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Claims  |
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What is claimed is:
1. A process of production of a pure homologous series of mono-,di-, tri-
and tetra- fatty acyl esters of sugar which comprises the following
successive steps:
a) mixing a fatty acyl chloride and an anhydrous sugar in a molar ratio of
about 3:1 in a first solvent containing pyridine, at a temperature
comprised between 60.degree. and 80.degree. C., for about eighteen hours,
whereby fatty acyl esters of sugar consisting of a homologous series of
mono- to octa- fatty acyl esters of sugars are produced;
b) evaporating said first solvent till the obtention of a dry residue;
c) extracting said dry residue with a second solvent that allows said first
solvent to solubilize while said fatty acyl esters of sugar remain in the
form of a precipitate, or
dissolving said dry residue in hot alcohol and pouring the solution so
obtained in a mixture of ice:water, whereby said first solvent solubilize
while the fatty acyl esters of sugar precipitate;
the steps a), b) and c) being followed by steps d) to g) or by steps d') to
f');
d) extracting said precipitate in a third solvent consisting of a hot
alcohol different from and more hydrophillic than the alcohol used in step
c), whereby the homologous series of mono- to tetra- fatty acyl esters of
sugar solubilize while the homologous series of penta- to octa- fatty acyl
esters of sugar remain in the form of a precipitate;
e) separating and cooling the so obtained solution down to O.degree. C.,
whereby said third solvent and remaining traces of said first solvent
solubilize while the homologous series of mono- to tetra- fatty acyl
esters of sugar precipitate;
f) extracting the precipitate so obtained in a fourth solvent of a
hydrophilicity such that the homologous series of di- to tetra- fatty acyl
esters solubilize while a precipitate consisting of a substantially pure
mono- fatty acyl ester of sugar remains in the precipitate in a
substantially pure form; and
g) fractionating and purifying the so obtained solution, whereby the pure
di-, tri- and tetra- fatty acyl esters of sugar are obtained separately;
d') extracting said precipitate in a fifth solvent of a hydrophilicity such
that the homologous series of di- to octa- fatty acyl esters of sugar
solubilize while the mono-fatty acyl ester of sugar remains in the form of
a precipitate, said precipitate being purified till a substantially pure
mono- fatty acyl ester of sugar is obtained;
e') submitting the so obtained solution to a fractionation step, first in a
sixth solvent of a hydrophilicity such that a first fraction containing
the homologous series of penta- to octa- fatty acyl esters of sugar is
obtained and second in a seventh solvent of a hydrophilicity such that a
second fraction containing the homologous series of di- to tetra- fatty
acyl esters of sugar is obtained; and
f') submitting said second fraction to a further fractionation step,
whereby the di-, tri- and tetra-fatty acyl esters of sugar are obtained in
separate fractions, which fractions are further purified to obtain the
substantially pure homologous series of di-, tri- and tetra- fatty acyl
esters of sugar.
2. A process according to claim 1 wherein said fatty acyl chloride is
selected from the group consisting of myristyl, lauryl, palmitoyl,
stearyl, oleyl, linolyl and linolenyl chlorides.
3. A process according to claim 1 wherein said sugar is selected from the
group consisting of glucose, arabinose, maltose, galactose, mannose,
cellobiose and lactose.
4. A process according to claim 2 wherein said fatty acyl chloride is
palmitoyl chloride.
5. A process according to claim 3 wherein said sugar is maltose.
6. A process according to claim 1 wherein said fatty acyl esters of sugar
are maltose palmitates.
7. A process according to claim 1 wherein said first solvent contains
dimethylformamide and pyridine and the temperature is 60.degree. C.
8. A process according to claim 1 wherein said first solvent consists of
pyridine and the temperature is 80.degree. C.
9. A process of production of a pure homologous series of mono-, di-, tri-
and tetra- palmitates of maltose which comprises the following successive
steps:
a) mixing palmitoyl chloride and an anhydrous maltose in a molar ratio of
about 3:1 in a first solvent consisting of pyridine, at 80.degree. C., or
of a mixture of dimethylformamide and pyridine at 60.degree. C., for
eighteen hours, whereby maltose palmitates consisting of a homologous
series of mono- to octa- palmitates of maltose are produced;
b) evaporating said first solvent till the obtention of a dry residue;
c) extracting said dry residue with diethyl ether or in a second solvent of
similar hydrophilicity that allows said first solvent to solubilize while
maltose palmitates remain in the form of a precipitate, or
dissolving said dry residue in hot ethanol and pouring the solution so
obtained in a mixture of ice:water, whereby said first solvent solubilize
while maltose palmitates precipitate;
the steps a), b) and c) being followed by steps d) to g) or by steps d') to
f');
d) extracting said precipitate in hot methanol, whereby the homologous
series of mono- to tetrapalmitates of maltose solubilize while the
homologous series of penta- to octa- palmitates of maltose remain in the
form of a precipitate;
e) separating and cooling the so obtained solution down to O.degree. C.,
whereby methanol and remaining traces of said first solvent are solubilize
while the homologous series of mono- to tetra- palmitates of maltose
precipitate;
f) extracting the precipitate so obtained in a fourth solvent consisting of
heptane:ethanol 95:5 (v:v) or a solvent of similar hydrophilicity, whereby
the homologous series of di- to tetra- palmitates of maltose solubilize
while maltose monopalmitate remains in the precipitate in a substantially
pure form; and
g) fractionating the so obtained solution by high-performance liquid
chromatography in a gradient solvent system of heptane:ethanol from 95:5
to 50:50 (v:v) or a gradient solvent system of similar hydrophilicity,
whereby separate fractions consisting of di-, tri- and tetra- palmitates
of maltose are obtained and further purified till the obtention of pure
homologous series of di-, tri- and tetra- palmitates of maltose;
d') extracting said precipitate in a fifth solvent consisting of
heptane:ethanol 95:5 (v:v) or a solvent of similar hydrophilicity, whereby
the homologous series of di- to octa- palmitates of maltose solubilize
while maltose monopalmitate remains in the form of a precipitate, said
precipitate being purified till a substantially pure maltose monopalmitate
is obtained;
e') submitting the so obtained solution to a fractionation step by flash
chromatography in a step gradient solvent system of
dichloromethane:methanol from 97:3 to 85:15 (v:v) or a gradient system of
similar hydrophilicity, whereby a first fraction containing the homologous
series of penta- to octa- palmitates of maltose is obtained and a second
fraction containing the homologous series of di- to tetra- palmitates of
maltose is obtained; and
f') submitting said second fraction to a further fractionation step by
high-performance liquid chromatography in a gradient solvent system of
heptane:ethanol from 95:5 to 50:50 (v:v) or a gradient solvent system of
similar hydrophilicity, whereby separate fractions consisting of di-, tri-
and tetrapalmitates of maltose are obtained and further purified till the
obtention of pure homologous series of di-, tri- and tetra- palmitates of
maltose.
10. A substantially pure homologous series of mono-, di-, tri- and tetra-
acyl esters of sugar prepared by the process of claim 1.
11. A substantially pure homologous series of mono-, di-, tri- and tetra-
palmitoyl esters of sugar prepared by the process of claim 4.
12. A substantially pure homologous series of mono-, di-, tri- and tetra-
palmitates of maltose prepared by the process of claim 6.
13. A substantially pure homologous series of mono-, di-, tri- and tetra-
palmitates of maltose prepared by the process of claim 9.
14. A composition for use in the treatment of an angiogenesis-dependent
pathology comprising the di-, tri- and tetra-acyl esters of sugar of claim
10.
15. A composition for use in the treatment of an angiogenesis-dependent
pathology comprising the di-, tri- and tetra-palmitates of sugar of claim
11.
16. A composition for use in the treatment of an angiogenesis-dependent
pathology comprising the di-, tri- and tetra-palmitates of maltose of
claim 12.
17. A composition for use in the treatment of an angiogenesis-dependent
pathology comprising the di-, tri- and tetra-palmitates of maltose of
claim 13.
18. A composition according to claim 16 comprising di-: tri-: tetra-
palmitates of maltose in the following respective percentages of weight
15-30: 25-50: 30-60.
19. A composition according to claim 17 comprising di-: tri-: tetra-
palmitates of maltose in the following respective percentages of weight
15-30: 25-50: 30-60.
20. A composition according to claim 14 further comprising an angiostatic
steroid.
21. A composition according to claim 15 further comprising an angiostatic
steroid.
22. A composition according to claim 16 further comprising an angiostatic
steroid.
23. A composition according to claim 17 further comprising an angiostatic
steroid.
24. A composition according to claim 18 further comprising an angiostatic
steroid.
25. A composition according to claim 19 further comprising an angiostatic
steroid.
26. A process of production of a pure homologous series of mono-, di-, tri-
and tetra- fatty acyl esters of a sugar which comprises the following
successive steps:
(a) mixing a fatty acyl chloride and an anhydrous sugar in a molar ratio of
about 3:1 in a first solvent containing pyridine, at a temperature between
60.degree. and 80.degree. C., for about eighteen hours, whereby fatty acyl
esters of sugar consisting of a homologous series of mono- to octa- fatty
acyl esters of sugars are produced;
(b) evaporating said first solvent till the obtention of a dry residue;
(c) extracting said dry residue with a second solvent that allows said
first solvent to solubilize while said fatty acyl esters of the sugar
remain in the form of a precipitate, or
dissolving said dry residue in hot alcohol and pouring the solution so
obtained in a mixture of ice:water, whereby said first solvent solubilize
while the fatty acyl esters of sugar precipitate;
(d) extracting said, precipitate with a third solvent consisting of a hot
alcohol different from and more hydrophillic than the alcohol used in step
(c), whereby the homologous series of mono- to tetra- fatty acyl esters of
the sugar solubilize while the homologous series of penta- to octa- fatty
acyl esters of the sugar remain in the form of a precipitate;
(e) separating and cooling the so obtained solution to 0.degree. C.,
whereby said third solvent and remaining traces of said first solvent
solubilize while the homologous series of mono- to tetra- fatty acyl
esters of the sugar precipitate;
(f) extracting the precipitate so obtained in a fourth solvent of a
hydrophilicity such that the homologous series of di- to tetra- fatty acyl
esters solubilize while a precipitate consisting of a substantially pure
mono- fatty acyl ester of the sugar remains in the precipitate in a
substantially pure form; and
(g) fractionating and purifying the so obtained solution, whereby the pure
di-, tri- and tetra- fatty acyl esters of the sugar are obtained
separately. |
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Claims |
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Description |
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Methods for the preparation of pure homologous series of mono to tetra
fatty acyl esters of sugars; characterization of one antitumor component
as maltose 1, 6, 6' tripalmitate; and pharmaceutical formulations useful
in the treatment of cancer.
BACKGROUND OF THE INVENTION
In 1982, a Canadian patent No. 1,120,399 entitled "Pharmaceutical
composition for treatment of tumor cells" by Nigam, Vijai N. and
Brailovsky, Carlos, A. was granted to Universit e de Sherbrooke. It
described the administration of certain fatty acid esters of mono and
disaccharides which surprisingly elicit an antitumor response as shown by
an enhancement of the host capacity to reject a large number of tumor
cells, to retard growth in tumor size and to induce hemorrhagic tumor
necrosis. Fatty acid esters of maltose, galactose, glucose, mannose,
arabinose, cellobiose and lactose were particularly useful when the fatty
acid comprised 12 to 18 carbon atoms.
At that time the method employed for the preparation of these compounds was
not patented since it consisted in a conventional methodology without
regards to providing stringent purity requirement and optimal yields. The
method that was based on the deployment of a known solvent system used for
thin layer chromatography (TLC) on silica gel G plates. It employed a
silica gel column instead of silica gel plates, thereby avoiding scraping
of bands from plates, and obtained larger amounts of the product. A
fraction isolated from the column that gave a thick band of Rf 0.68 on TLC
plates and a ratio of glucose to palmitic acid of approximately 0.5 was
referred to as maltose tetrapalmitate. The compound prepared accordingly
was used for numerous biological experiments.
It was further surprisingly noted that the fraction referred to as maltose
tetrapalmitate and used in biological investigation was indeed not pure
and, upon rechromatography on TLC plates in other solvent systems,
provided 2-3 bands.
In addition, it was further noted that the band of Rf 0.68 in CHCl.sub.3 :
MeOH:H.sub.2 O (60:25:4) solvent system, upon scraping from unstained
silica gel plates could be split into 3 bands upon rechromatography in
other solvent systems as well. In addition, it contained small amounts of
palmitic acid. These results which came as a surprise established that
there may be an association between the three components due to
hydrophobic interaction because of the presence of water in the solvent
system which was comprised of CHCl.sub.3 :MeOH:H.sub.2 O, 60:25:4. To our
satisfaction, the property of hydrophobic interactions between various
bacterial lipid As (which are structurally similar to maltose palmitates)
and the splitting of single bands upon rechromatography had been described
before (Chen et al. J. Infect. Dis. 128: 543-551, 1973).
The question confronting us was to find the identity of the three
components and to find out the most active antitumor component among them,
characterize it, and to see if associating them in various ratios provided
a more active product than individual components as far as its antitumoral
activity in vivo and its solubility in aqueous solvent were concerned. To
date, we have seen no report for the isolation of pure fractions of
maltose palmitates which have been structurally well characterized and
have been tested for their antitumor activity. One report based on our
initial finding described antitumor activity of maltose mono fatty acyl
esters which was superior to maltose poly fatty acyl esters but the
components were not well characterized and the purification procedure used
was column chromatography similar to the one used by us (Nishikawa, Y.,
Yoshimoto, K., Nishijima, M., Fukuoka, F. and Ikekawa, T. Chem. Pharm.
Bull. 29 (2): 505-513, 1981). Mono esters are mild detergents and could
lyse tumor cells at the high concentrations used by those researchers.
Another remarkable finding made by us (Anticancer Res. 9:1883-1888, 1989)
was that a combination of crude MTP with cortisone or .alpha.-OH
progesterone or tetrahydro S resulted in a high antitumor activity and it
was interpreted as being caused by the antiangiogenic activity of the
combination, rather than immune stimulating activity, since hydrocortisone
is known to be highly immunosuppressive.
It should be noted that both the Health Protection Branch (HPB) in Canada
and FDA in U.S.A. insist on the use of characterized products for human
use, especially when they are prepared by synthetic routes. The use of
uncharacterized mixtures alone or in formulations is not permitted. The
major problem in the use of uncharacterized partially purified mixtures of
substances is badge variation and the presence of impurities that may
remain associated within the mixture and elicit toxic reactions on dose
escalation and upon chronic use. In our case, once it became apparent that
our column prepared MTP (as described in our previous patent) was not a
single component, there was no excuse for not identifying and
characterizing the individual components and finding which was the active
one. Indeed, as detailed later, it became clear that the most active
component was maltose tripalmitate, rather than maltose tetrapalmitate (as
previously thought) and that maltose tetrapalmitate could not even be
administered due to its lack of solubility. Maltose tetrapalmitate when
emulsified could be injected intraperitoneally (ip) and its activity was
derived from its transformation to the maltose tripalmitate. Thus the
claim in our earlier patent stating that the active component was maltose
tetrapalmitate was only partly true.
It seemed to us that:
1) new methods of purification must be devised using HPLC, which would have
a high capacity of resolution and which can be adapted for future large
scale isolation of the components, especially with the new pilot plant
HPLC separation equipment provided by Waters Inc.;
2) the activities of purified components, individual maltose palmitates,
should be tested in the presence and absence of hydrocortisone to see if
their activity is based on immunological stimulation, or based on
antiangiogenic activity;
3) the active antitumor agents should be structurally characterized after
their separation on HPLC using chemical means; and
4) a pharmaceutical formulation should be made to take into account the
distribution of individual maltose palmitates in various organs, the rate
of degradation of higher palmitate esters into lower palmitate esters, and
a good solubility or dispersibility. This would lead to suggested optimum
dose and/or delivery rate devised specifically for cancers of different
organs.
The isolation of the three individual products contained in the previously
described MTP was attained in a long and painstaking manner. The use of
HPLC as a chromatographic tool for separation of closely related
substances is well known and the resolving power of HPLC surpasses those
of other chromatographic techniques. However, finding the appropriate
support systems and solvents requires numerous trials to arrive at the
most suitable combinations. Up to date, to our knowledge, fatty acyl
esters of sugar have not been subjected to rigorous separation and few, if
any, have been structurally characterized with respect to the position of
esterification. Most characterizations end up solely with the number of
fatty acid residue per mono or disaccharide molecule.
STATEMENT OF THE INVENTION
The systems developed by us for the separation of mixtures of fatty acyl
maltose are unique insofar they define a specific scheme used in their
separation. First, the dimethyl formamide use as a solvent has been
eliminated in Method 3 which also provides maximum maltose tripalmitate
yield. The mono acylated product is isolated by solvent fractionation and
only the di, tri and tetra acylated components require the use of HPLC The
solvent systems hereinbelow described for HPLC separation provide the most
effective method of separation of these three components.
The possibility that new commercially feasible solvent systems can be
developed in the future for the separation of fatty di, tri and tetra acyl
disaccharides by HPLC is remote since (i) the new solvents will be of
higher molecular weight and higher boiling point since we have already
tried low molecular weight low boiling point solvents acceptable to HPB,
Canada, (ii) they will not be cost effective with respect to availability,
price and their removal from isolated fatty acyl esters, (iii) their toxic
nature could be a barrier if traces remain associated with the purified
fatty acyl esters and, (iv) they have to be those which are approved by
HPB for use in drug purification.
DESCRIPTION OF THE INVENTION
The present invention is hereinbelow described in the following Figures and
specific embodiments, which purpose is to illustrate this invention rather
than to limit its scope.
METHOD 1
Improved and commercially applicable methods of preparation of maltose
palmitates.
Ten millimole (10 mmole) dry maltose (dried by trituration of commercial
maltose hydrate with distilled pyridine and evaporation of pyridine under
reduced pressure) was added to 40 ml distilled dimethyl formamide (DMF)
followed by 4 ml of distilled pyridine. Forty millimole (40 mmole)
palmitoyl chloride (Aldrich Chem. Co.) was added to the solution dropwise
with stirring. The reaction was allowed to proceed overnight (14-16 h)
with stirring at 60.degree. C. in the hood. Fifty ml toluene was added to
the reaction flask and the mixture was then rotary evaporated at
60.degree. C. which resulted in an azeotropic removal of excess pyridine
and DMF. This step was repeated with two (2) more 50 ml portions of
toluene. The crude reaction product (16.5 g) was dissolved in 75 ml
chloroform, followed by the addition of 50 g 70-230 mesh silica gel
(Merck). The mixture was rotary evaporated at 40.degree. C. to affect
suspension of the crude product on the solid silica base. The suspended
product was placed in a Buchner funnel and washed with 4 liters of warm
(30.degree. C.) distilled water. This step was required to remove pyridine
hydrochloride (4 g) which was a reaction product, free maltose (only
minute amounts were found), maltose mono palmitates, any remaining DMF and
pyridine and to convert any free palmitoyl chloride to palmitic acid. The
suspended product was then rotary evaporated at 40.degree. C. to remove
water, placed in a Buchner funnel and washed with chloroform to remove
palmitic acid (recovery 4.5 g), followed by elution of the product
(maltose palmitates referred to as glycolipids) with chloroform: methanol
(1:1). The dissolved product was filtered through a glass fiber filter to
remove particles of silica gel and rotary evaporated to dryness followed
by vacuum desiccation (48 h). Final traces of impurities were removed by
dissolving the product in hot (60.degree. C.) ethanol and cooling to
0.degree. C., which resulted in the precipitation of the product. The
product (glycolipid mixture of di, tri and tetra palmitates of maltose)
was recovered by suction filtration. This step was repeated once more. The
recovery was 7 g. The product gave 4 to 5 bands on TLC using chloroform:
methanol (9: 1) as the developing solvent.
Separation of glycolipids into groups of di, tri and tetra palmitoyl
maltose by flash chromatography.
A 5 cm i.d..times.45 cm glass column fitted with an air flow adapter was
packed with 20 cm of dry silica gel G (Merck 0.040-0.063 mm). Five mm of
washed sand was placed on top of the bed followed by 400 ml of chloroform
(CHCl.sub.3). Using an air pressure of 10 psi, the CHCl.sub.3 was washed
through the bed thus packing and equilibrating the column. Five (5.0) gm
maltose palmitate mixture prepared as described above was dissolved in 10
ml of chloroform and the dissolved sample was pushed into the top of the
bed. Fractions were eluted using 400 ml of each of the following solvents
in order of increasing polarity except for the first solvent which was 600
ml: (1) CHCl.sub.3 ; (2) CHCl.sub.3 :MeOH, (99:1); (3) CHCl.sub.3 : MeOH,
97:3; (4) CHCl.sub.3 : MeOH, 93:7; (5) CHCl.sub.3 : MeOH, 85:15; (6)
CHCl.sub.3 : MeOH, 65:35; and (7) CHCl.sub.3 : MeOH, 50:50. The volume of
each fraction was 30 ml and the number of fractions collected was 92. The
fractions were spotted on 20.times.20 cm glass supported silica gel 60
plates (0.25 mm layer--E. Merck) plate. Fractions 1-23 were developed in
CHCl.sub.3 : MeOH (99:1), fractions 24-46 in CHCl.sub.3 : MeOH (98:1) and
fractions 47-92 in CHCl.sub.3 : MeOH (90:10). Following development, the
plates were dried and sprayed with a solution of 0,025 M ceric sulfate and
0.02 M ammonium molybdate in 10% H.sub.2 SO.sub.4, followed by heating the
plates at 100.degree. C. for 5 min in an oven. The spray reagent reacted
with maltose palmitates to give a blue color when heated. Fractions with
identical Rf values were combined, and the combined fractions were
analysed on TLC alongside individually purified bands of determined
palmitic acid/maltose ratios. They were obtained upon scraping of single
bands and running in 2-solvents till no split of bands occurred. The
fractions were recrystallized in 95% ethanol, suction filtered and then
dried in a vacuum desiccator. The distribution of palmitate was as
described in Table 1.
Preparation of maltose tetra and tripalmitate mixture from crude maltose
palmitates
5 g maltose palmitates was applied to a flash chromatography column as
described above. The hexa and penta palmitates of maltose were eluted with
300 ml CHCl.sub.3, followed by eluting tetra and tri palmitates with 400
ml CHCl.sub.3 : MeOH (97:3), The di and mono palmitates were eluted with
400 ml CHCl.sub.3 : MeOH (50:50), The column was made ready for another
purification by reequilibrating with 400 ml CHCl.sub.3. The fractions were
filtered through a glass fiber filter and the products and solvents were
recovered by rotary evaporation. The mixture of maltose tetra and
tripalmitates was then subjected to purification by HPLC as described
below,
TABLE 1
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Flash chromatography of maltose palmitates (for details see text) The
fractions were combined on
the basis of their composition when chromatographed on TLC. Sample
applied was 5.5 g (NMR ratio 2.3)
Weight of Palmitate
Number product maltose
of tubes
Fraction
Eluting solvent(s)
recovered ratio by
combined
No. and their volume
(mg) % yield
NMR Interpretation
__________________________________________________________________________
1-32
1 CHCl.sub.3, (600 ml)
211 (3.83)
4.2 Mostly tetra +
CHCl.sub.3 :MeOH (99:1) (270 ml)
little hexa-penta
33-41
2 CHCl.sub.3 :MeOH (99:1) 130 ml +
565 (10.27)
3.6 Tetra + Tri
CHCl.sub.3 :MeOH (97:3) - 150 ml
42-43
3 CHCl.sub.3 :MeOH (97:3) (60 ml)
306 5.57 3.4 Tetra + Tri
44-49
4 CHCl.sub.3 :MeOH (97:3) (190 ml)
495 8.98 2.8 Tri + Di
50-52
5 CHCl.sub.3 :MeOH (97:3) (90 ml)
449 8.16 2.5 Tri + Di
53-56
6 CHCl.sub.3 :MeOH (97:3) (120 ml)
1376 25.03
2.3 Di + Tri
57-60
7 CHCl.sub.3 :MeOH (97:3) (120 ml)
437 8.86 2.2 Di + Tri
61-69
8 CHCl.sub.3 :MeOH (97:3) (70 ml)
554 10.08
1.8 Di + Mono
CHCl.sub.3 :MeOH (85:15) (210 ml)
70-75
9 CHCl.sub.3 :MeOH (85:15) (210 ml)
597 10.86
1.5 Di + Mono
76-77
10 CHCl.sub.3 :MeOH (85:15)
114 -2.07
--
78-79
11 CHCl.sub.3 :MeOH (85:15)
290 -5.27
1.3 Di + Mono
80-82
12 Wash CHCl.sub.3 :MeOH (1:1)
330 6.0 --
TOTAL 5724 104
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Isolation of MTP by high performance liquid chromatography (HPLC)
One hundred mg of mixed fraction No. 2 (tubes 33-41) giving a palmitate
maltose ratio of 3.6 (Table I) was subjected to HPLC. The equipment used
was a Waters model 6000A solvent delivery system. The column was a
stainless steel 0.5 cm.times.25 cm column packed with 5.mu. silica
supplied by YMC Inc., Morris Plains, N.J., U.S.A. The detector used was a
Varian model RI-3 refractive index detector. The column was washed and
equilibrated with chloroform methanol (99.5:0.5) and the flow rate was
adjusted to 4.0 mVmin. Fractions were collected in tubes at the top of the
peaks and at the drop in the peak or appearance of a shoulder. This
allowed pure as well as mixed fraction (of two components) to be
recovered. Thus 6-7 fractions were collected up to 11.5 min. The next was
the predominant fraction which had a peak at 14.5 min (FIG. 1). All the
fractions were evaporated and weighed. The major fraction (peak at 14.5
min) was 70 mg providing a yield of 70%. It gave a single band on TLC as
shown in FIG. 2. On NMR analysis the product gave a palmitate maltose
ratio of 3.7 (NMR determinations are usually underestimates of fatty acid
protons) allowing it to be designated MTP. The overall yield of MTP can be
regarded as 7 percent of the crude mixed maltose palmitates. This yield
can be increased to about 10 percent if fractions at the shoulder of MTP
peak are combined and then resubjected to HPLC.
METHOD 2
The individual steps are described below in greater detail.
Step 1. Dehydration of maltose and storage of the anhydrous maltose
Maltose monohydrate (Fisher M-75, Montr eal, Qu e.) is transformed into its
anhydrous form by triturating 500 g lots of maltose monohydrate with 100
ml of freshly distilled pyridine (Fisher Scientific, Montr eal, Qu e.).
The suspension is subjected to evaporation under reduced pressure in a
rotary evaporator at 40.degree. C. bath temperature. This procedure
results in the azeotropic removal of the water of crystallization along
with the pyridine into the distillate. The resulting dry maltose powder
can then be stored in a vacuum desiccator over drierite at room
temperature for lengthy periods (>2 years).
Step 2. Preparation of maltose palmitates
Anhydrous maltose (100 g=293 mmol) is added to a 2-l round bottomed flask
and 750 ml of distilled dimethyl formamide (Anachemia, Montr eal, Qu e.)
is gently poured into the flask followed by the addition of 250 ml of
distilled pyridine. The contents are kept over a heating pad at 60.degree.
C. to affect dissolution of maltose. A 400 ml separatory funnel is then
adjusted over the flask and to it is added 250 g (250/1.45=1712 ml) of a
mixture of palmitoyl chloride (Aldrich P. 78) and 100 ml of distilled
dimethyl formamide. The palmitoyl chloride is released drop by drop and
the contents stirred by holding the flask over a magnetic stirring device,
during a period of 30 min-1 hr. A fluffy precipitate that forms is allowed
to dissolve before allowing additional amounts of palmitoyl chloride into
the reaction mixture. When all the palmitoyl chloride has been added to
the flask it is transferred to the heating pad and the rheostat adjusted
so that the temperature is raised to 60.degree. C. The flask is then
shifted to an oven maintained at 60.degree. C. It is kept there for 18 h.
After standing at 60.degree. C. for 18 h, the contents are brought to room
temperature and gently added to ice-water mixture (2000 ml) in a 4-l
beaker. A large amount of precipitate is formed. The contents are then
transferred to a freezer at -20.degree. C. After 2 hours, the solids are
filtered over a cooled Bucher funnel and the material is copiously washed
with cold distilled water. The solid retained over the Buchner funnel is
allowed to dry and stored at 4.degree. C. in a desiccator. The yield of
the solids varies from 325 to 350 g. Analysis of the crude maltose
palmitates reveals at least 15 bands on TLC. The crude maltose palmitates
are stable and give the same profile on HPLC for at least 9 months, a
period during which we compared its stability.
Step 3. Pre-HPLC fractionation of crude maltose palmitates
Crude maltose palmitates contain a mixture of palmitates ranging from
maltose octa to mono palmitate as well as traces of dimethyl formamide
(DMF) and pyridine. The maltose tetra, tri and di palmitates required for
the drug formulation are isolated from the crude maltose palmitates by a
three-step process.
In the first step, 300 g are extracted with 1000 ml of hot (60.degree. C.)
methanol in which maltose octa to penta palmitates are insoluble and are
thereby eliminated. In the second step, the hot methanol extract is cooled
to 0.degree. C. which results in the precipitation of maltose tetra to
mono palmitates, while DMF, pyridine and any other methanol soluble
impurities remain in solution. The precipitate is filtered and then dried
by vacuum desiccation. In the third step, the precipitate is extracted
with heptane:ethanol (95:5) in which maltose mono palmitate is insoluble.
The extract is filtered and solvents are removed from the filtrate by
rotary evaporation. The recovered product weighs about 100 g (33%) and
contains approximately 95% of the maltose tetra, tri and di palmitates
present in the crude reaction product, and which are present in the ratio
50:35:15 based on HPLC integration data.
Step 4. Purification of maltose di, tri and tetrapalmitates by HPLC
A PREP LC 3000 80-ml P/N WAT088656 HPLC system with a M59OEEF advanced
solvent delivery system (P/N WAT089302), a fraction collector (P/N
WAT007441), a M1000 PREP PAK module (P/N WAT089592) and a variable UV
detector (P/NWAT098293) and Prepak Silica Cartridge and Column (P/N
WAT020732) constitute an entire HPLC purification system for separating
maltose palmitates on a semi pilot plant scale. This equipment can load 5
g of the mixture and separate it into its individual components in less
than 30 minutes and therefore can process 80 g of maltose palmitates per
day.
However, for dealing with small quantities of the material a 10 ml/min
delivery system is currently used for providing MTP formulation for animal
experiments. The procedure for purifications, which is now standardized is
as follows:
Materials: The High Performance Liquid Chromatography System used consists
of:
1 System controller (Waters)
1 Data module (Waters)
2 Model 6000 pumps (Waters)
1 U6K Sample injector (Waters)
1 Fixed Wavelength W detector (Waters)
1 10 mm.times.250 mm normal phase HPLC column (YMC A-023 Sil)
1 3.9 mm.times.150 mm reverse phase HPLC column (Waters Resolve C-18)
______________________________________
Solvents used are:
n-Heptane HPLC (Baker)
Absolute Ethanol
Methanol Accusolu (Anachemia)
______________________________________
Partly purified maltose palmitates were extracted with heptane: ethanol
(95:5) and the concentration of the extract was adjusted to 40 mg/ml.
The HPLC system was programmed to deliver the linear gradients shown in
FIG. 4 at a flow rate, of 2 ml/min.
The gradient is described below in the form of a table.
______________________________________
Time % %
(Minutes) Heptane Ethanol
______________________________________
0 95 5
22 93 7
30 85 15
38 50 50
39 95 5
______________________________________
A sample of 10 mg crude maltose palmitates (250 .mu.l heptane: ethanol
extract) was applied to the system which employed the use of
semipreparative silica gel column (described above), and fractions of the
major components were collected manually. The palmitate/maltose ratio of
the fractions was determined by NMR analysis as shown in the following
table:
______________________________________
Palmitoyl
Elution time (Minutes)
residues/maltose
Fraction of components unit
______________________________________
1 25.51 and 26.77
4
2 38.11 3
3 42.59 2
______________________________________
On the basis of the palmitate/maltose ratio fractions 1, 2 and 3 were
termed maltose tetrapalmitate, maltose tripalmitate and maltose
dipalmitate respectively. However, since the components of fraction 1 were
only partially resolved by this technique further purification of this
fraction by reverse phase chromatography was performed.
Reverse phase chromatography of fraction 1 was performed using methanol as
the eluent at a flow rate of 0.5 mL/minute while employing a C-18 column
(described above). Application of a 1 mg sample of fraction 1 in 250 .mu.l
methanol resulted in the separation of the two components or isomeric
maltose palmitates. The elution times of the completely resolved
components is shown in the following table:
______________________________________
Relative %
Elution time
(by integration
Component (Minutes) of peaks)
______________________________________
1 16.42 31.35
2 20.92 68.65
______________________________________
FLOW CHART FOR PREPARATION OF MALTOSE PALMITATES
Mixture of palmitoyl chloride and maltose in a 3:1 molar ratio.
1) stir in excess pyridine and dimethyl formamide at 60.degree. for 18 h.
2) evaporate in vacuo to remove solvents
Residue
1) dissolve in minimum amount of hot ethanol and pour into cold water
2) filter and dry the resulting precipitate H.sub.2 O precipitate filtrate:
water, dimethyl formamide and pyridine extract with hot (60.degree.)
methanol
______________________________________
methanol extract:
insoluble residue: maltose penta-hexa,
hepta- and octa palmitates
______________________________________
1) cool to 0.degree.
2) filter and dry precipitate
______________________________________
Methanol Precipitate:
Filtrate: methanol, traces of dimethyl
maltose mono, di, tri
formamide and pyridine
and tetra palmitates
______________________________________
extract with heptane: ethanol (95:5)
insoluble residue: maltose monopalmitate
heptane: ethanol extract: maltose tetra, tri and di palmitates
______________________________________
solvent 1) gradient HPLC: heptane:ethanol (95:5)
recovery to heptane:ethanol (50:50)
by 2) collect isometrically pure fractions
distillation of maltose tetra, tri and dipalmitate
3) remove solvent by evaporation in vacuo
4) crystallize in 95% ethanol
______________________________________
Formulation of tetra:tri:di palmitoyl maltose (30:50:20)
METHOD 3
Preparation of anhydrous maltose
It was prepared by dissolving commercial maltose hydrate (Fisher M-75) in
pyridine and evaporating the solution under reduced pressure in a vacuum
evaporator at 40.degree. C. to achieve azeotropic removal of water of
crystallization.
Palmitoyl chloride:
It is used as supplied by Aldrich Chem. Co., Cal. N2 P-78.
Procedure of preparation of maltose palmitates:
I. Anhydrous maltose (1 mmole) and palmitoyl chloride (3 mmole) are each
dissolved in a small volume of pyridine and the mixture is brought to
80.degree. C. and stirred for 18 hours under a moisture free nitrogen
atmosphere. At the end of 18 h, the mixture was subjected to evaporation
to remove the pyridine under reduced pressure.
II. The residue was extracted with diethyl ether and then filtered to
remove pyridine hydrochloride.
III. The filtrate was evaporated to dryness and the residue was extracted
with heptane-ethanol (95:5) and filtered to remove maltose mono palmitate
which forms 20 percent of the mixed maltose palmitates (w/w). This
procedure provides an easy method for the preparation of maltose
monopalmitate.
IV. The heptarte-ethanol extract was re-evaporated and the residue was
separated into two fractions using flash chromatography on a silica gel G
(Merck 9385) as the solvent. The first fraction (fraction A) was obtained
by elution of the column with dichloromethane: MeOH (97:3) ratio. This
fraction contains higher homologues of maltose palmitates namely maltose
octa, hepta, hexa and penta palmitates. The second fraction (fraction B)
was obtained after elution with dichloromethane:MeOH (85:15). It consists
of maltose tetra, tri and dipalmitates.
V. Fraction B was evaporated to dryness, then dissolved in heptane:ethanol
(50:50) and filtrated through a 0.5 micron membrane in order to remove any
dust particles and traces of higher homologous of maltose palmitates. The
filtered product is purified by gradient high performance liquid
chromatography using a silica gel column and a UV detection system at 214
mm. The solvent gradients system used are heptane: ethanol (95:5) to
heptane: ethanol (50:50). Isomerically pure fractions of maltose
tetrapalmitate, maltose tripalmitate and maltose dipalmitate, as described
previously in method 2, were collected and were simultaneously quantitated
using a Waters 730 data module. The fractions were evaporated to dryness
in vacuo and trace impurities removed by dissolving in hot 95% ethanol and
precipitation by cooling to 0.degree. C. followed by filtration. Under
these conditions maltose tripalmitate gives a crystalline product.
Other HPLC techniques that may also be used to purify refined maltose
palmitates are tabulated as follows:
__________________________________________________________________________
GRADIENT ISOCRATIC
Initial composition
Final composition
Composition
COLUMN
DETECTION
__________________________________________________________________________
Hexane:Ethanol
Hexane:Ethanol
-- Silica gel
UV
(98:2) (50:50) 214 or 229 nm
Pentane:Methanol
Pentane:Methanol
-- Silica gel
UV
(92:2) (50:50) 214 or 229 nm
-- -- Chloroform:
Silica gel
Refractive
Methanol index
98:.5:.5)
Pentane:Methanol
Pentane:Methanol
-- C-18 UV
(5:95) (25:75) 214 or 229 nm
Ethanol:Methanol
Ethanol -- C-18 UV
(20:80) (100%) 214 or 229 nm
Isopropanol:
Isopropanol C-18 UV
Methanol (15:85)
(100%) 214 or 229 nm
__________________________________________________________________________
Characterization of maltose tri and tetrapalmitates
The major peaks eluting in the HPLC employing either of the three methods
of HPLC separation were collected and analyzed for glucose to palmitic
acid ratio by colorimetric means after acid hydrolysis or maltose:
palmitic acid ratio by NMR. Depending on the ratio they were classified as
maltose di, tri, tetra etc. palmitates and described as pure or as
mixtures based on the ratio and the presence of peaks upon HPLC analysis
and thin layer chromatography. Special emphasis was placed on the
fractions that gave maltose: palmitic acid ratio of 2.8-3.0 and 3.8 to
4.0. These fractions were rechromatographed and in the case of the first
(i.e. maltose tripalmitate) a single peak in the HPLC was obtained. Thus
we could isolate pure maltose tripalmitate. Using the same criteria, a
double peak with glucose: palmitic acid ratio of 1.95 were isolated and
they could be separated into peak 1 and peak 2 when only top of the
peak-fractions were collected and the mixed fractions were discarded.
These were isomeric maltose tetrapalmitates.
Crystallization of maltose tri and tetra palmitates
The solutions of isomeric maltose tetrapalmitates were evaporated to
dryness and then dissolved in a minimum amount of hot 95 percent ethanol.
Upon cooling and allowing to stand at 4.degree. C., crystals of the two
maltose tetrapalmitates could be obtained. The tubes containing the
crystals were centrifuged in the cold and the crystalline products, washed
with cold 95 percent ethanol. The early peak maltose tetrapalmitate was
referred to as tetra 1 and the later peak as tetra 2.
Tetra 1 had amp of 87.degree.-91.degree. C. and an optical rotation of
[.alpha.].sub.D.sup.20 =43.degree. (C-0.5 in CHCl.sub.3). Tetra 2 gave a
mp of 105.degree.-107.degree. C. and an optical rotation
[.alpha.].sub.D.sup.20 =46.44 (C-0 9 in CHCl.sub.3). Each gave a single
band in TLC and a single peak in HPLC. The retention time was 11.6 min for
tetra 1 and for the other 11.8 in method 3 of HPLC purification.
The fraction containing maltose tripalmitate was also subjected to dryness
in vacuo, redissolved in hot 95 percent ethanol and then left at 4.degree.
C. It provided a crystalline product with amp of 159.degree.-162.degree.
C. It had a retention time of 13.42 as compared to 11.6-11.8 for maltose
tetrapalmitates. Its optical rotation was [.alpha.].sub.D.sup.20
=41.degree. (C-0.5 in CHCl.sub.3).
Structural studies on maltose tri and tetrapalmitates
Same procedures were employed for the characterization of the three
products.
1. All three failed to reduce Fehling solution and to reduce silver nitrate
showing that the reducing carbon C of maltose was esterified.
2. The products were subjected to periodate oxidation as described by R. G.
Spiro in Methods of Enzymology, Vol. 8, pp. 3-52, 1966. Periodate
consumption ind | | |
