessary, further diluted with 0.9% NaCl. The molar ratio ACV /L₃₀-HSA is determined by calculating the concentration of ACV at 255 nm (E 1% = 528.6), after having subtracted the albumin absorption at that wavelength (E 1% = 3.43). The albumin concentration is measured by the method of Lowry et al (J. Biol. Chem. 193, 265-275,1951). The molar ratio ACV/L₃₀-HSA in several conjugate preparations has been found to vary between 10 and 12:1. Without imposing undue limitations on the present invention, it seems plausible that the advantageous results achieved by the present invention are to be attributed to the lysosomatropic approach of the anti-viral chemotherapy (Balboni P.G. et al, Nature, 264, 181-183 (1976) and Fiume L. et al., FEBS Lett. 153, 6-10 (1983)). The conjugation of ACP-MP with L₃₀-HSA does not reduce, but on the contrary, for reasons presently unknown, increases the capacity of this carrier to interact with the receptor present on the surface of hepatocytes. As a matter of fact, as shown in the following table 1, the disappearance from mouse blood of [¹⁴C] marked asialofetuine (AF) is inhibited by the conjugate at a significant higher rate than in the case of an equal amount of non-conjugated L₃₀-HSA. (Fetuine is a glycoprotein of the bovine phoetal serum which, after removal of the sialic acid, penetrates selectively into the hepatocytes after having interacted with the receptor for proteins terminating with galactose residues). Fetuine was enzymatically desialated (Morrell A.G. et al, J. Biol. Chem. 241, 3745-3749 (1966)). Then it was marked with [¹⁴C] formaldehyde according to Cox. R.A. and Greenwell P., Biochem. J. 186, 861-872 (1980). Swiss female mice of 28-30 g were treated by the intravenous route with 2 µg/g of [¹⁴C] AF (4.9 x 10⁶ dpm/mg). L₃₀-HSA and the conjugate were intravenously administered at the dose of 2 µg/g simultaneously with [¹⁴C] AF. In all cases the injected volume was 10 µl/g. After 10 minutes blood samples were taken from the retroorbital plexus of the animals under ether-induced anaesthesia and the radioactivity of the plasma was measured. Each value represents the average (± standard error) of the results obtained in 5 animals. The difference between the results of animals treated with L₃₀-HSA and those of the animals administered with the conjugate, as evaluated by the Student t values, is significant (P <0.02). (For details of the Student t test see "Statistical Methods in Medical Research", 2nd Ed., Blackwell Scientific Publications, Oxford, 1987, p.98-100 and 186-192.) Table 2 shows the effect of ACV, ACV-MP and of the conjugate L₃₀-HSA -ACV-MP₁₀ on the incorporation of thymidine in the DNA of liver, intestine and medulla ossium of mice affected by hepatitis induced by Ectromelia virus. In these animals the incorporation of thymidine in the liver is caused by the synthesis of viral DNA (Fiume L. et al, FEBS Lett. 129-261-264, XX (1981)). The lowest dose of ACV causing a significant inhibition of the DNA synthesis in the liver was 10 µg/g, corresponding to that used in patients affected by chronic hepatitis B. At that dose, ACV also inhibits DNA synthesis in the intestine to a significant extent. ACV-MP in free form was not more active than ACV. ACV-MP conjugated with L₃₀-HSA, on the contrary, caused the inhibition of DNA synthesis in the liver at a dose of 0.6 µg/g only. Moreover no significant inhibition in the intestine and medulla ossium was induced. The experimental work has been carried out as described by Fiume L. et al, GUT 25, 1392-1398, (1984). The results (dpm/mg DNA) have been statistically evaluated by means of the Student t test and considered either significant (S) or not significant (NS) for P<0.05 or P>0.05, respectively. These observed data indicate that, after administration of the conjugate, ACV is concentrated in the liver wherein it is released in a pharmacologically active form. It seems, thus, experimentally confirmed that the conjugate of ACV-MP with lactosaminated human albumin may increase the therapeutical index of ACV in the treatment of chronic hepatitis B, both if administered alone and if administered in combination with interferon. Conjugates of ACV with lactosaminated albumin can also be obtained according to processes different from that described herein. ACV may be converted, by means of glutaric anhydride, to ACV-glutarate, or by means of the succinic anhydride to the ACV-succinate. Subsequently these derivatives can be bonded to the L-SA either through their hydroxysuccinimidic ester, or through the mixed anhydride method or by using carbodiimides. These processes have been already used for the bonding of ACV to bovine and rabbit albumins and to human immunoglobulins (Quinn, R.P. et al.,Analyt. Biochem. 98 , 319-328 (1979)). The resulting derivatives have been used as immunogens to obtain antibodies retaining ACV which are used for the radioimmunological dosing of this drug. By one of these processes, another anti-viral nucleoside, viz. adenine arabinoside (Ara-A), has been converted into ara-A-glutarate which was then bonded to the asialofetuin through its hydroxysuccinimidic ester. A conjugate ara-A-glut-AF has been obtained which proved to be pharmacologically active in inducing hepatic targeting of ara-A in the mouse (Fiume et al., FEBS Lett. 116 , 185-188 (1980)).

Claims: Conjugate of 9-(2-hydroxyethoxymethyl)-guanine with lactosaminated human albumin by means of a bridging unit selected from one of monophosphate, succinate and glutarate. Conjugate according to claim 1, characterized in that the molar ratio of 9-(2-hydroxyethoxymethyl)-guanine to lactosaminated albumin is between 10:1 and 12:1. A process for the preparation of the conjugate according to one or both of claims 1-2, characterized in that aqueous solutions of a derivative of 9-(2-hydroxyethoxymethyl)-guanine and of lactosaminated human albumin are reacted under stirring and in the dark. A process according to claim 3, characterized in that said derivative of 9-(2-hydroxyethoxymethyl)-guanine is selected from one of monophosphate, glutarate and succinate. A process according to claim 3, characterized in that said derivative of 9-(2-hydroxyethoxymethyl)-guanine is the monophosphate and the reaction with lactosaminated human albumin is carried out in the presence of 1-ethyl-3-(diethylaminopropyl)-carbodiimide at a pH higher than 6.5, at 25°C, in the dark and under stirring, for 24 hours. Pharmaceutical composition, characterized by containing, as the active ingredient, the conjugate product of claims 1 or 2 in combination with the usual excipients and vehicles. Pharmaceutical composition according to claim 6, useful for the therapy of chronic hepatitis B.

Inventors: BALDACCI, MASSIMO, BUSI, CORRADO, FIUME, LUIGI, MATTIOLI, ALESSANDRO
Applicants: LABORATORI BALDACCI SPA
Application year: 1986
Main classification: A61K47
IPC codes: A61K47, A61K38