FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10;rule 13)
"PROTEIN DRUG PROBIOTIC COMPLEX, PROCESS FOR PREPARATION AND PHARMACEUTICAL COMPOSITIONS
THEREOF"
Unichem Laboratories Ltd., a corporation organized and existing under the laws of India, of Mahalaxmi
Chambers, 22 Bhulabhai Desai Road, Mumbai- 400026, Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed:
2
FIELD OF INVENTION
The present application relates to water soluble pharmaceutical composition and process
for preparing the same. The application particularly relates to compositions containing
proteins and methods for preparing the same. 5 The invention more particularly relates to
Protein drug probiotic complex, Process for preparation and pharmaceutical compositions
thereof.
BACKGROUND OF INVENTION
10
Protein-drug interaction has been under extensive investigation as these biomolecules are
often the target of drug action. Any drug injected into animals first encounters serum
proteins of which albumin is a major protein, which exhibits affinity to a variety of drugs
that are not freely soluble in aqueous environment. Therefore there are a number of studies
15 reported on drug-albumin interaction. The use of albumin as a drug carrier is a wellestablished
especially using bovine serum albumin.
Similarly ovalbumins from hen eggs have also been studied for use as a drug carrier. Also
the cow milk protein, casein has been claimed to form complex with certain drugs.
20
Amphotericin-B is an effective antifungal agent against Candida species. In addition to its
use as an antifungal agent, the compound has been used as an anti-protozoan drug against
the parasite Leishmania, which causes leishmaniaisis
25 While amphotericin-B is an effective antifungal agent, its nephrotoxic side effect is a major
draw back. A few lipid-based formulations using cholesteryl sulphate and deoxycholate or
liposome appear to be less toxic than the free amphotericin-B. Even then they are still in
secondary use.
30 U.S patent 5,051,406 relates to a pharmaceutical composition using albumins as a carrier to
mitigate the undesirable side effects of drugs and to improve their absorption and light
sensitivity. However these high drug content albumin composition are all uniform
suspensions obtained at high centrifugal speed and meant for oral administration.
3
D. Romanini el al (1998) Chemico-biological interactions, 115, 247-260 discloses that
polyene antibiotics like amphotericin-B have been used as optical probe to detect
conformational changes in membranes and proteins like serum albumin. Such studies show
in the human serum albumin there is a high affinity site and a 5 secondary low affinity sites
for amphotericin-B. However in this study, there was no evaluation of the biological
activity of the complex.
A number of strategies have been tried to improve the solubility of amphotericin-B and to
10 reduce its toxicity. The use of bile salt deoxycholate solubilizes amphotericin-B generating
mixed micelles. Although such a preparation shows antifungal activity, its toxic side effects
still persists. Whereas colloidal dispersions obtained by inclusion of antibiotic with
cholesteryl sulfate or various liposomes or dimyristoyl phosphotidyl choline (PC) or
phosphotidyl glycerol (PG) significantly reduced toxicity.
15
Michael N. Oda et al (2006) Journal of Lipid Research Vol. 47, 260-267 shows that the
vesicle formed by phosphotidyl choline (PC) or phosphotidyl glycerol (PG) is solubilized
by apolipoprotein A-I even if the vesicles contain amphotericin-B. Such a reconstituted
high-density lipoprotein complex gives a clear solution when dissolved in water. The
20 amphotericin-lipid-protein complex shows much reduced toxicity.
Curcumin, the major constituent of turmeric is an important natural product having
multiple beneficial medicinal properties. Its wound healing, anti-inflammatory and
anticancer properties are all well documented. The anti-proliferative properties apparently
25 stems from its ability to induce apoptosis in tumor cells. Curcumin binds and modifies the
elastic property of the host lipid bilayers and has membranes thinning effect.
U.S patent 4,999,205 describes a curcumin complex with water-dispersible proteins,
Casein/ α-casein which will not precipitate upon standing in water or by centrifugation for
30 use as food additive. The claim relates to allowing the casein protein and curcumin allowed
to interact in aqueous solution at pH above 9.0, at which the curcumin is present in its
water-soluble red alkaline form, and then acidifying the solution to bring it below pH 8.0.
4
This results in curcumin forming complex with casein in its neutral yellow form, meant for
use as a colouring agent.
U.S Patent 6,652,875 relates to a formulation for delivery of bioactive constituents to
biological surfaces wherein said formulation 5 comprises a suspension or solution of at least
one isolated purified casein or its salt thereof, in water together with at least one bioactive
constituents including antimicrobial agent, halogenated diphenyl ether, benzoates and
quaternary organic salts. The formulations are in the form of gel or suspension for use in
dental and oral care.
10
Thus, there is a continuous need to develop compositions which improve the solubility and
reduce the toxicity of various drugs.
In view of the above the present invention discloses a pharmaceutical composition with a
15 water soluble drug protein complex. It is another embodiment of the present invention is to
provide a pharmaceutical composition which retains the antifungal activity. Still another
embodiment of the present invention provides a pharmaceutical composition which reduces
the toxicity of drugs.
20 SUMMARY OF INVENTION
The present invention discloses a water-soluble protein based pharmaceutical composition
and a process for the preparation the same comprising of the steps of solubilising a carrier
protein in a suitable buffer, adding the dissolved pharmaceutical agent/s to the solubilised
25 carrier protein and stirring at a defined temperature for a defined time, diluting the solution
with a buffer and concentrating by diafiltration, centrifuging the clear retantate solution
obtained after diafiltration and obtaining the clear supernatant, converting the clear
supernatant to powder form to obtain the drug-protein pharmaceutical composition.
30 The invention more particularly relates to Protein drug probiotic complex, Process for
preparation and pharmaceutical compositions thereof, the process comprising the steps of
adding the drug to carrier protein source at room temperature and stirring well, mixing
probiotic source, allowing it to stand for 3 hours to overnight, centrifuging the product to
5
separate solid protein drug complex, and incorporating the protein drug probiotic complex
prepared into oral therapy formulations. The process step may also include the step of
washing and lyophilizing the complex. The protein source could be naturally occurring
emulsion comprising oil and water phase or an emulsion derived from animal or milk
derived from animals. The pr 5 obiotic source can be yeast or bacteria.
The oral therapy formulations is preferably pharmaceutical dosage form such as tablet,
capsule, lozenge, bar, toffee, chewing gum, pill, pellet, cores, granules, powder, wafer,
film, lollypop, solution, suspension, emulsion and confectionary dosage form and is either
a homogeneous or heterogeneous system comprising of one or more than one phases.
10
BRIEF DESCRIPTION OF DRAWINGS
Figure 1a depicts the results of the hemolytic assay for blood group B+ using AB of
concentration 10.8μM after 0, 2 and 4 hours. The results are for the various samples:
A=DMSO; B=CR (6 μg /ml); C=AB; D=AB + CR (6 μg /ml); E=α-casein- AB; F=α-
15 casein-AB-CR; G=BSA-AB; H=BSA-AB-CR; I=HSA-AB; J=HSA-AB-CR
Figure 1b depicts the results of the hemolytic assay using AB of concentration 21.6μM
after 0, 2 and 4 hours: The results for the various samples are shown as follows: A=DMSO;
B=CR (12 μg /ml); C=AB; D=AB + CR (12 μg /ml); E=α-casein- AB; F=α-casein-AB20
CR; G=BSA-AB; H=BSA-AB-CR; I=HSA-AB; J=HSA-AB-CR
DETAILED DESCRIPTION
Pharmaceutical compositions using protein as carrier mitigate the undesirable side effects
25 of drugs. The present invention discloses of a water-soluble protein based pharmaceutical
composition and a process for the preparation the same comprising of the steps of
- solubilising a carrier protein in a suitable buffer of a defined pH,
- dissolving the pharmaceutical agent/s to be complexed in a suitable solvent
- adding the dissolved pharmaceutical agent/s to the solubilised carrier protein and
30 stirring
- diluting the solution preferably three times its volume with a buffer and
concentrating by diafiltration, thereafter repeated the step for at least two times.
6
- centrifuging the clear retantate solution obtained after diafiltration at 8000-15000
rpm for 5-20 minutes and obtaining the clear supernatant,
- converting the clear supernatant to powder form preferably by lyophilizing or
spray-drying
to obtain the drug-protein pha 5 rmaceutical composition.
The preferred embodiment of the invention is to have the carrier protein to be any of the
caseins and albumins. The carrier proteins may be truncated by treatment with proteases in
the complex before or after complexing with the drug.
10
The buffer used for solubilising the carrier protein includes PBS, Tris-HCl, Tris-acetate or
HEPES, preferably PBS and the pH of the above buffers range from 4.5 to 8.5, preferably
7.0. The same buffers may be used for dilution prior to diafiltration.
15 The drugs or the pharmaceutical agent for complexation can be selected from a group
consisting of amphotericin-B, itraconazole, flucaconazole, voraconazole, tomaxifen,
doxorubicin, taxol and curcumin. The pharmaceutical agents may be complexed with the
protein alone or along with other agents as well. A preferred combination is that of protein
along with curcumin and amphotericin-B. The combination compositions may be such that
20 individual protein-drug complex may be prepared and thereafter mixed together, or protein
may be directly complexed with one or more drugs. It is yet another embodiment of the
inventions that the pharmaceutical composition is crosslinked with crosslinking agents like
DCC.
25 Solvents to be used for solubilising the pharmaceutical agent may be selected from organic
solvents such as methanol, ethanol, isopropanol, n-butanol, iso-butanol, DMSO, diethyl
ether, tetrahydrofuran,1,4 dioxane and propylene glycol. The solvent used preferably is
DMSO.
30 The temperature for shaking the solution of the complex may vary from 20C to 40C and
the duration for shaking is in the range of 10 minutes to 3 hours. The most preferable
conditions for the process are 37 C for 30 minutes.
7
The ratio of the pharmaceutical agent to the carrier protein may vary from 1:1 to 1:50
(w/w). The most preferred ratio of the pharmaceutical agent to the carrier protein is 1:20
(w/w). Particularly the pharmaceutical agent-protein complex for curcumin-protein
complex may vary in the range of 1:1 to 1: 5.
5
The protein complex comprising amphotericin-B with or without curcumin is proposed for
treatment of Leshmaniais preferably the carrier protein being casein.
The following disclosure exemplifies the preferred embodiments of the instant invention:
10
EXAMPLES
One of the preferred embodiments is for a process for the complexation of the hydrophobic
antifungal drug, amphotericin-B (AmB) using proteins like casein, bovine serum albumin
(BSA) and human serum albumin (HSA) and the complex thereof. The drug-protein
15 complex thus formed is water soluble. Different casein preparations were used for forming
the complex with AmB and the complexes were found to be water soluble. The AmBcasein
complexes were found to retain the antifungal activity and hemolytic assay revealed
that the AmB-casein complex reduced the membrane damaging property of AmB.
20 Further another hydrophobic compound curcumin, was added to the AmB-Casein complex
to form a water soluble curcumin-casein-AmB complex. This complex also retained the
antifungal activity and helped in reducing the membrane damaging property of AmB
considerably than the AmB-casein complex and AmB alone. Similarly a water soluble
curcumin-casein complex was also prepared. This complex when mixed with casein-AmB
25 complex in appropriate ratio exhibited the same activity and membrane damage protecting
property as the curcumin-casein-AmB complex.
Similar complexes of AmB and curcumin were made using the serum albumins (BSA &
HSA) instead of casein. These complexes also showed the antifungal and membrane
30 protecting activities though to a lesser extent than the corresponding complexes with
casein. Both BSA and HSA formed ternary complex with AmB and Curcumin, ie AmBBSA-
curcumin and AmB-HSA-Curcumin. These ternary complexes and also the respective
8
individual complex mixtures (BSA-AmB + BSA-curcumin) and (HSA-AmB + HSAcurcumin)
are bioactive and showed considerable membrane damage reducing property.
Apart from AmB and curcumin other drugs like itraconazole, flucaconazole, voraconazole,
tomaxifen, doxorubicin, taxol etc. were also 5 complexed with the proteins casein, BSA and
HSA.
The above embodiments may be more clearly understood by way of experimental examples
provided herein:
10
1) a) Preparation of Soluble-Casein fraction:
To the boiled milk (80ml), HCl (1N) was added in drop-wise manner, until the protein
precipitated out. This precipitate formed was removed by centrifugation at 5000 rpm for 10
min using a Beckman Coulter J2MC Centrifuge. The pellet was washed twice with Milli-Q
15 water (200ml) and then solubilized in 250ml of 0.1M Tris/HCl (pH 7.0). The turbid
solution was centrifuged at 10000 rpm for 15 min at room temperature. The protein content
of the solution was determined to be 2g (250 ml). This was concentrated using Amicon
stirred cell setup under nitrogen pressure until the final volume was 100ml. Dialyzed
against Milli Q water (2 liters each time with 3 changes). The dialyzed sample had protein
conc. of 3.48mg/ml. This was lyophilized and stored at -20o20 C until further use.
1) b) Complexation of Soluble-casein with Amphotericin B (AB).
To a solution of soluble-casein (100mg) in 200ml PBS pH 7.0, Amphotericin B (5mg
dissolved in 500 l DMSO) was added and stirred well. The mixture was incubated at 37
o25 C for 30 min with constant shaking. After incubation the complex was diluted to 300ml by
addition of (100ml) PBS and subjected to diafiltration (Amicon-stirred cell setup) under
nitrogen pressure until the volume reduced to 100ml. The process of dilution and
concentration was repeated twice. The final volume of clear colored retentate was 100ml.
This was centrifuged at 12000 rpm for 10min and lyophilized. All the glasswares used for
30 handling the complex were wrapped in aluminium foil to prevent exposure to light.
Molar ratio:
9
The AB-soluble casein complex (2mg) was extracted with 1ml of DMSO until the pellet
was free of AB. The absorbance of the extract at 416nm was used to calculate the
concentration of AB (Molar Extinction coefficient (ε) =96290 M-1 cm-1). The molar ratio
between AB to soluble-casein was found to be 1.21. The complex was soluble to the extent
of 5 25mg protein per ml of water and contained 0.68 mg of AB.
Broth assay:
10 This AB-soluble casein complex was found to inhibit the growth of Candida species in the
broth assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
treated culture showed that, with complex less number of colonies were present (Table-1).
TABLE-1
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•Soluble-casein-AB
•Soluble-casein-AB
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
1.03 X10 4
1.40 X10 3
1.79 X10 3
3.30 X10 2
15 Similar results are observed with C glabrata and C krusei.
Hemolytic assay:
Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the ABsoluble
casein complex (AB 10.8μM) showed 50% lysis at the end of 2nd 20 hr and even after
4 hrs 25% cells remained intact. AB-soluble casein complex at a higher concentration
(21.6μM) lysed to the extent of about 70% at the end of 2 hrs. The results clearly indicate
that the membrane damage to the red cells is much reduced when AB is present in the form
of protein complex.
25
2) a) Purification of α-casein:
The casein (2g) from procedure-1a was taken and solubilized in 250ml 0.1M Tris/HCl pH
7.0 buffer containing 2M urea (GR grade) and 1M thiourea (GR grade) (Buffer-A)
10
[250ml]; adjusted to pH 7.0 using 1N HCl. Centrifuged at 10000 rpm for 20 min. The
supernatant was loaded on to DEAE cellulose (Whatman) (150ml) pre-equilibrated using
elution buffer. The bound protein was eluted using buffer-A containing NaCl salt at 75mM
and 200mM concentration. The fractions were analyzed on 15% SDS gel and the like
fractions pooled together. The α-casein containing 5 fraction was concentrated using Amicon
stirred cell setup (3kDa cutoff membrane) under nitrogen pressure until the final volume
was 100ml. Dialyzed against Milli Q water (2 liters each time with 3 changes). This was
lyophilized and stored at -20oC until further use. The HPLC profile showed a symmetric
sharp single peak at 35min. The mass spectrometric data shows that the purified protein has
10 a molecular mass of 23.7 kDa.
2) b) Complexation of α-casein with Amphotericin B (AB).
To a solution of α-casein (100mg) in 200ml PBS pH 7.0, AB (5mg dissolved in 500 l
DMSO) was added and stirred well. The mixture was incubated at 37 oC for 30 min with
15 constant shaking. After incubation the complex was diluted to 300ml by addition of
(100ml) PBS and diafiltrated to get 100ml. The process of dilution and concentration was
repeated twice. The final volume of clear colored retentate was 100ml. This was
centrifuged at 12000rpm for 10min and lyophilized.
20 Molar ratio:
The AB - α-casein complex (5mg) was extracted with 1ml of DMSO until the pellet was
free of AB. The absorbance of the extract at 416nm was used to calculate the concentration
of AB (Molar Extinction coefficient (ε) =96290 M-1 cm-1). The molar ratio between AB to
α-casein was found to be 1.2. The complex was soluble to the extent of 40mg protein per
25 ml of water and contained 1.89 mg of AB.
Broth assay:
This AB - α-casein complex was found to inhibit the growth of Candida species in the
broth assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
30 treated culture showed that, with complex less number of colonies were present (Table-2).
TABLE-2
11
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•-casein- AB
•-casein- AB
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
1.03 X10 4
1.40 X10 3
1.65 X10 3
8.50 X10 2
Similar results are observed with C glabrata and C krusei.
Hemolytic assay:
Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the α-casein-
AB complex (AB 10.8μM) showed 50% lysis at the end of 5 2nd hr and even after 4 hrs 50%
cells remained intact. AB - α-casein complex at a higher concentration (21.6μM) lysed to
the extent of about 70% at the end of 2 hrs. The results clearly indicate that the membrane
damage to the red cells is much reduced when AB is present in the form of protein complex
(Figure1a & b).
10
3) a) Purification of α-casein.
The casein (2g) from procedure-1a was taken and solubilized in 250ml 0.1M Tris/HCl pH
7.0 buffer (Buffer-A) containing 8M urea (GR grade) overnight at 4oC. Next day the
solution was brought to room temperature and diluted to final concentration of 3M urea
15 using buffer-A (416ml). The protein solution (1.65mg/ml) was treated with 1mM DTT for
1hr at room temperature and centrifuged. Loaded on to DEAE cellulose column (150ml)
pre-equilibrated with Buffer-A containing 3M urea. Wash was carried out with buffer-A
containing 3M urea, 0.05M NaCl and 2mM -Mercaptaethanol until the absorbance at
280nm had reached less than 0.1. The bound protein was eluted using buffer-A containing
20 0.4M NaCl and 3.5M urea. The α-casein containing fraction was diafiltrated until the final
volume was 100ml. Dialyzed against Milli Q water (2 liters each time with 3 changes). The
dialyzed sample had protein conc. of 3.48mg/ml. This was lyophilized and stored at -20oC
until further use. The yield of α-casein and beta casein is 31.6% and 9.5% respectively.
Analysis on a 15% SDS PAGE showed that α-casein was more than 90% pure. The HPLC
25 profile showed a symmetric sharp single peak at 35min. The mass spectrometric data
shows that the purified protein has a molecular mass of 23.7 kDa.
3) b) Complexation of α-casein with Amphotericin B (AB).
12
To a solution of α-casein (100mg) in 200ml PBS pH 7.0, Amphotericin B (5mg dissolved
in 500 l DMSO) was added and stirred well. The mixture was incubated at 37 oC for 30
min with constant shaking. After incubation the complex was diluted to 300ml by addition
of (100ml) PBS and the volume reduced to 100ml by diafiltration. The process of dilution
and concentration was repeated twice. The final 5 volume of clear colored retentate was
100ml. This was centrifuged at 12000rpm for 10min and lyophilized.
Molar ratio:
The AB- α-casein complex (5mg) was extracted with 1ml of DMSO until the pellet was
10 free of AB. The absorbance of the extract at 416nm was used to calculate the concentration
of AB (Molar Extinction coefficient (ε) =96290 M-1 cm-1). The molar ratio between AB to
α-casein was found to be 1.21. The complex was soluble to the extent of 40mg protein per
ml of water and contained 1.89 mg of AB.
15 Broth assay:
This AB- α-casein complex was found to inhibit the growth of Candida species in the broth
assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
treated culture showed that, with complex less number of colonies were present (Table-3).
20 TABLE-3
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•-casein- AB
•-casein- AB
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
2.34 X10 3
1.40 X10 3
3.37 X10 2
7.5 X10 1
Similar results are observed with C glabrata and C krusei.
Hemolytic assay:
Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the AB- α-
casein complex (AB 10.8μM) showed 15% lysis at the end of 2nd 25 hr and even after 4 hrs
50% cells remained intact. AB- α-casein complex at a higher concentration (21.6μM) lysed
to the extent of about 30% at the end of 2 hrs. The results clearly indicate that the
13
membrane damage to the red cells is much reduced when AB is present in the form of
protein complex (Figure1a & b).
4) a) Purification 5 of α-casein from skim milk powder.
Skim milk powder (2 g) was solubilized in 250ml 0.1M Tris/HCl pH 7.0 buffer (Buffer-A)
containing 8M urea (GR grade) overnight at 4oC. Next day the solution was brought to
room temperature and diluted to final concentration of 3M urea using buffer-A (416ml).
The protein solution (1.2 mg/ml) was treated with 1mM DTT for 1hr at room temperature
10 and centrifuged. Loaded on to DEAE cellulose column (150ml) pre-equilibrated with
Buffer-A containing 3M urea. Wash was carried out with buffer-A containing 3M urea,
0.05M NaCl and 2mM -Mercaptaethanol until the absorbance at 280nm had reached less
than 0.1. The bound protein was eluted using buffer-A containing 0.4M NaCl and 3.5M
urea. The α-casein containing fraction was diafiltrated until the final volume was 100ml.
15 Dialyzed against Milli Q water (2 liters each time with 3 changes).. The dialyzed sample
had protein conc. of 3 mg/ml. This was lyophilized and stored at -20oC until further use.
The yield of α-casein and -casein is 40% and 25% respectively. Analysis on a 15% SDS
PAGE showed that α-casein was more than 90% pure. The HPLC profile showed a
symmetric sharp single peak at 35min. The mass spectrometric data shows that the purified
20 protein has a molecular mass of 23.7 kDa.
4) b) Complexation of α-casein with Amphotericin B (AB).
To a solution of α-casein (100mg) in 200ml PBS pH 7.0, AB (5mg dissolved in 500 l
DMSO) was added and stirred well. The mixture was incubated at 37 oC for 30 min with
25 constant shaking. After incubation the complex was diluted to 300ml by addition of
(100ml) PBS and diafiltrated to get 100ml. The process of dilution and concentration was
repeated twice. The final volume of clear colored retentate was 100ml. This was
centrifuged at 12000rpm for 10min and lyophilized.
30 Molar ratio:
The AB- α-casein complex (2mg) was extracted with 1ml of DMSO until the pellet was
free of AB. The absorbance of the extract at 416nm was used to calculate the concentration
14
of AB (Molar Extinction coefficient (ε) =96290 M-1 cm-1). The molar ratio between AB to
α-casein was found to be 1.21. The complex was soluble to the extent of 40mg protein per
ml of water and contained 1.89 mg of AB.
5 Broth assay:
This AB-α-casein complex was found to inhibit the growth of Candida species in the broth
assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
treated culture showed that, with complex less number of colonies were present (Table-4).
10 TABLE-4
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•-casein- AB
•-casein- AB
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
2.6 X10 3
2.7 X10 2
5.6 X10 2
7.3 X10 1
Similar results are observed with C glabrata and C krusei.
Hemolytic assay:
15 Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the AB- α-
casein complex (AB 10.8μM) showed 15% lysis at the end of 2nd hr and even after 4 hrs
50% cells remained intact. AB- α-casein complex at a higher concentration (21.6μM) lysed
to the extent of about 30% at the end of 2 hrs. The results clearly indicate that the
membrane damage to the red cells is much reduced when AB is present in the form of
20 protein complex (Figure1a & b).
5) Complexation of α-casein with Curcumin (CR).
To a solution of α-casein (100mg) in 200ml PBS, pH 7.0, CR (5mg dissolved in 500l
DMSO) was added and stirred well. The mixture was incubated at 37oC for 30 min. with
25 constant shaking. The complex was diluted to 300ml by addition of (100ml) PBS and diafiltrated
to get 100ml. The process of dilution and concentration was repeated twice. The
final volume of clear colored retentate was 100ml. This was centrifuged at 12000rpm for
10min and lyophilized.
15
Molar ratio:
The CR- α-casein complex (2mg) was extracted with 2ml of DMSO until the pellet was
free of CR. The absorbance of the extract at 434nm was used to calculate the concentration
of CR (Molar Extinction coefficient (ε) =57560 M-1 cm5 -1). The molar ratio between CR to
α-casein was found to be 0.97. The complex was soluble to the extent of 20mg protein per
ml of water and contained 0.3 mg of CR.
6) Complexation of α-casein with Amphotericin B (AB) and Curcumin (CR).
10 To a solution α-casein (100mg) in 200ml PBS pH 7.0, AB (5mg dissolved in 500l
DMSO) was added and stirred well. Allowed to stir at room temperature for 10 min. Next
CR (5mg dissolved in 500l DMSO) was added and stirred. The mixture was incubated at
37oC for 30 min. with constant shaking. The complex was diluted to 300ml by addition of
(100ml) PBS and dia-filtrated to get 100ml. The process of dilution and concentration was
15 repeated twice. The final volume of clear colored retentate was 100ml. This was
centrifuged 12000rpm at 10min and lyophilized.
Molar ratio:
The α-casein-AB-CR complex (5mg) was extracted with 1ml of DMSO until the pellet was
20 free of AB and CR. The Absorbance of the extract was noted at 416nm (AB) / 434nm (CR)
and the molar ratio AB to α-casein was 0.09 and CR to α-casein was 0.98. The complex
was soluble to the extent of 40mg protein per ml of water and contained 0.14 mg of AB/
0.61 mg CR.
25 Broth assay:
The α-casein-AB-CR complex was found to inhibit the growth of Candida species in the
broth assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
treated culture showed that, with complex less number of colonies were present (Table-5).
30
16
TABLE 5
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•-casein- AB-CR
•-casein- AB-CR
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
1.03 X10
4
1.40 X10
3
4.52 X10
2
8.50 X10
1
Similar results are observed with C glabrata and C krusei.
5
Hemolytic assay:
Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the α-casein-
AB-CR complex (AB 10.8μM) showed 4% lysis at the end of 2nd hr and even after 4 hrs
94% cells remained intact. The α-casein-AB-CR complex (AB 21.6μM) at the end of 2hrs
10 lysed to the extent of about 7% and even after 4 hrs 75% cells remained intact (Figure1a &
b).
7) a) Complexation of α-casein-Amphotericin B (AB) using N, N1 Dicyclohexyl
carbodiimide (DCC).
15 To a solution of α-casein-AB complex (100mg) in 20ml, add DCC (2.5mM dissolved in
250l acetonitrile) and stir well. Allowed overnight at room temperature. Next day the
complex was centrifuged at 10000 rpm for 20 minutes inorder to remove any precipitate.
The supernatant was loaded on to PD-10 pre-packed column (Amersham), equilibrated
with purified water and eluted using (5ml) purified water. As the free drug is insoluble in
20 water, the eluted fraction depicts the drug-protein complex in soluble form. Lyophilized
and stored at –20oC until further use.
Molar ratio:
The prepared complex (3mg) was extracted with 1ml of DMSO until the pellet was free of
25 AB. The absorbance of the extract at 416nm was used to calculate the concentration of AB
(Molar Extinction coefficient (ε) =96290 M-1 cm-1). The molar ratio between AB to α-
casein was found to be 1.95.
17
7) b) Complexation of α-casein and AB using N, N1 Dicyclohexyl carbodiimide (DCC).
To a solution of α-casein (300mg) in 25ml MilliQ water, add DCC (30mM dissolved in
300l acetonitrile) and stir well. Next AB (60mg in 1.5 ml DMSO) was added as above.
Allowed overnight at room temperature. Care was taken 5 to protect the complex from direct
exposure to light. Next day the complex was centrifuged at 10000 rpm for 20 minutes
inorder to remove any precipitates. The supernatant was loaded on to PD-10 pre-packed
column (Amersham), equilibrated with purified water and eluted using (5ml) purified
water. As the free drug is insoluble in water, the eluted fraction depicts the drug-protein
complex in soluble form. Lyophilized and stored at –20o10 C until further use.
Molar ratio:
The prepared complex (3mg) was extracted with 1ml of DMSO until the pellet was free of
AB. The absorbance of the extract at 416nm was used to calculate the concentration of AB
(Molar Extinction coefficient (ε) =96290 M-1 cm-115 ). The molar ratio between AB to α-
casein was found to be 1.28. The complex was soluble to the extent of 30mg protein per ml
of water and contained 1.5 mg of AB
Broth assay:
20 This complex was found to inhibit the growth of Candida species in the broth assay (at an
AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the treated culture
showed that, with complex less number of colonies were present (Table-6).
TABLE-6
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•-casein- AB -DCC
•-casein- AB -DCC
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
9.8 X10
3
8.5 X10
2
5.0 X10
2
4.0 X10
1
25
8) a) Complexation of BSA with Amphotericin B.
18
To a solution of BSA (100mg) in 200ml PBS, pH 7.0, Amphotericin B (5mg dissolved in
500l DMSO) was added and stirred well. The mixture was incubated at 37oC for 30 min
with constant shaking. The complex was diluted to 300ml by addition of (100ml) PBS and
dia-filtrated to get 100ml. The process of dilution and concentration was repeated twice.
The final volume of clear colored retentate was 5 100ml. This was centrifuged at 12000rpm
at 10min and lyophilized.
Molar ratio:
The BSA -AB complex (3mg) was extracted with 1ml of DMSO until the pellet had no
10 bound AB. The absorbance of the extract was noted at 416nm for AB (Molar Extinction
coefficient (ε) =96290 M-1 cm-1) and the molar ratio AB to BSA was found to be 1.73. The
complex was soluble to the extent of 10mg protein per ml of water and contained 0.24mg
of AB.
15 Broth assay:
This BSA-AB complex was found to inhibit the growth of Candida species in the broth
assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
treated culture showed that, with complex less number of colonies were present (Table-7).
20 TABLE 7
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•BSA-AB
•BSA-AB
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
1.03 X10
4
1.40 X10
3
1.88 X10
3
1.33 X10
2
Similar results are observed with C glabrata and C krusei.
Hemolytic assay:
25 Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the BSA-AB
complex (AB 10.8μM) showed 95% lysis at the end of 2nd hr and even after 4 hrs 3% cells
19
remained intact. The BSA-AB complex (AB 21.6μM) at the end of 2hrs lysed to the extent
of about 98% (Figure1a & b).
8) b) Complexation of BSA with Curcumin (CR).
To a solution of BSA (100mg) in 200ml PBS, 5 pH 7.0, CR (5mg dissolved in 500l DMSO)
was added slowly and stirred well. The mixture was incubated at 37oC for 30 min. with
constant shaking. The complex was diluted to 300ml by addition of (100ml) PBS and diafiltrated
to get 100ml. The process of dilution and concentration was repeated twice. The
final volume of clear colored retentate was 100ml. This was centrifuged 12000 rpm for
10 10min and lyophilized.
Molar ratio:
The BSA-CR complex (2mg) was extracted with 6ml of DMSO until the pellet had no
bound CR. The absorbance of the extract at 434nm was used to calculate the concentration
of CR (Molar Extinction coefficient (ε) =57560 M-1 cm-115 ). The molar ratio between CR to
BSA was found to be 3.59. The complex was soluble to the extent of 10mg protein per ml
of water and contained 0.2 mg of CR.
8) c) Complexation of BSA with Amphotericin B (AB) and Curcumin (CR).
20 To a solution of BSA (100mg) in 200ml PBS, pH 7.0, AB (5mg dissolved in 500l DMSO)
was added and stirred well. Allowed to stir at room temperature for 10 min. Next CR (5mg
dissolved in 500l DMSO) was added and stirred. The mixture was incubated at 37oC for
30 min. with constant shaking. The complex was diluted to 300ml by addition of (100ml)
PBS and dia-filtrated to get 100ml. The process of dilution and concentration was repeated
25 twice. The final volume of clear colored retentate was 100ml. This was centrifuged and
lyophilized.
Molar ratio:
The BSA-AB-CR complex (4mg) was extracted with 2ml of DMSO until the pellet had no
30 bound AB and CR. The absorbance of the extract was noted at 416nm (AB) / 434nm (CR)
and the molar ratio AB to BSA was 0.46 and CR to BSA was 5.29. The BSA-AB-CR
20
complex was soluble to the extent of 10mg protein per ml of water and contained 0.065 mg
of AB/ 0.29mg CR.
Broth assay:
This BSA-AB-CR complex was found to inhibit the 5 growth of Candida species in the broth
assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
treated culture showed that, with complex less number of colonies were present (Table-8).
TABLE 8
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•BSA-AB-CR
•BSA-AB-CR
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
0.7 X10
4
6.0 X10
2
1.4 X10
3
2.0 X10
2
10
Hemolytic assay:
Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the BSA-ABCR
complex (AB 10.8μM) showed 11% lysis at the end of 2nd hr and even after 4 hrs 40%
15 cells remained intact. The BSA-AB-CR complex (AB 21.6μM) at the end of 2hrs lysed to
the extent of about 16% and even after 4 hrs 30% cells remained intact (Figure1a & b).
9) a) Complexation of HSA with Amphotericin B (AB).
To a solution of HSA (100mg) in 200ml PBS, pH 7.0, AB (5mg dissolved in 500l
DMSO) was added and stirred well. The mixture was incubated at 37oC for 30 min. with
20 constant shaking. The complex was diluted to 300ml by addition of (100ml) PBS and diafiltrated
to get 100ml. The process of dilution and concentration was repeated twice. The
final volume of clear colored retentate was 100ml. This was centrifuged at 12000rpm for
10min and lyophilized.
25 Molar ratio:
The HSA-AB complex (2mg) was extracted with 1ml of DMSO until the pellet had no
bound AB. The absorbance of the extract was noted at 416nm (AB-Molar Extinction
21
coefficient (ε)=96290 M-1cm-1) and the molar ratio AB to HSA was found to be 3.6. The
complex was soluble to the extent of 10mg protein per ml of water and contained 0.5mg of
AB.
5 Broth assay:
This HSA-AB complex was found to inhibit the growth of Candida species in the broth
assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
treated culture showed that, with complex less number of colonies were present (Table-9).
TABLE 9
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
AB
AB
HSA-AB
HSA-AB
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
1.10 X10 4
0.7 X10 3
1.4 X10 3
2.4 X10 2
10
Hemolytic assay:
Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the HSA-AB
complex (AB 10.8μM) showed 94% lysis at the end of 2nd hr and even after 4 hrs 2% cells
15 remained intact. The HSA-AB complex (AB 21.6μM) at the end of 2hrs lysed to the extent
of about 100% (Figure1a & b).
9) b) Complexation of HSA with Amphotericin B (AB) and Curcumin (CR).
20 To a solution of HSA (100mg) in 200ml PBS, pH 7.0, AB (5mg dissolved in 500l
DMSO) was added and stirred well. Allowed to stir at room temperature for 10 min. Next
CR (5mg dissolved in 500l DMSO) was added and stirred. The mixture was incubated at
37oC for 30 min. with constant shaking. The complex was diluted to 300ml by addition of
(100ml) PBS and dia-filtrated to get 100ml. The process of dilution and concentration was
25 repeated twice. The final volume of clear colored retentate was 100ml. This was
centrifuged at 12000rpm for 10min and lyophilized.
Molar ratio:
22
The HSA-AB-CR complex (3mg) was extracted with 3ml of DMSO until the pellet had no
bound AB and CR. The absorbance of the extract was noted at 416nm (AB) / 434nm (CR)
and the molar ratio AB to HSA was 0.35 and CR to HSA was 4.04. The complex was
soluble to the extent of 10 mg protein per ml of water and contained 0.05 mg of AB/ 0.22
5 mg CR.
Broth assay:
The HSA-AB-CR complex was found to inhibit the growth of Candida species in the broth
assay (at an AB concentration of 0.33 μg/ml). Subsequent dilution and plating of the
10 treated culture showed that, with complex less number of colonies were present (Table-10).
TABLE 10
Culture used/ Sample tested Conc. of AB CFU/ml
Candida albicans (M71)
•AB
•AB
•HSA-AB-CR
•HSA-AB-CR
0.33 μg/ml
0.83 μg/ml
0.33 μg/ml
0.83 μg/ml
0.8 X10 4
1.02 X10 3
1.6 X10 3
3.0 X10 2
Hemolytic assay:
Use of AB (10.8μM) lysed the red cells completely in about 30min. Whereas the HSA-ABCR
complex (AB 10.8μM) showed 23% lysis at the end of 2nd 15 hr and even after 4 hrs 25%
cells remained intact. The HSA-AB-CR complex (AB 21.6μM) at the end of 2hrs lysed to
the extent of about 16% and even after 4 hrs 21% cells remained intact (Figure1a & b).
10) Preparation of casein using Probiotics:
20
Fresh cow milk (200ml) (Source: Commercially available) was taken into a clean glass
beaker and boiled. Brought to room temperature and fresh inoculum of Lactobacilli added.
Allowed overnight to ferment at room temperature. Next day, the well-formed curd was
centrifuged at 10000 rpm for 20 minutes and the pellet separated from the whey. The pellet
25 was washed with Milli Q water (400ml) twice and the pellet lyophilized. This vacuum
dried powder was stored at –20oC until further use. The yield was 15g. The α-casein can be
purified from this starting material as well.
23
11) Preparation of Casein-Amphotericin B complex:
Fresh Cow milk (200ml) was taken into a clean glass beaker and boiled. Brought to room
temperature and 10mg of amphotericin-B powder (Himedia) was directly added and stir
well. Next, fresh inoculum of Lactobacilli added. Left at room temperature for overnight
period. Next day, the well-formed curd was 5 centrifuged at 10000 rpm for 20 minutes and
the pellet separated from the whey. The pellet was washed with Milli Q water twice and
then lyophilized. This vacuum dried powder was stored at –20 oC until further use. The
yield of dry powder was about 15g. This material may also be used in oral therapy
formulations.
10
The term “oral therapy formulation” covers all pharmaceutically acceptable dosage forms
including confectionary dosage forms. Further, pharmaceutically acceptable dosage forms
includes without any limitations, tablet, capsule, lozenge, bar, toffee, chewing gum, pill,
pellet, cores, granules, powder, wafer, film, lollypop, solutions, suspensions and emulsions.
15
Moreover the “protein drug probiotic complex” prepared in the invention relates to the
formation of protein drug probiotic complex as illustrated in this particular example which
explains that the adduct of protein and drug with probiotic is either lyophilized or freeze
dried or vacuum dried once formed.
20
Protein drug and probiotic complex is a novel complex and the protein drug complex
prepared according to example 11 in the presence of a probiotic source i.e. bacteria
contains a detectable amount of probiotic source i.e. bacteria along with protein drug
complex. The final product is protein drug probiotic complex.
25
The process disclosed herein is for preparation of a protein drug probiotic complex based
pharmaceutical composition comprising of the steps of adding the drug to carrier protein
source at room temperature and stirring well, mixing probiotic source, allowing it to stand
30 for 3 hours to overnight, centrifuging the product to separate solid protein drug complex,
and incorporating the protein drug probiotic complex prepared into oral therapy
formulations. The process step may also include the step of washing and lyophilizing the
complex. The protein source could be naturally occurring emulsion comprising oil and
24
water phase or an emulsion derived from animal or milk derived from animals. The
probiotic source can be yeast or bacteria.
The oral therapy formulations is preferably pharmaceutical dosage form such as tablet,
capsule, lozenge, bar, toffee, chewing gum, pill, pellet, cores, granules, powder, wafer,
film, lollypop, solution, suspension, emulsion and confectionary 5 dosage form and is either
a homogeneous or heterogeneous system comprising of one or more than one phases.
12) HPLC analysis of -casein:
10 The HPLC analysis was carried out as follows: 1mg of -casein sample was solubilized in
200 l of 0.1M Tris pH 7.5 containing 8M urea, 0.3% -mercaptathanol and 1.3% sodium
citrate (reducing buffer) for 1h at 37oC. The samples were then diluted (1:5 v/v) with 6M
urea and 0.1% trifluroacetic acid and filtered through 0.45filters. The product was
analyzed on HPLC system using C4- reverse phase column. The mobile phase was made
15 up of 0.1% TFA (v/v) in MilliQ water (Eluant A) and gradient was developed using 0.1%
TFA in 90% (v/v) acetonitrile in water (Eluant B). The run program consists of linear
gradient, starting at 30% to 50%, Eluant B from 0 to 40 minutes; 50% to 100% B from 40
to 46 minutes; 100% B from 46 to 48 minutes; 30% B from 48 to 55 minutes. The flow rate
was 0.8 ml/min and analyzed at 220nm and 280 nm. A symmetric sharp single peak eluted
20 at 35min indicates the sample was essentially pure.
13) Analysis of antifungal activity of protein-drug complexes against Candida species.
The protein-drug samples were solubilized in water and filter sterilized using 0.45μ sterile
syringe filters. Different concentrations; namely, 0.33g/ml and 0.83g/ml AB were
25 pipetted into sterile micro-centrifuge tubes and made up to 1ml using sterile water. This
sample in turn was transferred aseptically into 5ml sterile SD broth. An inoculum of
Candida spp. overnight culture was added such that the final OD600nm was 0.1. Also
Amphotericin B [Himedia] and DMSO (Merck, GR grade) were taken separately as
controls. The culture tubes were incubated overnight at 37oC. Next day the O.D600nm was
30 noted.
The overnight incubated samples were pipetted 200l X 3 times, into sterile microcentrifuge
tubes and centrifuged at 10000 rpm for 10 minutes at room temperature. The
25
supernatant removed and to the pellet 600l sterile SD broth was added, vortexed, from
this 50l sample was spread on to the SD agar plate and incubated overnight at 37oC. Next
day the number of colonies appeared were counted.
14) Hemolytic assay (5 in vitro membrane damage assay):
Preparation of Erythrocytes:
2 ml Human blood of different blood groups (from healthy donors) were collected into
sterile test tube containing 0.2M EDTA (0.2ml). Centrifuged at 2400 rpm for 10 minutes
10 and the plasma; buffy coat removed. Erythrocytes were then washed thrice with PBS
(containing 155mM NaCl, 7mM Na2HPO4, 3mM KH2PO4, adjusted to pH 7.4), suspended
in PBS at a hematocrit of 40% and used for the assay.
(Aliquote of Erythrocytes used for the assay would upon total hemolysis of the cells give
rise to an absorbance of 0.45 to 0.55 at 560 nm).
15
Hemolytic Assay:
9ml of PBS was added to clean glass test tube. Then protein-drug complex and AB
(dissolved in DMSO) were added to respective tubes, such that the final concentration of
AB was 10.8μM & 21.6μM and the buffer volume made up to 10ml. Control tubes
containing PBS and DMSO were also taken. The tubes were then pre-incubated at 37 o20 C for
10 minutes. After pre-incubation, RBC was added and immediately 1ml aliquot was taken
as 0th hr sample. The remaining was incubated and aliquoted at intervals of 0.25, 0.5, 1,
1.5,2, 3 and 4 hrs. The aliquoted samples were centrifuged at 2400 rpm for 10 minutes and
the supernatant read at 560nm.
25
A Control (Total hemolysis) was taken, where RBC was added to 10ml Milli-Q water and
vortexed vigorously, centrifuged and O.D noted. The percent hemolysis was calculated
taking total hemolysis (Control) as 100%.
30 While the present invention has been described with respect to certain preferred
embodiments, it will be apparent to those skilled in the art that various changes and
modifications may be made without departing from the scope of the invention as defined in
the following claims.

We claim:
1. A Protein drug probiotic complex and oral therapy formulations thereof, wherein the protein is casein.
2. The pharmaceutical composition comprising Protein drug probiotic complex as claimed in claim 1,
3. A process for preparation of a Protein drug probiotic complex and oral therapy formulations thereof, comprising of the steps of:
i. adding drug to carrier protein source at room temperature and stirring ii. mixing with a probiotic source, iii. allowing to stand for 3 hours to overnight,
iv. centrifuging at 8000 - 15000 rpm for 5-30 minutes preferably at 12000 rpm for 10 minutes and more preferably at 10000 rpm for 20 minutes to separate solid protein drug probiotic complex, v. optionally washing and optionally lyophilizing the solid protein drug probiotic
complex, and vi. incorporating the protein drug probiotic complex prepared in step iv, or v into oral therapy formulations.
4. The process as claimed in claim 3 step (i) wherein said protein source is milk derived from animals or naturally occurring casein source or casein solution.
5. The process as claimed in claim 3 step (ii) wherein said probiotic source is yeast or bacteria or more preferably Lactobacilli.
6. The process as claimed in claim 3 step (vi) wherein said oral therapy formulation is selected from pharmaceutical dosage forms.
7. The process as claimed in claim 6 wherein pharmaceutical dosage form is selected from tablet, capsule, lozenge, bar, toffee, chewing gum, pill, pellet, cores, granules, powder, wafer, film, lollypop, solution, suspension, emulsion and confectionary dosage form.
8. The process as claimed in claim 3 step (vi) wherein said oral therapy formulation is either a homogeneous or heterogeneous system comprising of one or more than one phases.
9. The Protein drug probiotic complex as claimed in claim 1 wherein the drug is Amphotericin-B.