Antibiotic M43D, pharmaceutical compositions and method of use

Antibiotic M43D, a new glycopeptide antibiotic of the vancomycin class, is produced by Nocardia orientalis NRRL 2450. M43D has excellent antibacterial activity comparable to that of vancomycin.

SUMMARY OF THE INVENTION 
Antibiotic M43D is a new glycopeptide antibiotic produced by Nocardia 
orientalis NRRL 2450. Antibiotic M43D and its salts have excellent 
activity against gram-positive microorganisms. 
DETAILED DESCRIPTION 
This invention relates to a new antibiotic called M43D, and to its salts. 
M43D has structural formula 1: 
##STR1## 
Antibiotic M43D and its salts have excellent antibacterial activity. The 
pharmaceutically acceptable salts of M43D are especially useful. 
New, improved antibiotics are continually in demand, particularly for the 
treatment of human diseases. Increased potency, expanded spectrum of 
bacterial inhibition, increased in vivo efficacy, and improved 
pharmaceutical properties (such as greater oral absorption, higher blood 
or tissue concentrations, longer in vivo half life, and more advantageous 
rate or route of excretion and rate or pattern of metabolism) are some of 
the goals for improved antibiotics. 
In the search for new antibiotics, structural modification of known 
antibiotics is attempted whenever possible. This approach is limited, 
however, to modifications which retain the desired activity. Many 
antibiotics, including the glycopeptides, have such complex structures 
that even small changes can be difficult to make by chemical means. The 
discovery of new antibiotics produced by fermentation processes continues, 
therefore, to be of great importance even in cases where the antibiotic, 
once recognized, is quite similar to a previously known antibiotic. 
Antibiotic M43D is a newly discovered member of the glycopeptide group of 
antibiotics. Closely related members of this group include vancomycin 
(see, for example, U.S. Pat. No. 3,067,099), ristocetin, antibiotic A51568 
factors A and B (see the copending applications of M. M. Hoehn and G. G. 
Marconi, Ser. No. 562,255, filed Dec. 16, 1983, and LaVerne D. Boeck et 
al., Ser. No. 561,008, filed Dec. 13, 1983). 
In U.S. Pat. No. 3,067,099, McCormick et al. described the preparation of 
vancomycin. Three strains of Streptomyces orientalis, one of which was 
numbered M43-05865, were disclosed as being capable of making vancomycin. 
The three cultures were deposited at what was then the Northern Regional 
Research Laboratories at Peoria, Ill., the S. orientalis M43-05865 culture 
being given the accession number NRRL 2450. Later, the organism 
designation for the strains was changed from Streptomyces orientalis to 
Nocardia orientalis. 
The vancomycin described in U.S. Pat. No. 3,067,099 became an important, 
commercially available antibiotic. The N. orientalis culture used to 
prepare commercial product was N. orientalis strain M5-18260 (NRRL 2452) 
or its progeny. 
The structure of a closely related derivative of vancomycin was determined 
by Sheldrick et al [G. M. Sheldrick, P. G. Jones, O. Kennard, D. H. 
Williams and G. A. Smith, Nature 271 (5642), 223-225 (1978)]; later, the 
structure of vancomycin itself was found by Harris et al. [C. M. Harris 
and T. M. Harris, J. Am. Chem. Soc. 104, 4293-4295 (1982)] to be that 
shown in formula 2: 
##STR2## 
M43D is a minor factor produced by the N. orientalis M43-05865 (NRRL 2450) 
strain. The antibiotic complex produced by this strain, the M43 antibiotic 
complex, contains two major factors--antibiotic M43A and vancomycin. 
Antibiotic M43A is the subject of a copending application of Harvey M. 
Higgins, Mack H. McCormick and Kurt E. Merkel, entitled ANTIBIOTIC M43A, 
Ser. No. 600,729, filed herewith this even date. In addition, the M43 
complex contains a number of other minor factors. Among the minor M43 
factors are (1) A51568 factor A of Hoehn et al., supra, and possibly 
A51568 factor B of Boeck et al., supra; (2) the compounds designated 
agluco-A51568A, aglucovancomycin and agluco-M43A, and possibly 
desvancosaminevancomycin, all of which are disclosed in the copending 
application of R. Nagarajan and A. Schabel entitled NOVEL GLYCOPEPTIDE 
ANTIBIOTICS, Ser. No. 600,727, filed herewith this even date; and (3) 
antibiotic M43C and possibly antibiotic M43B, both of which are disclosed 
in the copending application of Karl H. Michel entitled ANTIBIOTICS M43B 
AND M43C, Ser. No. 600,726, also filed herewith this even date. The method 
of producing M43D and the other new M43 factors by fermentation of N. 
orientalis NRRL 2450 is described in the copending application of M. M. 
Hoehn and R. Nagarajan entitled METHODS FOR PRODUCING M43 ANTIBIOTICS, 
Ser. No. 600,728, filed herewith this even date. 
The structural relationships of this group of antibiotics are provided in 
formulas 1-12 which follow: 
__________________________________________________________________________ 
##STR3## 
##STR4## 
Compound No. 
Compound R R.sub.1 
R.sub.2 
R.sub.3 
n R.sub. 4 
__________________________________________________________________________ 
1 M43D H CH.sub.3 
CH.sub.3 
CONH.sub.2 
1 vancosaminyl-Oglucosyl 
2 Vancomycin H H CH.sub.3 
CONH.sub.2 
1 vancosaminyl-Oglucosyl 
3 M43A CH.sub.3 
CH.sub.3 
CH.sub.3 
CONH.sub.2 
1 vancosaminyl-Oglucosyl 
4 M43B CH.sub.3 
CH.sub.3 
CH.sub.3 
COOH 1 vancosaminyl-Oglucosyl 
5 M43C CH.sub.3 
CH.sub.3 
CH.sub.3 
CONH.sub.2 
1 glucosyl 
6 Agluco-A51568A H H H CONH.sub.2 
1 H 
7 Aglucovancomycin 
H H CH.sub.3 
CONH.sub.2 
1 H 
8 Agluco-M43A CH.sub.3 
CH.sub.3 
CH.sub.3 
CONH.sub.2 
1 H 
9 Desvancosamine-A51568A 
H H H CONH.sub.2 
1 glucosyl 
10 Desvancosamine-vancomycin 
H H CH.sub.3 
CONH.sub.2 
1 glucosyl 
11 A51568A H H H CONH.sub.2 
1 vancosaminyl-Oglucosyl 
12 A51568B H H H CONH.sub.2 
2 vancosaminyl-Oglucosyl 
__________________________________________________________________________ 
M43D, the new glycopeptide antibiotic of this invention, is very close in 
structure and activity to vancomycin and is, therefore, a valuable 
addition to this group of antibiotics. 
M43D is shown in formula 1 as a zwitterion. Those in the art will 
recognize, however, that M43D has a carboxyl group, two amino groups and 
three phenolic groups which can react to form various salts. All such 
forms of M43D are part of this invention. M43D salts are useful, for 
example, for separating and purifying M43D. In addition, the salts have an 
improved solubility in water. 
M43D salts are prepared using standard procedures for salt preparation. For 
example, the M43D zwitterion can be neutralized with an appropriate acid 
to form an M43D acid addition salt. 
The acid addition salts of M43D are particularly useful. Representative 
suitable salts include those salts formed by standard reactions with both 
organic and inorganic acids such as, for example, sulfuric, hydrochloric, 
phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, cholic, 
pamoic, mucic, D-glutamic, d-camphoric, glutaric, glycolic, phthalic, 
tartaric, formic, lauric, stearic, salicylic, methanesulfonic, 
benzenesulfonic, sorbic picric, benzoic, cinnamic and like acids. 
Pharmaceutically acceptable acid addition salts are an especially preferred 
group of salts of this invention. 
Antibiotic M43D is prepared by culturing Nocardia orientalis NRRL 2450, or 
an M43D-producing variant, mutant or recombinant thereof, under submerged 
aerobic conditions in a suitable culture medium until a substantial amount 
of M43D is produced. The culture medium used to grow Nocardia orientalis 
NRRL 2450 can be one of a number of media. For economy in production, 
optimal yield, and ease of product isolation, however, certain culture 
media are preferred. Thus, for example, preferred carbon sources include 
carbohydrates such as dextrin, dextrose, glucose and glycerol. Preferred 
nitrogen sources include enzyme digests of casein, cottonseed meal, 
soybean grits, protein peptones and the like. Among the nutrient inorganic 
salts which can be incorporated in the culture media are the customary 
soluble salts capable of yielding iron, potassium, sodium, magnesium, 
calcium, ammonium, chloride, carbonate, sulfate, nitrate, and like ions. 
Essential trace elements necessary for the growth and development of the 
organism should also be included in the culture medium. Such trace 
elements commonly occur as impurities in other constituents of the medium 
in amounts sufficient to meet the growth requirements of the organism. It 
may be necessary to add small amounts (i.e. 0.2 ml/L) of an antifoam agent 
such as polypropylene glycol (M.W. about 2000) to large-scale fermentation 
media if foaming becomes a problem. 
For production of substantial quantities of antibiotic M43D, submerged 
aerobic fermentation in tanks is preferred. Small quantities may be 
obtained by shake-flask culture. Because of the time lag in antibiotic 
production commonly associated with inoculation of large tanks with the 
spore form of the organism, it is preferable to use a vegetative inoculum. 
The vegetative inoculum is prepared by inoculating a small volume of 
culture medium with the spore form or mycelial fragments of the organism 
to obtain a fresh, actively growing culture of the organism. The 
vegetative inoculum is then transferred to a larger tank. The medium used 
for the vegetative inoculum can be the same as that used for larger 
fermentations, but other media can also be used. 
N. orientalis NRRL 2450 can be grown at temperatures between about 
25.degree. and about 37.degree. C. Optimum antibiotic production appears 
to occur at temperatures of about 30.degree. C. 
As is customary in aerobic submerged culture processes, sterile air is 
bubbled through the culture medium. For efficient antibiotic production 
the percent of air saturation for tank production should be about 50% or 
above (at 30.degree. C. and about 5 psi of back pressure). 
Antibiotic production can be followed during the fermentation by testing 
samples of the broth against organisms known to be sensitive to antibiotic 
M43D. One useful assay organism is Staphylococcus aureus NRRL B313. In 
addition, antibiotic production can be monitored by HPLC with UV 
detection. 
Following its production under submerged aerobic fermentation conditions, 
antibiotic M43D can be recovered from the fermentation medium by filtering 
the broth to remove mycelia and purifying the filtered broth by a series 
of adsorptions on suitable adsorbents, such as an ion-exchange resins, 
chemically modified hydrophobic inorganic supports used in high 
performance reversed-phase liquid chromatography, and high porosity 
polymers, eluting the M43D in each case with a suitable solvent such as 
aqueous acetonitrile. 
M43D inhibits the growth of a broad spectrum of pathogenic bacteria, 
especially gram-positive bacteria. Table I summarizes the minimal 
inhibitory concentrations (MIC's) at which M43D inhibits certain 
organisms, as determined by standard agar-dilution assays. In Table I the 
activity of M43D (phosphate salt) is compared with that of vancomycin 
(free base). 
TABLE I 
______________________________________ 
In Vitro Activity of M43D 
MIC (mcg/ml) 
Organism Vancomycin M43D 
______________________________________ 
Staphylococcus aureus NRRL B313 
0.5 1 
Staphylococcus aureus V41 
0.5 1 
Staphylococcus aureus X400 
1 2 
Staphylococcus aureus S13E 
1 1 
Staphylococcus epidermidis EPI1 
2 4 
Staphylococcus epidermidis 222 
1 2 
Streptococcus pyogenes C203 
0.5 0.5 
Streptococcus pneumoniae Park 1 
0.125 0.25 
Streptococcus faecium ATCC 9790 
1 2 
Streptococcus sp. group D 2041 
4 4 
Haemophilus influenzae C.L. 
64 &gt;64 
Haemophilus influenzae 76 
128 &gt;64 
Escherichia coli N10 
&gt;128 &gt;64 
Escherichia coli EC14 
&gt;128 &gt;64 
Escherichia coli TEM 
64 &gt;64 
Klebsiella pneumoniae X26 
&gt;128 &gt;64 
Klebsiella pneumoniae X68 
&gt;128 &gt;64 
Klebsiella pneumoniae KAE 
&gt;128 &gt;64 
______________________________________ 
Pharmaceutical formulations of M43D or salts of M43D (an M43D compound) are 
also part of this invention. M43D, preferably as a pharmaceutically 
acceptable salt, can be formulated for oral or parenteral administration 
for the therapeutic or prophylactic treatment of bacterial infections. For 
example, an M43D compound can be admixed with conventional pharmaceutical 
carriers and excipients and used in the form of tablets, capsules, 
elixirs, suspensions, syrups, wafers and the like. The compositions 
comprising an M43D compound will contain from about 0.1 to about 90% by 
weight of the active compound, and more generally from about 10 to about 
30%. The compositions may contain common carriers and excipients, such as 
corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, 
kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. 
Disintegrators commonly used in the formulations of this invention include 
croscarmellose sodium, microcrystalline cellulose, corn starch, sodium 
starch glycolate and alginic acid. Tablet binders that can be included are 
acacia, methylcellulose, sodium carboxymethylcellulose, 
polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, 
starch and ethylcellulose. Lubricants that can be used include magnesium 
stearate or other metallic stearates, stearic acid, silicone fluid, talc, 
waxes, oils and colloidal silica. Flavoring agents such as peppermint, oil 
of wintergreen, cherry flavoring or the like can also be used. It may be 
desirable to add a coloring agent to make the dosage form more esthetic in 
appearance or to help identify the product. 
For intravenous (IV) use, a water soluble form of the antibiotic can be 
dissolved in one of the commonly used intravenous fluids and administered 
by infusion. Such fluids as, for example, physiological saline, Ringer's 
solution or 5% dextrose solution can be used. 
For intramuscular preparations, a sterile formulation of a suitable salt 
form of M43D, for example the hydrochloride salt, can be formulated in a 
pharmaceutical diluent such as Water-for-Injection, physiological saline, 
5% glucose or as a suspension in an aqueous base or a pharmaceutically 
acceptable oil base, e.g. an ester of a long chain fatty acid such as 
ethyl oleate. 
For oral use, a sterile formulation of a suitable salt form of the 
antibiotic, for example, the hydrochloride salt, formulated in a diluent 
such as distilled or deionized water, is particularly useful. 
Alternatively, the unit dosage form of the antibiotic can be a solution of 
the antibiotic or preferably a salt thereof in a suitable diluent in 
sterile, hermetically sealed ampoules. The concentration of the antibiotic 
in the unit dosage may vary, e.g. from about 1 percent to about 50 percent 
depending on the particular form of the antibiotic and its solubility and 
the dose desired by the physician. 
In a further aspect, this invention provides a method for treating or 
controlling infectious diseases, especially those caused by gram-positive 
microorganisms, in animals. This method comprises administering to the 
animal an effective dose of an M434D compound. An effective dose is 
generally between about 0.5 and about 100 mg/kg of M43D or a 
pharmaceutically acceptable salt of M43D. A preferred dose is from about 
10 to about 60 mg/kg of M43D compound. A typical daily dose for an adult 
human is from about 250 mg to about 1.0 g. 
In practicing this method, the antibiotic can be administered in a single 
daily dose or in multiple doses per day. The treatment regime may require 
administration over extended periods of time, e.g., for several days or 
for from two to three weeks. The amount per administered dose or the total 
amount administered will depend on such factors as the nature and severity 
of the infection, the age and general health of the patient, the tolerance 
of the patient to the antibiotic and the microorganism or microorganisms 
involved in the infection. 
A convenient method of practicing the treatment method is to administer the 
antibiotic via IV infusion. In this procedure a sterile formulation of a 
suitable soluble salt of the antibiotic is incorporated in a solution of a 
physiological fluid, such as 5% dextrose solution, and the resulting 
solution is infused slowly IV. Alternatively, the piggy-back method of IV 
infusion can be used. 
In another embodiment, this invention relates to methods of increasing 
feed-utilization efficiency in poultry, swine, sheep and cattle, of 
promoting growth rates in cattle raised for meat production and of 
enhancing milk production in lactating ruminants. For increasing feed 
utilization efficiency and promoting growth, an M43D compound is 
administered orally in a suitable feed in an amount of from about 2 to 
about 200 grams per ton of total feed. For beef cattle, for example, a 
range of about 12 to 3000 mg/head/day is suitable. For enhancing milk 
production in lactating ruminants, oral administration of a daily amount 
of from about 0.04 to about 16 mg/kg of body weight (or about 25 to about 
5000 mg/ruminant/day) is suggested. 
The following examples are provided to illustrate this invention:

EXAMPLE 1 
Shake-flask Fermentation of N. orientalis M43-05865 to Produce M43D 
A lyophilized pellet of Nocardia orientalis M43-05865 (NRRL 2450) is 
dispersed in 1-2 ml of sterilized water. This solution (&lt;0.1 ml) is used 
to inoculate an agar slant having the following composition: 
______________________________________ 
Ingredient Amount (g/L.) 
______________________________________ 
Dextrin 10 
Enzymatic hydrolysate of casein.sup.a 
2 
Beef extract 1 
Yeast extract 1 
Agar 20 
Distilled water q.s. to 1 liter 
______________________________________ 
.sup.a N-Z Amine A, Humko Sheffield Chemical, Lyndhurst NJ 
The inoculated slant is incubated at 30.degree. C. for 4-6 days. The mature 
slant culture is covered with sterile distilled water and scraped with a 
loop to loosen the spores. The resulting spore suspension (1 ml) is used 
to inoculate 100 ml of a vegetative medium having the following 
composition: 
______________________________________ 
Ingredient Amount (g/L.) 
______________________________________ 
Glucose 15 
Soybean meal 15 
Cornsteep solids 
5 
CaCO.sub.3 2 
NaCl 5 
Tap H.sub.2 O q.s. to 1 liter 
______________________________________ 
The inoculated vegetative medium is incubated in a 500-ml Erlenmeyer flask 
for 24-48 hours at 30.degree. C. on a reciprocal shaker with a 2-inch 
stroke at 108 RPM or on a rotary shaker operating at 250 RPM. 
This incubated vegetative medium (5 ml) is used to inoculate 100 ml of a 
sterilized (120.degree. C. for 30 minutes) production medium having the 
following composition: 
______________________________________ 
Ingredient Amount (g/L.) 
______________________________________ 
Glucose 10 
Edible molasses 
20 
Peptone.sup.a 5 
CaCO.sub.3 2 
Tap H.sub.2 O q.s. to 1 liter 
______________________________________ 
.sup.a Bacto (Difco Laboratories, Detroit, MI) 
The inoculated fermentation medium is incubated in a 500-ml Erlenmeyer 
flask at 25.degree.-30.degree. C. for 72-96 hours on either a rotary 
shaker operating at 250 RPM or a reciprocal shaker operating at 108 
strokes per minute. The pH of the uninoculated medium varies with the 
medium used for production, but the production media of Examples 2-5 have 
an initial pH range of 6.0 to 7.5 and a harvest pH range of 6.5 to 8.0. 
EXAMPLE 2 
Isolation of Antibiotic M43D 
A. Isolation of M43 Complex 
Whole broth (2 L.), prepared as described in Example 1, was filtered. The 
filtrate was treated with a cation exchange resin (Dowex 50W-X4, H.sup.+, 
NH.sub.4.sup.+, pH 5.0), using 100-ml of resin and stirring batchwise for 
30 minutes. The effluent was decanted and discarded. The resin was washed 
thoroughly with water, and the water wash was discarded. The resin was 
then eluted batchwise with 1N NH.sub.4 OH (250 ml and 175 ml per batch). 
The eluates were combined and concentrated under vacuum to a volume of 
about 50 ml. An aliquot (2 ml) was removed for assay, and the remaining 
concentrate was lyophilized to give 300 mg of M43 complex. 
B. Analytical Separation of M43D 
M43 complex is examined by analytical HPLC, using the following system: 
______________________________________ 
Column: Beckman Ultrasphere (5.mu. particle size) 
ODS, 25 cm 
Mobile Phase: 
Solvent A: CH.sub.3 CN/TEAP (5:95) 
Solvent B: CH.sub.3 CN/TEAP (2:3) 
[TEAP = 0.5% aqueous triethylamine 
adjusted to pH 3 with conc phosphor- 
ic acid] 
Gradient: 9% B to 70% B over a 40-min period, 
then hold for 5 min. at 70% B 
Flow Rate: 1.0 ml/min. 
Detection: UV at 254 nm 
______________________________________ 
M43A Factor Retention Time (min.) 
______________________________________ 
A51568 factor B.sup.a 
5.92 
A51568 factor A 8.96 
vancomycin 12.23 
desvancosamine-A51568A 
17.59 
M43D 19.96 
desvancosamine-vancomycin.sup.a 
20.38 
M43A 24.26 
M43B.sup.a 25.46 
M43C.sup.a 29.58 
agluco A51568A 36.97 
aglucovancomycin 37.72 
agluco-M43A 39.79 
______________________________________ 
.sup.a trace amount 
C. Preparative Separation of M43D from Vancomycin 
Vancomycin hydrochloride (1.5 g) was dissolved in deionized water. The 
resulting solution was diluted with deionized water to a volume of 10 ml. 
This solution was passed through a 0.45-.mu.m membrane filter.sup.(1). The 
filtrate was chromatographed in 2-ml portions through a column 
(37.times.350 mm) containing Li Chroprep RP-18 (15-25 .mu.m).sup.(2) as 
the stationary phase. The column was eluted with an aqueous acetonitrile 
gradient containing 7.5-20% (v/v) of acetonitrile. The aqueous phase of 
the gradient contained triethylamine (0.2%) and was adjusted (prior to the 
acetonitrile addition) to pH 3 with 10% aqueous phosphoric acid. The 
column effluent was monitored by UV activity and separated into fractions 
based on this activity. The M43D-containing fractions from forty such 
separations were combined. The acetonitrile was removed by evaporation 
under high vacuum. Inorganic salts were removed by adsorption of M43D on 
Diaion HP-20 resin.sup.(3). The HP-20 resin was washed with water and 
subsequently eluted with water containing methanol (25-50% methanol v/v). 
The methanol was removed under high vacuum, and the resulting aqueous 
solution was freeze-dried to give M43D as a amorphous white solid (0.107 
g). 
FNT (1) Millex-HA 0.45 .mu.m filter unit, Millipore Corporation, Bedford, MA 
01730 
FNT (2) Art. #13901, E. Merck Darmstadt-Germany 
FNT (3) Mitsubishi Chemical Industries Limited, Tokyo 100, Japan 
Characteristics of M43D 
M43D has an integer molecular weight of 1461 as determined by 
fast-atom-bombardment mass spectrometry. The molecular weight of M43D is 
14 units higher than that of vancomycin (m.w. 1447). 
The proton NMR assignments for M43D are summarized in Table II. The 
chemical shifts listed in Table II were obtained in DMSO-d.sub.6 solution 
at 60.degree. C. and at 360 MHz proton frequency. The numbering scheme 
used is shown in formula 13: 
##STR5## 
TABLE II 
______________________________________ 
Proton NMR Assignments for M43D.sup.a 
Assignment Chem. Shift 
______________________________________ 
A-NH 6.51 
A-2' 4.19 
A-1' 5.12 
A-2 7.85 
A-5 7.30 
A-6 7.45 
B-NH 7.96 
B-1' 5.68 
B-2 5.57 
B-6 5.24 
C-NH 7.45 
C-2' 4.85 
C-1' 5.19 
C-2 7.52 
C-3 7.22 
C-6 7.30 
D-NH 8.34 
D-1' 4.45 
D-2 6.32 
D-4 6.38 
Asn-NH 6.63 
Asn-.alpha. 4.45 
Asn-.beta.'s 2.16 and 
Asn-.beta.'s 2.34 
2.31 
Leu-.alpha. 3.05 
Leu-.beta.'s 1.57 and 
1.39 
Leu-.gamma. 1.65 
Leu-67 's 0.87 and 
0.87 
Glucose 
#1 5.35 
#2 3.60 
#3 3.48 
#4 3.30 
#5 NA.sup.b 
#6 3.69 and 
3.58 
Vancosamine 
#1 5.24 
#2 1.80 and 
1.62 
#3(CH.sub.3) 1.24 
#4 NA 
#5 4.65 
#6(CH.sub.3) 1.07 
______________________________________ 
.sup.a Phenols not yet assigned 
.sup.b NA = not assigned 
EXAMPLE 3 
Antibiotic M43D prepared according to the method of Example 1, but using 
the following production medium: 
______________________________________ 
Ingredient Amount (g/L.) 
______________________________________ 
Glucose 10 
Yeast 5 
Distillers solubles 
5 
KCl 4 
CaCO.sub.3 1 
Tap H.sub.2 O q.s. to 1 liter 
______________________________________ 
EXAMPLE 4 
M43D prepared by the method of Example 1, but using the following 
production medium: 
______________________________________ 
Ingredient Amount 
______________________________________ 
Casamino acids 5 g/L 
Dextrin 5 g/L 
Glycerol 5 g/L 
Blackstrap molasses 
10 g/L 
Yeast 5 g/L 
K.sub.2 HPO.sub.4 
1 g/L 
Mineral Stock 5 ml 
Tap H.sub.2 O q.s. to 1 liter 
______________________________________ 
EXAMPLE 5 
M43D prepared by the method of Example 1, but using the following 
production medium: 
______________________________________ 
Ingredient Amount (g/L.) 
______________________________________ 
Soybean meal 15 
Casein 1 
NaNO.sub.3 3 
Glucose syrup 20 
Tap H.sub.2 O q.s. to 1 liter 
______________________________________ 
EXAMPLE 6 
M43D Tablet Formulation 
Preparation of tablets containing 250 mg of M43D: 
______________________________________ 
Ingredient Weight 
______________________________________ 
M43D diphosphate 283.2 mg 
Microcrystalline cellulose 
101.1 mg 
Croscarmellose sodium 
12.0 mg 
Providone 12.0 mg 
Magnesium stearate 3.0 mg 
Stearic acid 4.0 mg 
Purified water 0.16 ml 
______________________________________ 
Add M43D diphosphate, a portion of the microcrystalline cellulose and a 
portion of the croscarmellose sodium to a suitable container and blend 
until homogenous. Prepare a solution of Povidone in water, and add the 
Povidone solution to the blended powders. Granulate the resulting mixture, 
size if necessary and dry. Add the remaining microcrystalline cellulose 
and croscarmellose sodium to the dried mxiture and blend. Add magnesium 
stearate and stearic acid, and blend the mixture. Compress the resulting 
powder blend into tablets with a theoretical weight of 415 mg. Each tablet 
contains M43D diphosphate equivalent to 250 mg of M43D. 
EXAMPLE 7 
M43D Capsule Formulation 
______________________________________ 
Ingredient Weight 
______________________________________ 
M43D dihydrochloride 
262.3 mg 
Corn starch flowable powder 
137.65 mg 
Silicone fluid 350 centistokes 
2.75 mg 
Corn starch 147.1 mg 
______________________________________ 
Blend M43D dihydrochloride, starch flowable powder, silicone fluid 350 
centistokes and starch powder in a suitable mixer until homogeneous. Fill 
into appropriate size hard gelatin capsules to a net fill weight of 550 
mg. Each capsule contains M43D dihydrochloride equivalent to 250 mg of 
M43D. 
EXAMPLE 8 
M43D Liquid Formulation 
Prepare a sterile insoluble form of M43D by crystallization or 
precipitation. Mill or screen to a particle size suitable for suspension. 
Suspend the M43D in the following vehicle. 
______________________________________ 
Ingredient Amount 
______________________________________ 
Lecithin 1% 
Sodium citrate 2% 
Propylparaben 0.015% 
Water for injection 
q.s. to desired volume 
______________________________________ 
The suspension may be manufactured in bulk and filled into vials or may be 
prepared extemporaneously by adding the vehicle to the M43D in the vial.