USE OF INHIBITORS OF VACUOLAR-TYPE H<+> ATPASES

Abstract: The activity of inhibitors of vacuolar-type H<+>-ATPases in the inhibition of vacuolation induced by Helicobacter pylori is disclosed. Preferred agents are the macrolide antibiotics bafilomycins A1, A2, B1, B2, C1, C2 and D, especially bafilomycin A1. The use of such compounds in the treatment of diseases, including peptic ulceration, is claimed.

Description: USE OF INHIBITORS OF VACUOLΛR-TYPE H+ ATPASES. The present invention relates to inhibitors of cellular vacuolation in pathological conditions. More particularly, the present invention relates to the use of anti-vacuolating agents in the treatment of pathology induced by infection with Helicobacter pylori . Helicobacter pylori is a Gram-negative bacterium commonly found in the human stomach. Although the majority of H. pylori infections are asymptomatic, infection by this organism is statistically linked with the incidence of peptic ulceration, non-autoimmune chronic gastritis and gastric adenocarcinoma (Bartlett, 1988, Gastroenterology, 94.: 229-232; Blaser & Brown, 1989, Advances in Internal Medicine, 3_4: 21-42; Blaser, 1990, Journal of Infectious Diseases, 161: 626-633) . H. pylori infection may lead to vacuolation of infected cells in the gastric mucosa, a process wherein vacuoles appear in the cytoplasm and expand by absorbing fluid. Vacuoles derive from a still undefined intracellular compartment whose lumen is acidic, as indicated by the vital staining with neutral red and the synergistic vacuolating effect of ammonium ions added to the medium of cultured cells or produced by a bacterial urease (International patent application WO90/04030; Cover, et al., 1991, Infect. Immun. , 5_9: 1264-1270; Segal, et al . , 1992, Infect. Immun. , 60; 1883-1889) . Cell vacuolation is known to play a major role in cell death (Figura et al , 1990, Pathogenic Mechanism of Helicobacter pylori : Production of Cytotoxin. In Helicobacter pylori , gastritis and peptic ulcer, Malfertheimer and Dischneit (Eds.), Berlin: Springer-Verlag, pp. 86-95) . The vacuolating influence of H. pylori has been ascribed to both a vacuolating cytotoxin (Cover and Blaser, 1992, JBC, 267: 10570-10575) and to a urease enzyme (Xu et al. 1990, Journal of Infectious Diseases, 161: 1302-1304) . The vacuolating toxin and urease enzymes have been shown to induce vacuolation of eukaryotic cells in vitro (see Cover and Blaser, Op. Cit. ; Cover et al . , 1991, Op. Cit.). Bafilomycins are a class of macrolide antibiotics isolated from the organism Streptomyceε griseus , which demonstrate both antifungal and antibacterial activity (Werner et al , Journal of Antibiotics, 37: 110-117) . The antibacterial activity of bafilomycins against H. pylori is known to be very poor. Bafilomycin-Al has been shown to be a specific inhibitor of vacuolar-type H + -ATPase (V-ATPase) (Bowman et al , 1988, PNAS, f$5: 7972-7976) , a proton-pumping ATPase capable of forming a proton gradient across a cell membrane. Such ATPases have been purified from the membranes of fungal and plant vacuoles, coated vesicles, chromaffin granules and the Golgi complex. The interior of such intracellular compartments is known to be acidic and it is postulated that V-ATPase is responsible for the acidification thereof (Umata et al , JBC, 1990, 265: 21940-21945). Summary of the Invention It is an object of the present invention to provide an agent effective to prevent pathological vacuolation of cells. It has surprisingly been found that inhibitors of V-ATPases are able to not only prevent such vacuolation, but also to reverse it. According to a first aspect of the present invention, therefore, there is provided the use of an inhibitor of vacuolar-type H + ATPase (V-ATPase) in the preparation of a composition for use in the treatment of a disease involving pathological cellular vacuolation. SUBSTITUTE SHEET Detailed Description of the Invention Vacuolation is a physiological process which occurs in all cells in the absence of disease. By "pathological vacuolation", it is intended to refer to all vacuolation events which manifest themselves as part of, or in connection with, a pathological condition of a cell. Such events include, but are not limited to, bacterial infection and its side-effects, exposure to cytotoxic chemicals, the side effects of drugs and neurological degeneration such as that which occurs during aging. In particular, it is intended to refer to the vacuolation of gastric tissues which is believed to be induced by Helicobacter pylori infection. Preferably, therefore, the pathological vacuolation is associated with H. pylori . It has surprisingly been found that inhibitors of V-ATPase are effective both to inhibit and reverse the vacuolation process. By "inhibitors of V-ATPase", it is intended to refer to agents which inhibit the activity of V-ATPase. This enzyme is known to be active in the transport of protons across cell membranes. The activity of this enzyme which is inhibited by V-ATPase inhibitors therefore includes, but is not limited to, the proton transport activity. A number of V-ATPase inhibitors are known (see Uchida et al, 1985, JBC, 260: 1090-1095; Nelson, 1989, J.Bioenerg. Biomem. , 2_1 : 553-571; and Stone et al , 1989, J.Bioenerg. Biomem., 2 \ 605-620). Examples of such agents include N, N'-dicyclohexylcarbodiimmide (DCCD) , 7-chloro-4- nitrobenzoxadiazole (NBD-C1) and N-ethylmaleimmide (NEM) , as well as bafilomycins, for example bafilomycins Al, A2, Bl, B2, Cl, C2 and D (Werner et al , Op. Cit.). These, and other inhibitors of V-ATPases are envisaged for use in the present invention. Bafilomycins Al, Bl and Cl have been shown to be highly SUBSTITUTE SHEET specific inhibitors of V-ATPase which are highly effective in the invention. Therefore, the use of bafilomycins Al, Bl or Cl is preferred. Advantageously, bafilo ycin-Al is used. However, it is envisaged that the differing pharmacological properties of the other bafilomycins and other inhibitors of V-ATPases may make a different agent the agent of choice in a particular circumstance. For example, different anti-V- ATPase agents will present different side-effects and will require administration according to different administration regimes. Therefore, the most appropriate agent should be selected according to the particular circumstances of the case. The invention envisages the use of modified bafilomycins wherein the properties thereof, such as their V-ATPase inhibitory properties, their toxicity and other side effects, have been altered in order to provide a more effective agent. Such modified bafilomycins may be the product of natural mutations or may be engineered into the molecules artificially. The effective dose of the agent to be administered will depend to a large extent upon the agent itself and will be determined in each case by a physician according to standard protocols used in the art. However, it is an advantage of bafilomycin-Al that it may be used at extremely low doses, being extremely specific in its inhibition of V-ATPase. Furthermore, it presents very few drawbacks in terms of toxicity. Preferably, bafilomycin-Al is administered in pharmaceutically effective doses, advantageously comprising doses of between 1 mg and 1 gram, preferably between 10 mg and 300 mg. The extremely low toxicity of bafilomycin-Al allows doses as high as 18.75 mg/kg, corresponding to an approximate concentration of 0.3 mM in vivo, to be administered without toxic effects. SUBSTITUTE SHEET The agent of the invention is preferably administered by the oral route, in combination with pharmaceutically acceptable excipients, diluents or carriers commonly used for the oral administration of antibiotics. Alternatively, the agent may be administered by injection or in a suppository, according to protocols known in the art for antibiotic adminstration. Preferably, the dose is repeated three or four times per day or as required, as established by the physician. The present invention .further provides a method for the treatment of a disease involving pathologic cellular vacuolation comprising the administration of a pharmaceutically effective amount of a V-ATPase inhibitor. In the present invention, "treatment" is intended to denote both prophylaxis and therapy of a disease. It has been shown that V-ATPase inhibitors are effective not only in the prevention, but also the reversal of vacuolation induced by H. pylori . Therefore, the administration of an agent according to the invention is effective at a later stage in the disease, as well as at the onset of the disease and indeed before the disease manifests itself. Accordingly, persons known to be at risk from a disease involving cellular vacuolation may receive a prophylactically effective amount of the agent of the invention in order to discourage or prevent onset of the disease. Likewise, patients presenting with an advanced stage of the disease may be effectively treated according to the invention because the invention acts to reverse as well as arrest cellular vacuolation. Pathological vacuolation is known to be associated with H. pylori infection in a number of diseases. Preferably, the invention is used in the treatment of a disease selected from the group consisting of chronic gastritis, peptic TE HEET ulceration and gastric adenocarcinoma. The use in the treatmant of peptic ulceration is especially preferred. The present invention further provides an agent according to the invention packaged in a form convenient for the administration to patients in admixture with a pharmaceutical carrier, prepared according to conventional pharmaceutical compounding techniques. For example, such a form may take that of an injectable dose of the agent, a tablet, a pill, a suppository, a suspension, syrup or mixture, a slow-release vehicle or the like. For oral administration, the agents of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension. Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil. For intramuscular, intraperitoneal, subcutaneous and SUBSTITUTE SHEET intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl- pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. For the treatment of H. pylori disease, the use of tablets, pills or other preparation to be taken orally is preferred. The invention further provides a method for the manufacture of a composition for the treatment of a disease involving pathological cellular vacuolation comprising combining a pharmaceutically effective dose of an inhibitor of V-ATPase with a pharmaceutically acceptable excipient, diluent or carrier. The V-ATPase inhibitor may be derived from natural sources, or may be prepared by synthetic processes. For example, where the inhibitor is a bafilomycin, it may be obtained from the organism Streptomyces griseus as described in the art (Werner et al . , Op. Cit.). Alternatively, it may be generated by processes of chemical synthesis which are well known in the art. Where the bafilomycin is a modified bafilomycin, it is advantageously produced by chemical synthesis. Although natural modifications of bafilomycins may occur, it is envisaged that advantageous modifications to the properties of bafilomycins, such as their V-ATPase inhibitor activity, their toxicity and other side-effects, may be introduced during chemical synthesis of the molecule. The invention is described below, for the purposes of illustration only, by means of the following examples. Brief Description of the Drawings Figure 1 shows the molecular structure of bafilomycins Al, Bl, Cl and D; Figure 2 shows the protective effect of bafilomycin-Al on the vacuolation of HeLa cells induced by Helicobacter pylori cell-free extract both by the number of vacuolated cells (Figure 2A) and the percentage uptake of neutral red (Figure 2B) . The values indicate^ are the mean of three different experiments shown in triplicate; Figure 3 illustrates the dose dependence of the protective effect of bafilomycin-Al, showing the percentage of vacuolated cells and the percentage uptake of neutral red; Figure 4 demonstrates the rescue of Helicobacter pylori vacuolated cells by bafilomycin-Al. The same cluster of HeLa cells are shown at different times after addition of H. pylori and bafilomycin-Al, l\ hours (A) , 3 hours (B) and 6 hours (C) after treatment with H. pylori extract and l hours (D) , 3 hours (E) and 4 hours (F) after treatment with both H. pylori extract and bafilomycin-Al; Figure 5 demonstrates the reversal of H. pylori induced vacuolation by bafilomycin-Al as a function of time; Figure 6 shows the use of different V-ATPase inhibitors on the vacuolating activity of H. pylori extract and on HeLa cell vacuolation. (A) the bacterial extract was treated with the indicated inhibitor as specified in the experimental section and then added to HeLa cells; (B) HeLa cells were treated with the specified inhibitor of V-ATPase at the same concentration and under the same conditions as the bacterial extract; HEET Figure 7 shows the dose response of bafilomycin inhibition of HeLa cell vacuolation comparing bafilomycin-Al, bafilomycin-Bl, bafilomycin-Cl and bafilomycin-D; Figure 8 shows the reversal of cell vacuolation by the different bafilomycins. Experimental Procedures Materials: Bafilomycins Al, Bl, Cl and D were provided by of Prof. K.H. Altendorf (University of Osnabruk, Germany). They were dissolved in DMSO at the concentration of 1.5 mM and stored in aliquots at -20°C. Their concentration was determined spectrophotometrically using an extinction coefficient of 25,000 M "1 cm "1 at 245 nm in methanol (Bowman et al, 1988, PNAS, 85: 7972-7976). Neutral red, Mg-ATP and N-ethylmaleimmide (NEM) were from Sigma; N, N 1 - dicyclohexylcarbodiimmide (DCCD) was from Fluka and 7- chloro-4-nitrobenzoxadiazole (NBC-C1) was from Koch-Light. Sterile disposable material, media for cell culture and fetal calf serum (FCS) were from Flow. Bacterial Extract: Helicobacter pylori cytotoxic strain CCUG 17874 and non cytotoxic strain G-21 were grown in Brucella broth containing 0.2% cyclodextrin and Skirrow selective supplement in a microaerobic environment, at 37°C for three to four days. The bacterial cultures were centrifuged at 16,000 x g for 30 minutes. The pellet was resuspended in PBS and sonicated 5 times for 30 seconds at 4°C with a Soniprep model 150 ultrasonic generator. The material was centrifuged at 40,000 x g for 20 minutes and the supernatant was sterilized by filtration through 0.22 μm cellulose filters. The extract was frozen in liquid nitrogen and stored at - 80°C at a protein concentration of 26 mg/ l. Cells:HeLa cells were cultured as monolayers in plastic flasks in Earle-modified minimal essential medium (MEM) , containing 10% FCS, in a 5% C0 2 atmosphere at 37°C. 24 hours before experiments, cells were suspended with trypsin- EDTA and seeded in 96, 48 or 24 wells titration plates in MEM, 10% FCS at a density of 30 x 10 3 /cm 2 . Assay of cell vacuolation and cytotoxicity: HeLa cells were incubated with different concentrations of bafilomycins (0.001-1.0 uM) in MEM, 2% FCS and Helicobacter pylori extract was added (final concentration ranging between 0.5 and 2.6 mg/ l of bacterial protein). Alternatively, the cells were incubated with the bacterial extract and 1 μM or 12.5 nM bafilomycins added to different samples at different times (1-48 hours) after the extract addition. The extent of cell vacuolation was estimated from at least three independent microphotographs per sample (248 x magnification with a phase interference inverted microscope) by counting the number of cells having more than 5 vacuoles per cell and reporting this number as percentage of the total number of cells present in the picture. Alternatively, vacuolation was measured as neutral red uptake as described (Cover et al, 1991, Op. Cit.) with minor modifications. Briefly, cells in 96 titer plates were incubated for 8 minutes at 25°C with 0.1 ml of 0.05% neutral red in 125 mM NaCl, 5 mM KCl, 50 mM NaPi, pH 7.4 (PBS), and washed three times with 0.2 ml of 0.2% BSA in PBS. After addition of 0.1 ml of 70% ethanol in water containing 0.37% HC1 absorbance at 530 nm was measured with a Packard Argus 400 Micropiate Reader. Treatment of Helicobacter pylori extract and HeLa cells with inhibitors of V-ATPase: Helicobacter pylori bacterial extract was treated with inhibitors, before addition to HeLa cells, as it follows: DCCD 10 μMolar for 1 hour at 30°C; NBD-C1 100 μMolar for 1 hour at 30°C and the reaction was blocked with glycine 10 mMolar final concentration; NEM 275 μMolar for 1 hour at 30°C and the reaction was blocked by addition of ,9-mercaptoethanol 275 μM; Mg-ATP 14 mMolar for 1 hour at 0°C; 100 mMolar KN0 3 and 14 mMolar Mg-ATP for 1 SUBSTITUTE SH hour at 30°C; NaC0 3 100 mMolar, pH 11 for l hour at 0°C. The bacterial extract was then added to cells with a forty fold dilution at a final concentration of 0.65 mg/ml. Controls were made with HeLa cells incubated with untreated bacterial extracts and with cell treated with inhibitors under the same conditions as bacterial extracts at the same concentrations or after a forty fold dilution. The vacuolating activity of the bacterial extracts was assayed as above. Example 1: In order to demonstrate the role of V-ATPase proton pumping in the acidification of vacuoles, bafilomycin-Al, a specific inhibitor of V-ATPase, was used to study the kinetics of cell vacuolation induced in HeLa cells exposed to H. pylori extracts. The results of this experiment are shown in Figure 2. HeLa cells were treated for 30 minutes with or without one mMolar baf-Al in NEM, 2% FCS and further incubated with H. pylori extract (1.3 mg/ml final protein concentration) in the presence or in the absence of baf-Al. At the indicated times, the number of vacuolated cells was counted visually, or the amount of neutral red taken up in the cell vacuoles was measured spectrophotometrically. The two methods give a similar profile characterised by a lag phase of about 90 minutes, followed by a rapid vacuolation which features its maximum value after 5 hours of incubation. Figure 2 also shows that baf-Al, at a concentration at which it affects only V- ATPases, abolishes the vacuolating effect of the H. pylori extracts, independently of the method used for the evaluation of the effect. Control experiments showed that vacuolation was absent in cells not treated with the extract, in cells treated with baf-Al alone or in cells treated with a non cytotoxic H. pylori strain (G-21) . In addition, vacuolation was caused also by partially purified bacterial extract and could be inhibited with high titer anti H. pylori human sera (Figura et al , 1990, Op. Cit.). SUBSTITUTE S Example 2: The dose dependence of the protective effect of baf-Al was investigated. The results, shown in Figure 3, demonstrate that baf-Al shows a 50% effect at a concentration as low as 4 nMolar and a complete inhibition of the bacterial induced vacuolation at 12.5 nMolar. HeLa cells were pretreated with the indicated concentration of baf-Al as described in Example 1, incubated for 6 hours with H. pylori extract (1.3 mgs per ml final concentration) , and the percentage of vacuolated cells and the uptake of neutral red were determined as described. The values quoted are the mean of two different experiments run in triplicate, plus or minus standard deviations. Example 3: Having determined that baf-Al strongly inhibits the formation of vacuoles, the effect of baf-Al in the reversal of cell vacuolation was investigated. Figure 4 shows a sequence of photomicrographs taken on the same cluster of HeLa cells, demonstrating that not only does baf- Al prevent vacuolation, but also that it is able to restore the normal physiological appearance of previously vacuolated cells. Baf-Al was also effective on cells which remained in the maximally vacuolated condition for many hours, which is shown in Figure 5, wherein the effect of 1 mMolar baf-Al administered after the stated period of time is shown. It is noteworthy that the kinetics of baf-Al induced recovery of the normal physiological appearance is the same independently of the time spent by cells in the fully vacuolated condition. The time needed for the disappearance of all vacuoles did not show a strict dependence on baf-Al concentration; the reversal to normal physiological conditions only took three times longer with 12.5 nMolar baf-Al than with 1 mMolar baf-Al. Cells rescued by baf-Al were able to proliferate and showed the same mitotic index as control cells not treated with the bacterial extract. SUBSTITUTE SHEET Example 4: The activity of V-ATPase inhibitors other than bafilomycins was investigated. Figure 6A shows the effect of treating H. pylori extracts with the inhibitors N, N'- dicyclohexylcarbodiimmide (DCCD) , 7-chloro-4- nitrobenzoxadiazole (NBD-C1) , N-ethylmaleimmide (NEM) , Mg- ATP, high pH and nitrate ions. None of these agents or V- ATPase disaggregating treatments affected the vacuolating activity of bacterial extract. This suggests that the vacuolating activity is not due to a V-ATPase produced by Helicobacter pylori , but may be due to a V-ATPase produced by the cell. The implication is that H. pylori produces an agent which modifies cellular V-ATPases. This hypothesis is supported by the results shown in Figure 6B, wherein administration of the inhibitors to HeLa cells was effective in the prevention of Helicobacter pylori-induced vacuolation. This suggests a role for the HeLa cell V- ATPase in vacuole development. Example 5: In order to investigate the effectiveness of bafilomycins Bl, Cl and D, HeLa cells were preincubated for 60 minutes with different concentrations of these bafilomycins and subsequently treated with Helicobacter pylori extract as described in Example 1. After 6 hours, the extent of vacuolation, measured as neutral red absorption, was determined. The results are shown in Figure 7. All bafilomycins effectively counteracted the formation of vacuoles in the cells at concentrations lower than 2 mMolar. This was also apparent from a semi-quantitative estimation of the number of vacuoles per cell by microscopy. The ID 50 values reported in Figure 7 indicate that the relative potency of the various bafilomycins, using baf-Al as a standard at ascribing it a value of 100, is: baf-Al 100, baf-Bl 26, baf-Cl 17 and baf-D2. In Figure 8, the effect of bafilomycins Al, Bl, Cl and D SUBSTITUTE SHEET in reversing the vacuolation induced by H. pylori is shown. Reversal of vacuolation was also shown in cells kept in a vacuolated condition for 24 hours.

Claims: CLAIMS 1. The use of an inhibitor of vacuolar-type H + ATPase (V- ATPase) in the preparation of a composition for use in the treatment of a disease involving pathological cellular vacuolation. 2. The use of claim 1 wherein the pathological cellular vacuolation is induced by a bacterial infection, exposure to cytotoxic chemicals, drugs or neurological degeneration. 3. The use of claim 2 wherin the pathological cellular vacuolation is induced by a bacterial infection. 4. The use of claim 3 wherein the bacterial infection is a Helicobacter pylori infection. 5. The use of any preceding claim wherin the V-ATPase inhibitor inhibits the proton-pumping activity of V-ATPase. 6. The use of any preceding claim wherin the V-ATPase inhibitor is N, N'-dicyclohexylcarbodiimmide (DCCD) , 7- chloro-4-nitrobenzoxadiazole (NBD-C1) and N-ethylmaleimmide (NEM) or a bafilomycin. 7. The use of claim 6 wherein the V-ATPase inhibitor bafilomycin-Al, bafilomycin-Bl or bafilo ycin-Cl. 8. The use of claim 7 wherein the bafilomycin is bafilomycin-Al. 9. The use of claim 8 wherein bafilomycin-Al is administered in a dose of between lμg and lg. 10. The use of claim 9 wherein bafilomycin-Al is administered in a dose between lOμg and 300μg. 11. The use of claim 9 or claim 10 wherein the dose is SUBSTITUTE SHEET administered repetitively. 12. The use of any preceding claim in the treatment of a disease which is in an advanced stage. 13. The use of any one of claims 1 to 11 which is prophylactic. 14. A method for the treatment of a disease involving pathologic cellular vacuolation comprising the administration of a V-ATPase inhibitor. 15. A method according to claim 14 wherein the disease is associated with Helicobacter pylori infection. 16. A method according to claim 15 wherein the disease is selected from the group consisting of chronic gastritis, peptic ulceration and gastric adenocarcinoma. 17. A method according to claim 16 wherein the disease is peptic ulceration. 18. A pharmaceutical preparation comprising a pharmaceutically effective amount of a V-ATPase inhibitor together with a pharmaceutically acceptable excipient, diluent or carrier for use in the treatment of a disease involving pathological cellular vacuolation. 19. The preparation of claim 18 comprising bafilomycin- Al. 20. The preparation of claim 19 comprising between lOμg and 300μg of bafilomycin-Al. 21. The preparation of claim 18, 19 or 20 adapted for oral administration. 22. A method for the preparation of a composition for the SUBSTITUTE SHEET treatment of a disease involving pathological cellular vacuolation comprising combining a pharmaceutically effective dose of a V-ATPase inhibitor with a pharmaceutically acceptable excipient, diluent or carrier.

Inventors: BUGNOLI MASSIMO, MONTECUCCO CESARE, PAPINI EMANUELE, RAPPUOLI RINO, BUGNOLI, MASSIMO, MONTECUCCO, CESARE, PAPINI, EMANUELE, RAPPUOLI, RINO
Applicants: BUGNOLI MASSIMO, MONTECUCCO CESARE, PAPINI EMANUELE, RAPPUOLI RINO, SCLAVO BIOCINE SPA, BIOCINE SCLAVO S.P.A.
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, A61K45, A61P31, A61P43, C07D207, C07D271, C07D313, C07D315