Field of the Invention

This invention relates to a process for producing polyketide pigmented antibiotics from Streptomyces venezuelae ISP5230 (ATCC no. 10712) , and to novel polyketide pigmented antibiotics.

Description of the Prior Art

The study of the effects of environmental stress on the production of antibiotics by Streptomyces has concen¬ trated largely on the role of nutrition in determining the onset of secondary metabolism. It has been observed in a variety of systems that growth suppression due to depletion of carbon, nitrogen, or phosphate from the culture medium often allows for expression of the genetic information for antibiotic biosynthesis (Doull, J.L. and Vining, L.C. ^l ) . In some cases, this expression has been linked mechanistically to regulons such as the Pho system respon¬ sible for detecting and adapting the cell to available sources of phosphate in the environment. (Liras, P. et al ² ) .

Although there appears to be considerable overlap between genetic systems which react to nutrient depletion and those effecting responses to other forms of stress such as high temperature, the association of the latter with antibiotic synthesis has not been investigated. The discovery of multiple groEL like-genes in a variety of streptomycetes however has prompted the suggestion that heat-shock induced chaperonins might play a role in the assembly of multienzyme complexes for antibiotic biosynthesis (Guglielmi, G. et al ³ ) . Recent studies have also indicated that various highly-conserved proteins associated with heat stress, such as DnaK, may be involved in the morphological development process in Streptomyces (Bucca, G. et al. ) .

It is generally known that cultures of Streptomyces venezuelae ISP5230 in a glucose-is leucine medium at 28°C, produce the broad spectrum antibiotic chloramphenicol.

It has also been reported by applicant, in Tetrahedron Letters, Vol. 32, No. 44, pp 6301-6304, 1991, that when glucose is replaced by galactose and the temperature is raised to 37°C, the Streptomyces venezuelae ISP5230 cultures produce a group of pigmented antibiotics, including a 8H- benz[b]oxazalo[3,2-f]phenanthridine, which applicant has called Jadomycin.

This pigmented compound has a novel benzoxazolophenan- thridine structure, unusual in that it incorporates a nitrogen atom into an angled pentacyclic framework. Somewhat similar metabolites produced by Streptomyces murayamaensis are the anthracyclines kinamycin A-D (Cone M.C et al. ⁵ ) . Inspection of the structure of Jadomycin indicates that it is very likely derived from a polyketide intermediate. This provides a possible explanation for the report by Malpartida F. et al ⁶ that genomic DNA from a related strain, S. venezuelae UC2374, hybridizes to a polyketide synthase (act I) probe.

The structure of Jadomycin was determined by spectral means. Unfortunately, only very small amounts of this pigmented antibiotic are produced by this process. More- over, its antibiotic activity was not found to be that significant.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a novel process for the preparation of polyketide pigmented antibiotics, including Jadomycin, from bacteria in significant quantities.

It is another object of the invention to produce novel polyketide pigmented antibiotics, including one of higher antibiotic activity than Jadomycin, called Jadomycin B.

According to one embodiment of the invention, a process for the production of a polyketide pigmented antibiotic of structural formula I,

I

Wherein R, is H or

Wherein R, R ₂ and R ₃ are H or lower alkyl

and pharmaceutically acceptable salts thereof is provided, comprising:

(a) culturing Streptomyces venezuelae ISP5230 in a suitable growth medium, pH 7 to 8.5 at a tempera¬ ture of about 27 to 37°C, for 2-17 hours,

(b) applying means for inducing a heat-shock response in the bacteria,

(c) culturing as in step (a) for a further 3 to 96 hours,

(d) separating the antibiotic of structural formula I so formed, and where required,

(e) reacting the antibiotic of structural formula I with a suitable acid or base, to form a pharma- ceutically acceptable salt.

According to another embodiment of the invention, a novel polyketide pigmented antibiotic of structural formula II is provided, comprising

π

and pharmaceutically acceptable salts thereof, optionally including a pharmaceutically acceptable adjuvant, carrier or diluent.

According to yet another embodiment of the invention, a method of treating a microbial or helminthic infection in animal subjects is provided, comprising treating a subject in need of such treatment with a therapeutically effective amount of a novel polyketide antibiotic of structural formula II π

Wfcorain 2 a-αd M→ ars H or lower al-kyl and pharmaceutically acceptable salts thereof, optionally

including a pharmaceutically acceptable adjuvant, carrier or diluent.

The term "lower alkyl" refers to a straight or branched chain saturated hydrocarbon group having from 1 to 4 carbon atoms e.g. methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and tert-butyl.

The term "pharmaceutically acceptable salt" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phos¬ phoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) salts with polyvalent metal cations such as zinc, calcium, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed from N,N ^, -dibenzylethylenediamine or ethylenedia ine; or (c) combinations, of (a) and (b) , e.g. a zinc tannate salt and the like.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1A is a graph illustrating the effects of heat on Jadomycin B production.

Figure IB is a graph illustrating the effects of heat on growth of Streptomyces venezuelae .

Figure 2 is a graph illustrating the effects of heat shock on Jadomycin B production at different times, post inoculation.

Figure 3A is a graph illustrating the effects of ethanol treatment on Jadomycin B production.

Figure 3B is a graph illustrating the effects of ethanol on growth of Streptomyces venezuelae . Figure 4 is a graph illustrating the effects of ethanol treatment on Jadomycin B production at different times, post inoculation.

Figure 5 is a graph illustrating the effects of the initial pH of growth medium on Jadomycin B production.

Figure 6 is a graph illustrating the effect of Jadomycin B on various bacteria and yeast species.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In making the invention, applicant has found that the production of Jadomycin and Jadomycin B (i.e. the compound of structural formula II in which R is sec-butyl and Rj is methyl) , at high temperature and their virtual absence from cultures growing at 27°C, suggested that the regulation of these compounds might somehow be linked to the heat-shock response, a universal cellular stress response character¬ ized by the production of a specific set of proteins, the heat-shock proteins (HSPS) .

Various known inducers of heat-shock response can be employed in applicant's process. For example, elevation of temperature for a short period of time, alcohol treatment and virus infection e.g. by a bacteriophage.

Regarding the elevated temperature treatment, the temperature is elevated to 42 to 50°C, for 0.5 to 2 hours, with agitation. Typically, the temperature is elevated to about 42°C for about 1 hour, under agitation.

With respect to the alcohol treatment, the alcohol is added to provide a concentration of 4 to 8 % v/v, preferab¬ ly, about 6 % v/v.

Respecting the phage treatment, the SV phages e.g. SV1 to SV3 and SV9 to SV12, may be used.

Suitable growth media for use with either elevated temperature or alcohol treatment include galactose- isoleucine medium (pH 7.3). This medium comprises galactose 0.17M, L-isoleucine 60mM, phosphate 15mM, MgSo ₄ .7H ₂ 00.81mM, and trace minerals. Other carbon sources e.g. starch or glucose may be substituted for the galactose, although the production is not as good. Other amino acids such as tryptophan, glycine and L-leucine may be substituted for the L-isoleucine. However, Jadomycin and Jadomycin B production requires isoleucine. A supplement of 5 mL of MYM medium ( pH 7) is typically used with the virus.

Preferably, (when using the alcohol treatment) the starting pH of the growth medium is in the range of 7 to 8.5. During growth, the pH drops about one pH unit, although the phosphate present acts to some extent as a buffer.

Suitable alcohols include Cl to C3 alcohols, with ethanol being preferred.

In Process step (a) , the bacteria are preferably cultured at a temperature of about 27°C. Further, when the heat shock stress inducing means is elevated temperature, the culturing time is preferably about 9 hours; when the stress inducing means is ethanol treatment, the preferred culturing time is 9-14 hours, and when the stressor is bacteriophage SV1, the culturing time is preferably about 2 hours. All culturing is effected under agitation.

In Process step (c) , the culturing is preferably continued for about a further 12 hours in the case of the elevated temperature treatment, and for about a further 48 hours in the case of the ethanol and bacteriophage treat- ments.

The pharmaceutically acceptable salts are prepared by conventional means by reacting with a suitable acid e.g. HC1 and H ₂ S0 ₄ , or a suitable base such as KOH or NaOH.

CULTURE AND GROWTH CONDITIONS Streptomyces venezuelae substrain HP-1 (a single colony isolate of ISP5230) used in this present study was preserved as spore suspensions at -20°C in 20% glycerol. Vegetative inocula were prepared by incubating 50 μL of spore stock in 100 mLs of MYM medium (Stuttard, C. ⁷ ) for 20-24 hours with shaking (250 rpm) at 27°C. Unwashed mycelium (5 mL) from these cultures was then added to 50 mL of galactose L-isoleucine medium (pH 7.3), which contained (per litre): galactose, 30 g; L-isoleucine, 4.0 g; potassium dihydrogen phosphate, 0.5 g; dipotassium hydrogen phosphate, 1.2 g; anhydrous magnesium sulfate, 0.2 g; calcium chloride, 90 mg; sodium chloride, 90 mg; ferrous sulfate heptahydrate, 9 mg; and a mineral solution that supplied: zinc sulfate heptahydrate, 4 mg; cupric sulfate pentahydrate, 0.18 mg; manganous sulfate monohydrate, 27 mg; boric acid, 26 mg; and ammonium molybdate tetrahydrate , 17 mg.

Following growth for 6-9 hours (unless otherwise stated) at 27°C in the above medium, one of the following treatments was applied:

i) heat shock- cultures were placed at 42°C for 1 hour with shaking (250 rpm) and then shifted back to 27°C for 3 to 72 hours.

ii) ethanol treatment - ethanol was added 3 to 14 hours after inoculation to a final concentration of 6% (v/v) and the cultures were grown for 48-96 hours at 27 to 37°C. Both vegetative and Jadomycin B production cultures were grown in 250 mL flasks.

(iii) Spore suspensions (80 μL) were added to 50 mL of galactose-isoleucine medium containing 5 mL of MYM medium pH 7. After 2 hours growth at 27°C, a suspension of phage SVl (10 ⁸ pfu/mL, 20 μL) was added and the culture incubated for 48 hours.

ANALYSES

Values are averaged from 3-12 replicated cultures. Growth was measured as the dry weight of cell material collected from samples by filtration.

To prepare samples for Jadomycin B assay, 3 mL por¬ tions were removed from cultures, and filtered through Whatman(trademark) I paper (4.25 cm) in order to remove mycelium. They were then refiltered using Millex- GV(trade- mark) 0.22 mm cartridge units and 1 mL portions were vacuum-evaporated overnight using a

Savant(trademark) Speed Vac Concentrator. The residues were resuspended in 200 μL of methanol/water (1:1 v/v), sonicated for three 30 s bursts using a Bronson(trademark) 3200 sonicator water bath and finally filtered again using Ultrafree-MC (trademark) 0.45 mm filtration units (Millipore) (trademark) in a Millipore microfuge. The samples were then placed in a Hewlett Packard 1090 Autosampler(trademark) and assayed using high pressure liquid chromatography (HPLC) . Samples (20 μL) were applied to a Vydac(trademark) C column (250 mm x 2 mm internal diameter) and Jadomycin B was eluted at a flow rate of 0.2 mL/min with a gradient of acetonitrile/ water/trifluroacetic acid (49.5:49.5:1) to 99%

acetonitrile 1% TFA over 20 minutes. Detection was at 313 n . The column was calibrated using a purified Jadomycin B standard.

RESULTS EFFECTS OF ELEVATED TEMPERATURE ON JADOMYCIN B PRODUCTION Since Jadomycin and Jadomycin B production was previously shown to be associated with growth of S. venezuelae at high temperatures, preliminary experiments were performed in order to investigate whether production of these compounds could be initiated as a result of a 1 hour, 42°C heat shock.

Figure 1A shows a comparison of Jadomycin B production in heat-shocked cultures with production in cultures that were shifted to 37°C and 42°C and maintained at the higher temperature after 6 hours of growth in galactose-isoleucine medium at 27°C. Control cultures were maintained at 27°C for the duration of the experiment. Jadomycin B production was highest in the heat-shocked cultures, which showed a distinct orange tint corresponding to the presence of the antibiotic. Maximum titres (27 μg/mL) were detected 12 hours following heat shock, and after 18 hours Jadomycin B concentrations showed a rapid decline. Lower levels of Jadomycin B accumulated in cultures maintained at high temperatures for the duration of the growth period, with slightly higher titres detected at 42°C compared with 37°C. Essentially no Jadomycin B was produced at 27°C. Although growth appeared to be unaffected by heat shock, progressively diminished growth was seen in cultures grown at 37°C and 42°C, respectively (Figure IB) .

Because it was evident from the previous experiment that higher titres of Jadomycin B accumulated when cultures were heat-shocked rather than maintained at high temperature, the effect of heat-shocking at different times following inoculation was investigated.

Figure 2 shows Jadomycin B titre obtained in S. venezuelae cultures heat-shocked at 3, 6, 9, 12, and 15 hours after inoculation into galactose-isoleucine medium. Values shown were those detected 12 hours after heat- shocking. In this experiment, the optimum time for heat- shocking was at 9 hours, post inoculation. After this time, heat-shocking was associated with diminishing Jadomycin B titres.

EFFECTS OF ETHANOL TREATMENT ON JADOMYCIN B PRODUCTION The effects on Jadomycin B production of ethanol exposure were examined. Ethanol was added 6 hours after inoculation to a total volume of 6%. These cultures were incubated for a further 4 days at 27°C in the presence of the ethanol (ethanol treatment) . Results are shown in Figure 3A. It was found that ethanol-treated cultures produced high titres of Jadomycin B which peaked on day 2 at 31 μg/ml, thereafter gradually decreasing for the duration of the experiment. Production of Jadomycin B was maintained for a longer period when compared with heat-shocked cultures in previous experiments and production levels did not decline as rapidly. As in previous experiments, Jadomycin (aglycone) was present in very low amounts.

In order to determine the best time after inoculation for ethanol treatment, ethanol was added to S. venezuelae cultures at 6, 9, 13, 17, 24, 48, 72, and 96 hours after inoculation into galactose-isoleucine medium. Cultures were incubated at 27°C for a further 4 days from the time that they were supplemented with ethanol. Samples were taken daily during this period. Maximum values were obtained by 48 hours in all cultures (Figure 4) . Cultures that were ethanol treated at 13 hours after inoculation produced the highest titres of Jadomycin B, 44 μg/ml by day 2. Cultures that were ethanol treated 48-96 hours after inoculation failed to produce measurable amounts of Jadomycin B despite the fact that they had accumulated a higher biomass by the time of ethanol addition. Ethanol treatment resulted in the cessation of growth regardless of the pretreatment incubation time and resultant biomass accumulation.

EFFECT OF PH ON JADOMYCIN B PRODUCTION WITH ETHANOL TREATMENT In order to determine the effects of pH on Jadomycin

B production, S. venezuelae was inoculated into galactose-isoleucine medium with starting pH values of 6, 6.5, 7, 7.5 and 8. Ethanol was added 9 hours after inoculation and Jadomycin B production was monitored over 4 days. Maximum titres were obtained in all cultures by day 2 (Figure 5) . It was noted that cultures with starting pH's of 7.5-8 produced the most Jadomycin B. Negligible amounts were produced in the cultures with a lower (6, 6.5) pH. A drop of approximately 1 pH unit was detected in all cultures by the end of a 4-day incubation period.

EFFECT OF PHAGE INFECTION ON JADOMYCIN B PRODUCTION

When SVl phage was added to liquid galactose- isoleucine cultures 2 hours following inoculation with S. venezuelae HP-1 spores, increased orange pigmentation was noted after 48 hours corresponding to an accumulation of 23 μg/ml Jadomycin B. This was accompanied by extensive cell lysis. No pigmentation was detected in uninfected controls. Similarly, increased pigmentation was observed following phage SVl infection of HP-1 mycelium on solid medium prepared by adding agar (1.6%, starch 1.5% and yeast extract 0.2%) to galactose-isoleucine medium. The pigment was extracted and shown to correspond to Jadomycin B by HPLC analysis. No pigmentation was seen on uninfected control plates.

DISCUSSION

Of the variety of treatments known to induce a heat- shock response in prokaryotic cells, three treatments specifically, elevated temperature for a short-time period, C1-C3 alcohol treatment, and phage infection, have all been found to favour production of the antibiotic Jadomycin B. In the absence of these treatments, very little production is seen in cultures growing at 27°C. This accounts for the failure to detect Jadomycin-type compounds during growth of S. venezuelae under chloramphenicol production conditions. Although some Jadomycin B is accumulated during growth at 37°C and 42°C, substantially higher amounts are detected within 13 hours following a 1 hour 42°C heat-shock. Thereafter titres diminish throughout the following 4 days.

The production pattern for Jadomycin B following heat-shock is unusual in that the antibiotic accumulates only during the early growth phase. A more generally observed pattern of antibiotic production is characterized by accumulation at the end of a period of growth. However, on

poorly assimilated carbon and nitrogen sources, production of antibiotics such as chloramphenicol is seen throughout the growth period. Since both galactose and isoleucine are non-preferred nutrient sources, growth in the Jadomycin B production medium may thereby provide sufficient nutritional stress to "prime" the cells for antibiotic production. Subsequent heat- shocking may impose the additional physiological stress required to permit expression of the Jadomycin B biosynthetic genes.

Although a 1-hour exposure of 5. venezuelae cells to ethanol was associated with a short period of Jadomycin B production, much higher levels were accumulated when cells were exposed to prolonged ethanol-treatment over a 2-day period. Thereafter Jadomycin B titres began to decline. Biomass accumulation was minimal throughout the 4-day incubation in the presence of ethanol. Although the mechanism whereby ethanol treatment induces the production of Jadomycin B has not been specifically determined, we suggest that it relates to the demonstrated ability of this treatment to trigger a heat- shock response.

An interesting feature seen with both the heat-shock and ethanol exposure was that the timing of the treatment application following inoculation into galactose- isoleucine medium was critical. Treatments given after 17 hours post inoculation were ineffective in initiating Jadomycin B production. Perhaps significantly, the level of heat-shock protein induction following heat stress has been frequently reported to show growth or development phase dependence. If the initiation of Jadomycin B production is linked to the heat-shock response, a similar association is perhaps to be expected.

Regarding treatment by viral infection, our observa¬ tions were that infection with SVl is associated with increased Jadomycin B production. This result appears attributable again to induction of a heat-shock response.

RESULTS - EFFECTS OF OTHER ALCOHOLS (ALCOHOL TREATMENT) Standard conditions for Jadomycin B production used except methanol or isopropanol (6%) substituted for ethanol.

Results: methanol isopropanol ethanol

Concentration of Jad B

(mg/L) 24 hours 13 9

28

48 hours 6 7

31

72 hours 8 7

28

Conclusion: There is some induction of Jadomycin B production with other alcohols but they are not as effective as ethanol.

RESULTS - BIOASSAY OF JADOMYCIN B BIOACTIVITY

A total of 300 ug of Jadomycin B (dissolved in chloroform) was spotted onto 6.4 mm disks, the chloroform was evaporated, and the disks applied to plates (brain heart infusion agar) spread with various Gm ⁺ and Gm_- bacteria as well as yeast (Presquisle cultures, Pennsylvania) . Incubated 24 hours at 37°.

Results: Activity against all microbes tested. Activity was greatest against Gram ⁺ bacteria.

Large zones of growth inhibition against: Micrococcus luteus (Gm ⁺ ) Bacillus subtilis (Gm ⁺ ) Streptococcus faecalis (Gm ⁺ ) Streptococcus aureus (Gm ⁺ )

Saccharomyces cerevisiae (yeast, eukaryotic) Smaller zones:

Escherichia coli (Gm ^* ) Pseudomonas aeruαinosa (Gm") Enterobacter aeroσenes (Gm")

Aeromonas hvdrophilus (Gm") PROTOCOL FOR QUANTITATIVE ASSESSMENT OF JADOMYCIN ACTIVITY IN LIQUID MEDIA (AND AGAR)

400μg of Jadomycin B (dissolved in chloroform) was spotted onto 12.5 mm sterile test disks and the chloroform was evaporated off in air. Test disks containing the Jadomycin B were then placed in either nutrient broth or in the centre of nutrient agar pour plate cultures.

Pour plates of each test microorganism were prepared by the addition of 0.2 mL of a 24 hour culture to 50 mL of molten nutrient agar (Difco Ltd.). Similarly, 0.1 mL of each test microbe was added to 10 mL nutrient broth (Difco Ltd.), prior to the addition of a Jadomycin B disk. Duplicate broth cultures of each microbe treated with Jadomycin B disks were incubated along with duplicate broth cultures lacking Jadomycin B disks.

After 48 hours incubation (37°C for bacteria, 25°C for the yeast) , the absorbency at 686 nm of the broth cultures was determined using a Spectronic(trademark) 20. The cultures grown in the absence of Jadomycin B were standardized, (against growth of the microbe on nutrient agar) , as colony forming units per mL (CFU/mM) . The absorbency of the microbes grown in the presence of Jadomycin B disks was compared to cultures

grown in the absence of Jadomycin B. The degree of inhibition of each microbe in the presence of Jadomycin B was expressed as the decrease in CFU/ml compared to the untreated cultures and expressed as a percentage. (i.e. percent Inhibition ■ [100 - [[CFU/ml of the microbe grown without Jadomycin B - CFU/ml of the microbe grown with Jadomycin B disks]xl00]]) .

The impact of Jadoymycin B on the growth of the Gram negative bacteria E. coli, P. aeruginosa, E. aerogenes, and A. hydrophilus was low. However, marked decreases in the growth of both Gram positive bacteria and yeast occurred in broth cultures. (See Table below and Figure 6).

The agar biossay method gave results similar to those noted above and confirms the liquid media results.

Broth Culture Agar Plates Radius

Microorganism % Inhibition * Inhibition Zone (mm)

M. luteus 91 29 BB.. ssuubbttiilliiss 9911 2 S. faecalis 19 11

S. aureus 79 9

S. cerevisiae 13 9

S. carlsbergensis 24 8

C. utilis 46 by 400 μg/lOmL Jadomycin B

TOXICOLOGY

The active compounds are suitable for treatment of microbial or helminthic infections, particularly bacterial and yeast infections. Although a general antibacterial activity has been shown, the novel compounds, in particular Jadomycin B, are most effective against Gram positive bacteria.

The new active compounds and/or their pharmaceutically acceptable salts, can be converted in a known manner into the customary formulations such as tablets, dragees, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert non- toxic, pharmaceutically acceptable ajuvants, carriers, excipients or solvents. The therapeutically active compound should in each case be present here in a concentration of about 0.5 to 90% by weight of the total mixture, that is to say in amounts which suffice to achieve the dosage range, hereinafter indicated.

The formulations are prepared, for example, by extending the active compounds with solvents and/or excipients, optionally with the use of emulsifiers and/or dispersing agents, and for example, when using water as a diluent, organic solvents can optionally be used as auxiliary solvents.

Examples of auxiliary substances which may be men¬ tioned are: water, non-toxic organic solvents, such as paraffins (for example petroleum fractions) , vegetable oils (for example groundnut oil/sesame oil) , alcohols (for example ethyl alcohol and glycerol) , excipients, such as, for example, ground natural minerals (for example kaolins, aluminas, talc and chalk), ground synthetic minerals (for example highly disperse silica and silicates) and sugars (for example sucrose, lactose and glucose) , emulsifiers (for example polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, alkyl-sulphonates and arylsulphonates) , dispersants (for example Lignin, sulphite waste Liquors, methylcellulose, starch and polyvinylpyrrole) and Lubricants (for example magnesium stearate, talc, stearic acid and sodium sulphate) .

Administration is effected in the customary manner, preferably orally or parenterally, particularly prelingually or intravenously. In the case of oral use, the tablets can, of course, also contain, in addition to the excipients mentioned, additives such as sodium citrate, calcium carbonate and dicalcium phosphate, together with various additional substances, such as starch preferably potato starch, gelatine and the like. Furthermore, Lubricants, such as magnesium stearate, sodium lauryl sulphate and talc, can be used concomitantly when making tablets. In the case of aqueous suspensions, the active compounds can be mixed with various flavour-improving agents or colorants in addition to the above-mentioned auxiliary substances.

In the case of parenteral use, solutions of the active compounds, using suitable liquid excipients, can be employed.

In general, it has proved advantageous, in the case of intravenous administration, to administer amounts of about 0.001 to 1 mg/kg, preferably about 0.01 to 0.5 mg/kg, of body weight to achieve effective results, and in the case of oral administration, the dosage is about 0.01 to 20 mg/kg, preferably 0.1 to 10 mg/kg of body weight.

Nevertheless, it may be necessary, under certain circumstances, to deviate from the amounts mentioned, and in particular to do so as a function of the body weight or of the nature of the administration method, of the individual behaviour towards the medicament, the nature of its formulation, and the time or interval over which the administration takes place. Thus, it can in some cases be sufficient to manage with less than the above- mentioned minimum amount, whereas in other cases the upper limit

mentioned must be exceeded. In the case of administration of larger amounts, it may be advisable to divide these into several individual administrations over the course of the day.

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