Sustained Release Drug Delivery System Comprising A Sporogenous Probiotic Bacterium

Abstract

A sustained release drug delivery system comprising spores from sporogenousprobiotic bacteria and drug, wherein said spores from sporogenous probiotic bacteriaact as carrier for sustained release of said drug and a process of preparation of the same.The present invention further provides a sustained release dosage form having a corecomprising spores from sporogenous probiotic bacteria, drug and mucoadhesivesubstance, preferably exopolysaccharides wherein said spores from sporogenousprobiotic bacteria act as carrier for sustained release of said drug; and a further coatingon the core comprising mucoadhesive substance, preferably exopolysaccharides,binder, lubricant, diluent and disintegrating agent and a process for preparation of thesustained release dosage form.

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Specification

Field of Invention
The present invention relates to sustained release drug delivery system. More
specifically the present invention relates to sustained release drug delivery system
where spores of probiotic bacteria, act as carrier for drug-molecules.
Background and prior art
Probiotics are known for their protecting effect against potentially harmful pathogens.
Several other beneficial effects, like antihyperglycemic, antidiarrhoeal, acute
pancreatitis, antihypercholestemia, activity against H. pylori infection, irritable bowel
syndrome, food related allergy, urinary tract infection bladder cancer and colon cancer
have also been attributed to their beneficial effects. There are plenty of drugs available
in the market specific for each of the diseases mentioned above. Probiotics are as such
not drugs, but they confer positive effect on normal well being of the patients.
Spores of Bacillus species are being used commercially as Probiotics and competitive
exclusion agents. Unlike the more commonly used Lactobacillus-type Probiotics,
spores are dormant life forms. The use of Bacillus spp. as Probiotics is not restricted to
a single species of these genera, but includes B. subtilis, B. cereus, B. coagulans, B.
clausii, B. megaterium and B. licheniformis (Green, D.H., Wakeley, P.R., Page A.,
Barnes A., Baccigalupi L., Ricca E., Cutting S.M. (1999) Characterization of two
Bacillus probiotics, Appl. Env. Microbiol. 65(9): 4288-91). The administration of
many spore-based products by the oral route (also topical product by Ganeden
Biotech.) is considered safe due to the widespread consumption of Bacillus spp.
through many foods. The bacillus spore has been proved to be having the antioxidant
and free radical scavenging activity (Kodali, V.P. and Sen, R. (2008) Antioxidant and
free radical scavenging activities of an exopolysaccharide (EPS) from a probiotic
bacterium. Biotechnol. J. 3: 245-251). There are many other spore-forming Bacillus
spp. showing immunostimulating and immunomodulating activity, which are however
devoid of pathogenicity. US publication 20090186057 discloses compositions
including an isolated Bacillus species, spores or an extracellular product of B.
coagulans, suitable for topical application, for inhibiting growth of yeast, fungus,
bacteria or Herpes simplex virus. It discloses that growth of yeast, fungus, bacteria or
Herpes simplex virus can be inhibited by topical application of compositions that
include an isolated Bacillus species, spores or an extracellular product of a B.
coagulans strain.
The Bacillus sp. spore coat appears as a series of concentric layers. The number and
fine structure of the layers differ for different species. Biochemical experiments
demonstrated that the coat is composed largely of protein with minor amounts of
carbohydrate and lipid. The spore is made up of the core, cortex and spore coat. The
spore coat is the outermost covering of the spore (Aronson, A.I., Pandey, N.K. (1978)
Comparative structural and functional aspects of spore coats. In: Chambliss, G.,
Vary, J.C. (eds) Spores VII. Am. Soc. for Microbiol., pp 54-61). Spore coat is made up
of the different layers of proteins. Spore coat is the highly resistant material to different
chemicals, heat, radiations etc. (Henriques, A.O., Moran, C.P. (2000) Structure and
Assembly of the Bacterial Endospore Coat, Methods 20.1: 95-110). It has been
found that the surface of the spore is having the negative charge due to the carboxyl
groups of aspartic acid and glutamic acid (Tsuzuki, T., Ando, Y., (1985). Chemical
Studies on the Spore Coat Protein of Clostridium perfringens type A. Agric. Biol.
Chem. 49: 3221-3225). The spore coat protein layers are linked with each by the cross
linking and the cross linkage is due to the disulphide bonds like the keratin protein.
About 35% of the dry wet of the spore was found to be coat protein. There are different
drugs that have shown the ability to bind with keratin like protein. The antimalarial
drug Primaquine diphosphate shows binding with keratin protein of the bovine horn.
Primaquine diphosphate on transdermal administration binds to the keratin protein of
the skin and drug release study shows the sustained release type of profile (Heard,
CM., Monk, B.V., Modley, A.J., 2003, Binding of primaquine to epidermal
membranes and keratin, Int. I. Pharmaceutics 257: 237-244). There are other
examples of spore and dye interactions as well. Auramin O is used to stain spores
fluorescently (Bartholomew JW, Lechtman MD, Finkelstein H. (1965), Differential
spore and lipid staining at room temperature by using fluorescent dye, J Bacteriol
90: 1146-47). These drug/dye molecules have amino group side chain in common.
Exopolysaccharides are high-molecular-weight polymers that are composed of sugars
residues and are secreted by a microorganism into the surrounding environment.
Microorganisms synthesize a wide spectrum of multifunctional polysaccharides

including intracellular polysaccharide, structural polysaccharide and extracellular
polysaccharide or exopolysaccharide (EPS) (Anita Suresh Kumar and Kalpana Mody.
Book Chapter 10 (pp 229-254): Microbial Exopolysaccharides: Variety and Potential
Applications in the Book entitled: Microbial Production of Biopolymers and Polymer
Precursors: Applications and Perspectives (2009) edited by Bernd H. A. Rehm.
Publisher: Caister Academic Press). Exopolysaccharides generally constitute of
monosaccharides and some non-carbohydrate substituents (such as acetate, pyruvate,
succinate, and phosphate). Many microbial EPS provide properties that are almost
identical to the gums currently in use. Probiotics like Lactobacillus, Bacillus coagulans
etc. shows production of exopolysaccharide.
US7632520 discloses Lactobacillus sporogenes spores included in an antibacterial
sustained release formulation. The sustained release in the formulation of US7632520
is achieved by incorporation of Cloxacillin in HPMC matrix and a binder in the core.
The immediate release part of the formulation is composed of cloxacillin, cefixime, L.
sporogenes spores, a lubricant, a disintegrant. However the sporogenous probiotic
bacterium has been used in the patent only for synergistic benefit and not for drug
delivery. The mechanism and range of drugs that can be used in this formulation is
entirely different as it focuses on synthetic polymer for slower drug delivery.
Oral Vaccine Delivery by Recombinant Spore Probiotics, International Reviews of
Immunology, December 2009, Vol. 28, No. 6, Pages 487-505, by Simon M. Cutting,
Huynh A. Hong, Loredana Baccigalupi and Ezio Ricca teaches the use of bacterial
spores as a delivery system for mucosal immunizations. The journal provides an
analysis of the nature of the interaction between wild type spores and the gut-associated
lymphoid tissue and then addresses the immune responses that are induced by oral
immunizations with recombinant spores displaying heterologous antigens. However the
bacterial spores in this journal were not reported for delivery of any non-peptide drug.
In this case, the mechanism is totally different in the way that the peptide was
expressed on the spore coat-surface as integral part of the bacterial genome, for it was
genetically modified. Further USFDA does not allow recombinant (genetically
engineered) microorganisms for oral consumption.
US 6156330 teaches chitin beads having a uniform and fine particle size and
comprising microsporidian spores whose principal cell wall substance is chitin;
chitosan beads comprising N-deacetylated chitin and having a uniform fine particle
size; methods for preparing these beads; carriers comprising these beads; and a method
for preparing microsporidian spores. The beads are used as carriers for the
immobilization or encapsulation of a variety of substances such as an antibiotic and a
microorganism. The, the beads can be used as carriers for sustained release of these
substances. However the patent does not use spore for direct drug delivery, but one of
its component chitin and its chemically modified form chitosan. US 6156330 also
includes production method of the spore as a source of the above two components. US
6156330 does not claim that any spore can be used as such for drug delivery and have
removed protein part during purification of chitin. In contrast, the present sustained
release drug delivery system uses the whole bacterial spore for delivery without
requiring removal of any of its part. In the present invention presence of spore-coat
protein is responsible for drug adsorption on the spore coat.
Object of the invention
It is an object of the present invention to provide a sustained drug delivery system in
which the vehicle is a spore of probiotic bacteria.
It is another object of the present invention to provide a sustained drug delivery system
in which the vehicle itself has health benefits.
It is yet another object of the present invention to provide long protective effect even
after the symptoms of the disease has diminished.
It is further object of the present invention to reduce drug dose and prolong beneficial
activity.
It is also an object of the invention to provide easy production of drug-delivery system
with cheap raw materials.
Summary of the Invention
It is aspect of the present invention to provide a sustained release drug delivery system
comprising spores from sporogenous probiotic bacteria as carrier and active drug.
It is another aspect of the present invention to provide a sustained release dosage form
having a core comprising spores from sporogenous probiotic bacteria as carrier, drug
active and mucoadhesive substance; and a further coating on the core comprising
mucoadhesive substance, binder, lubricant, diluent and disintegrating agent.
It is a further aspect of the invention to provide a process for preparation of a sustained
release drug delivery system comprising steps of
(i) treating spores from sporogenous probiotic bacteria and
mucoadhesive substance with drug dissolved in a solvent to obtain a
suspension,
(ii) incubating the suspension of step (i) with constant shaking,
(iii) centrifuging the suspension after step (ii) and further washing with
deionized water to obtain a sediment.
It is yet another aspect of the invention to provide a process for preparation of a
sustained release dosage form comprising steps of
(i) treating spores from sporogenous probiotic bacteria and
mucoadhesive substance with drug dissolved in a solvent to obtain a
suspension,
(ii) incubating the suspension of step (i) with constant shaking,
(iii) centrifuging the suspension after step (ii) and further washing with
deionized water to obtain a sediment,
(iv) mixing the sediment with mucoadhesive substance, binder, lubricant,
diluent and disintegrating agent to obtain a wet mass,
(v) processing the wet mass obtained in step (iv) by wet granulation and
further compressing into tablets.
Brief Description of Accompanying figures
Fig. 1 illustrates the FT-IR data showing binding of drug (metformin) to spore-coat
protein: (A) only metformin (MET), (B) spore coat protein (PROTEIN), (C) metformin
bound to spore coat protein (METPRO).
Fig. 2 illustrates the Langmuir adsorption isotherm.
Fig. 3 illustrates the Fluoroscence microscope image of the probiotic spore bound with
metformin.

Fig. 4 illustrates the Comparison of viability of the probiotic: Control - only probiotic
spore powder without drug, spore plus adsorbed drug after washing of
exopolysaccahrides (EPS) with saline; sustained release drug delivery formulation
containing spores and unwashed EPS.
Fig. 5 illustrates the HPLC profiles of model drug — metformin (control) and drug
after release from sustained release dosage form in phosphate buffer saline
Fig. 6 illustrates the dissolution profile conventional metformin tablet, sustained release
dosage forms without EPS and with EPS.
Fig. 7 illustrates the Comparison release profile of the dosage forms (containing EPS
and without EPS) with ideal sustained release Higuchi model in % drug release vs.
square root of time (h) plot.
Fig. 8 illustrates the Scanning electron microscopy images of (a) lyophilized probiotic
spore powder showing individual endospores, (b) lyophilized probiotic spores with
drug adsorbed.
Description of the Invention
The present invention provides a sustained release drug delivery system, where spores
of probiotic sporogenous probiotic bacteria act as carrier for sustained release of drug-
molecules. The spores of bacteria have spore-coat proteins on its exterior, which binds
to the drug molecules by strong hydrogen bonding.
The present invention utilizes spores of probiotic sporogenous bacteria as carrier for
drug-molecules and in the development of formulations of the spores, drugs and
unwashed mucoadhesive substance preferably exopolysaccharide (EPS) as a sustained
release drug delivery system without any harmful effect. The presence of EPS in the
formulation enhances storage stability and spore viability. EPS also acts as
mucoadhesive for these bacteria so that they can colonize GIT of host (Sen R; Pal D;
Kodali VP; Das S and Ghosh, SK (2010) Molecular characterization and in vitro
analyses of a sporogenous bacterium with potential probiotic properties. Probiotics &
Antimicrobial Proteins {In press), DOI: 10.1007/s 12602-010-9049-0). Development of
formulations of probiotic spores with adsorbed drug molecules and unwashed
exopolysaccharide (EPS), produced by the same before sporulation, provides sustained
release on application.
Though any other mucoadhesive substance such as carbopol, polyacrylates, chitosan
and its derivatives, Eudragit etc, (Rajput GC, Majumdar FD, Patel JK, Patel KN,
Thakor RS, Patel BP, Rajgor NB. 2010. Stomach specific mucoadhesive tablets as
controlled drug delivery system-a review work Int. J. Pharm. Biol. Res., 1(1), 30-40.)
can be used in the present invention; EPS is the preferred one as EPS increases survival
of the bacteria. Thus EPS released by corresponding sporogenous bacteria is preferred
for the present formulations. The mucoadhesive substance may be present in an amount
0.1% to 1% by weight.
The various sporogenous probiotic bacteria whose spores can be employed for the
present invention are B. coagulans, B. subtilis, B. cereus, B. clausii, B. megaterium and
B. licheniformis. The spore from sporogenous probiotic bacteria in the sustained
release system is present in an amount 1% to 20% by weight, preferably 2 % to 10 %
by weight.
The drugs, which are suitable for the present invention, but not limited to, are drugs
having amino (e.g., primaquin, cyclobenzaprine etc.), imino (e.g., metformin,
phenformin, amiloride etc.), amide (e.g., piroxicam, pyrazinamide, all peptide
antibiotics), sulphonyl/sufoxide (sulindac, dimethyl sulfoxide) sulfonyl (e.g.
indapamide, chlorthalidone, all suphonamides etc.), aromatic nitrogen (all p-lactam
antibiotics, milrinone, flecainide, clotrimoxazole etc), hydroxyl (e.g., nadolol,
metoprolol, procyclidine, biperiden hydrochloride, isosorbide, aurothio glucose etc.)
thiol (captopril), cyano (pinacidil), oxo (e.g. taxol, atracurium, chloroxazone,
spironolactone, griseofulvin etc.), carboxyl (e.g. vecuronium bromide, ethacrynic acid,
probenecid, flurbiprofen etc.), sulphide (e.g. polythiazide etc.), nitro (e.g. dantrolene,
nitroglycerine etc.), sulphonic acid (suramin), iodo (iodoquinol) groups in the side-
chain. However the affinity of these drugs to spore coat of Bacilli varies. Practically all
drugs except ionic salts such as magnesium hydroxide, calcium carbonate, sodium
bicarbonate, etc., are good candidates for the sustained release dosage form of the
present invention, provided their biological half life is less (<10h). Any drug (except
the salts) is adsorbed on the surface of said spores from sporogenous probiotic bacteria.
The drug binds to the spore-coat protein on the surface of said spores from sporogenous
probiotic bacteria. These drugs may be antidiabetic, anti-allergy, antihyperglycemic or
anticancer.

The sustained release drug delivery system may be formulated into various dosage
forms, preferably a tablet, utilizing various conventional pharmaceutical excipients.
Accordingly the sustained release dosage form may be formed of a core comprising
spores from sporogenous probiotic bacteria, drug and mucoadhesive substance and a
further coating on the core comprising binder, lubricant, diluent, mucoadhesive
substance and disintegrating agent. The spores from sporogenous probiotic bacteria act
as carrier for sustained release of said drug.
Drug actives may be present in an amount 0.1% to 20% by weight, preferably 1 % to
10% by weight.
The sustained release dosage form of the present invention contains spore from
sporogenous probiotic bacteria in an amount 1% to 20% by weight, preferably 2 % to
10 % by weight. Generally drug : probiotic spore=1:1 (w/w, when spore concentration
is 1011 g-1. If spore concentration increases less weight of spore would be used).
In a preferred embodiment the sustained release dosage form comprises
exopolysaccharides in an amount 0.1% to 1 % by weight. Probiotics : EPS=10:l(w/w,
when spore concentration is 1011 g-1. If spore concentration increases less weight of
spore would be used and proportionally less amount of EPS would be needed). This
may be present in the core. The amount of EPS present outside the core is generally
double of that present inside the core.
Binders that can be utilized in the present invention can be starch, acacia, tragacanth,
microcrystalline cellulose, sodium alginate and alginate derivatives, gelatin, or any
other binder and adhesive. The amount of binder varies from 10 % to 25 % by weight.
The lubricants utilized in the present invention are talc, cornstarch, colloidal silicas,
stearates, surfactants, polyethylene glycols or any other lubricant. They are present in
an amount 0.5 % to 10 % by weight.
The sustained release dosage form of the present invention also comprises
disintegrating agent like seralite resin, starch, modified starches clays etc.
Disintegrating agents are present in an amount 1% to 20 % by weight.
Various diluent may be present in the formulation of the present invention namely

lactose, microcrystalline cellulose, mannitol, sorbitol, sucrose based materials etc.
Diluent is present in an amount 0 % to 90 % by weight.
The sustained release dosage form of the present invention may further contain drug in
the loading dose for immediate release from the EPS covered spore surface. This may
be due to the phenomenon of simple gel entrapment of the excess amount of the drug in
the loading dose that was not adsorbed by the spores (Figure 8).
The present invention also provides a process for preparation of a sustained release
drug delivery system of the present invention comprising steps of
(i) treating spores from sporogenous probiotic bacteria and mucoadhesive
substance preferably exopolysaccharides with drug dissolved in a solvent to
obtain a suspension,
(ii) incubating the suspension of step (i) with constant shaking at a
temperature around 37°C,
(iii)centrifuging the suspension after step (ii) and further washing with
deionized water to obtain a sediment.
The present invention further provides a process for preparation of a sustained release
dosage form of the present invention comprising steps of
(i) treating spores from sporogenous probiotic bacteria and mucoadhesive
substance, preferably exopolysaccharides with drug dissolved in a solvent to
obtain a suspension,
(ii) incubating the suspension of step (i) with constant shaking,
(iii)centrifuging the suspension after step (ii) and further washing with
deionized water to obtain a sediment,
(iv)mixing the sediment with mucoadhesive substance, preferably
exopolysaccharides, binder, lubricant, diluent and disintegrating agent to
obtain a wet mass,
(v) processing the wet mass obtained from step (iv) by wet granulation and
further compressing into tablets using suitable tablet compression machine.
The solvent for the drug that can be used in the present invention can be propylene
glycol in water, butanol/water, glycerol/water, mineral oil/water, and n-hexane/water.
To prepare the dosage form of the present invention spore powder and mucoadhesive

substance, preferably exopolysaccharide were treated with drug in solvent in an
alkaline pH or acidic pH depending upon the nature of the drug, which then forms a
suspension upon incubation. (Alkalinity is essential for basic drugs and acidic pH for
acidic drugs so as to give an isoelectric pH environment to the drug).
The suspension is continuously stirred on the shaker to separate all clumps and allowed
to suspend each spore individually. Freebase of metformin is further added to the
suspension and kept it in shaking incubator for few hours. (In course of transit through
GIT the tablet has to be disintegrated into small particles first as the tablet was formed
in such a way and then into individual spores. The total surface area of these particles
are lesser than total surface area of spores altogether. Hence from particle surfaces drug
would leach at a slower rate than from the spores after total disintegration of the dosage
form. Moreover this extra amount of drug acts as loading dose. This amount of
metformin actually exceeds the maximum binding capacity of the spores. After
administration this extra amount of unbound drug will quickly increase blood plasma
drug concentration up to its threshold level. The bound drug is released slowly to
maintain plasma drug concentration). After incubation the suspension is centrifuged for
few minutes and the sediment of spore is separated and washed with deionized water.
The resulting composition forms uneven microparticles. These microparticles in the
form of powder was formulated into dosage form with further addition of remaining
amount of drug for immediate release and mucoadhesive substance, preferably
exopolysaccharide, binder, lubricant, disintegrating agent, diluent to make final weight
of the dosage form. Ideally for immediate release the drug should be equal to one single
dose of the drug (loading dose). For subsequent release within 24h range the required
dose has to be used for sustained release (that would be bound to spore).
The delivery system of the present invention shows drug-release from the formulation
over longer period of time. As the drug delivery system of the present invention
involves spore-bearing probiotic bacteria, which are resistant to environmental stress
conditions, drugs adsorbed onto the spores shows high physical stability. The final
formulations can be stored at room temperature and there is no need of refrigeration.
The present invention utilizes a sustained release drug delivery system, in which the
vehicle itself has some health benefits. Thus once the symptoms of the disease are
diminished, the vehicle, which is actually constituted of probiotic bacteria, goes on
exerting protective effect in the long run. These bacteria colonize in gastro intestinal
tract and exert its prolonged benefits, which is an added advantage. Such probiotic does
not produce adverse effect even in high concentration in terms of colony forming units.
If a proper drug is combined with the probiotic, the doses of the drug can be reduced
substantially. By the term "proper drug" it is meant that the drug has common
pharmaceutical effect that the probiotic also exerts. Further utilization of probiotic
spores as vehicles provides non-toxic and non-pathogenic formulations and also
provides ease in the production of such formulations. Further the spores being a cheap
raw material the process for preparation of the formulations as are also reduced.
The present invention is now illustrated using non-limiting examples.
Example 1
Preparation of drug adsorbed on spores of probiotic sporogenous probiotic bacteria
Materials and Method: 10mg of Bacillus coagulans RK-02 spore powder is treated with
a half of the model drug (e.g. 5mg free base of metformin) in the 5 ml of 30%
propylene glycol in water and kept it in 37°C shaking incubator at 120 rpm for 1 hour.
Drug binds to spore individual spore surface during this process. To the above
suspension the other half of total free base of metformin (5mg) plus 0.1 mg
exopolysaccharide was added and kept it in 37°C shaking incubator at 120 rpm for 1
hour (total 2h incubation). After 2 hours of incubation the suspension is centrifuged at
7000 rpm for 15 minutes and the sediment of spore was separated and washed with
deionized water.
Example 2
1g metformin, 52.5ml glycerol, 7ml phenol, and 35ml Millipore™ water were mixed
and incubated at 60°C overnight. Bacillus coagulans RK-02 spores were heat-fixed on
slide. The smear was flooded with meformin solution and was kept in dark for half an
hour at 37°C. Next the slide was washed with water, followed by 0.5% acid alcohol
repeatedly until no further dye comes out of smear. The slide is washed with water and
then flooded with 0.5% potassium permanganate for 2min. The slide is then washed
with water and is air-dried. (Bartholomew JW, Lechtman MW and Finkelstein H
(1965) Differential Spore and Lipid Staining at Room Temperature by Use of
Fluorescent Dye. / bacterid 90(4):\ 146-1147). The slide is observed under microscope
at excitation wavelength 450nm (Hassan S.S.M. Mahmoud W.H.; Elmosallamy

M.A.F.; Othman A.H.M. 1999, Determination of metformin in pharmaceutical
preparations using potentiometry, spectrofluorimetry and UV-visible
spectrophotometry Analytica Chimica Acta 378(1, 4): 299-311(13)
The binding/adsorption of drug to the spore coat proteins, as obtained by the process in
Example 1, was confirmed by Fluorescence Microscopy. Fluorescence microscope
(Olympus; Model- IX 51) photographs of smear of probiotic spores stained with
fluorescent dye were taken at 100X magnification and shown in Fig. 3.
Example 3
It is observed under Scanning Electron Microscope that the resulting composition
forms uneven microparticles as obtained by the process in Example 1. Scanning
electron microscope photograph of powdered probiotic spore with drug, wetted
probiotic spore and dried thick probiotic film were taken at different magnifications.
Scanning electron microscopy was done to visualize gloomy sticky structure of the
probiotic spores due to the presence of exopolysaccharide. Samples were gold plated
for the scanning electron microscopy. These microparticles were formed due to the
presence of the exopolysaccharides as evident in Fig 8(b). Figure 8(a) demonstrated
that such microparticles were not observed in the absence of EPS and rather individual
endospores were present. White dots shown in Fig 8(b) are excess drug (finer particles)
equivalent to loading dose entrapped between microparticles. The unbound drug may
be in the inter-particulate or intra-particulate spaces and the same is visible as white
dots. Free drug-molecules form very small nano-size particles and place themselves in
between the particles (intra-particulate spaces).
Example 4
Experiment to confirm that the drug is attached on the spore coat protein
Lysis of the PSC protein and separation of the drug molecule:
Probiotic spores bound with drug were suspended in the lysis buffer containing 50 mM
Tris base (pH 10), 8 M urea, 50 mM DTT and 1% SDS. The suspension is incubated at
60°C for 90 minutes with vigorous vertexing at 10-minute interval (Riesenman et al.,
1999). The lysis product was centrifuged at12000 g for 15 minutes. The supernatant
was treated with 0.1 M HCl and acetonitrile, vortexed for 30 seconds and centrifuged at
1763 g for 5 minutes. The supernatant was washed with the dichloromethane by vortex
mixing for 30 seconds and centrifuged at 1763 g for 5 min. The aqueous layer is used
for drug assay.
FTIR analysis:
FTIR analysis of free drug, spore coat protein and drug bound spore coat protein were
performed in Nexus-890 Fourier Transform Infrared Spectrophotometer (Thermo
Electron Co., Yokohama, Japan).
Results and discussion:
FT-IR data (Figure 1) showed binding of drug (metformin) to spore-coat protein: (A)
only metformin (MET), (B) spore coat protein (PROTEIN), (C) metformin bound to
spore coat protein (METPRO). Same peaks at 3100-3500 cm-1 for primary and
secondary amines (-NH-), 3360-3400 cm-1, 1630-1680 cm-1, 1500-1580 cm-1 for free
metformin (A) and metformin bound to spore-coat protein (C) confirmed binding of
drug to spore-coat by hydrogen bonding and no new covalent bond was created. Peak at
3620 cm-1 was due to monomeric -OH of propylene glycol solvent.
Example 5
Preparation of the sustained release dosage form
The microparticles as obtained by the process in Example 1 are then formulated into
tablet dosage form with further addition remaining amount (50% of total drug). 5mg of
drug for immediate release and 0.1 mg exopolysaccharide, 5% starch as binder, 1% talc
as lubricant, 2% Seralite resin as disintegrating agent. Lactose was used as diluent to
make final weight of a tablet 500 mg.
Example 6
Tablet was then subjected to dissolution testing in simulated intestinal fluid as per US
Pharmacopoeia. Drug release pattern followed Higuchi model for sustained release.
It is found out that a tablet in presence of dissolution medium is disintegrated into a
sticky mass due to presence of exopolysaccharides, from which diffusion dependent
drug release occurs slowly. Further it is observed that after about 6-8h the sticky mass
disintegrated into small particles, from which remaining drug releases slowly. This
extends the time of release of the drug.
Example 7
Test of viability of probiotic spores:

0.1 g accurately measured amount of probiotic spores ((1) Control - only probiotic
spore powder without drug, (2) spore plus adsorbed drug after washing of
exopolysaccahrides (EPS) with saline; (3) sustained release drug delivery formulation
containing spores and unwashed EPS) were suspended in the 2 ml of saline water and
incubated in water bath at 70°C for 30 minutes. (The heat shock kills contaminants (if
any) and also activates spores to germinate.) The above suspension was cooled to 45°C
and the spores were inoculated in serial dilution technique in GYEA-agar plate by pour
plate method.
It was observed that sustained release drug delivery formulation containing spores and
unwashed EPS showed more viability than drug bound spores without EPS (Fig 4).
Therefore use of EPS helped to protect the loss of immature endospores from death!
Hence use of EPS in the present formulations is indispensable.
Example 8
Lysis of the PSC protein and separation of the drug molecule:
Probiotic spores bound with drug were suspended in the lysis buffer containing 50 mM
Tris base (pH 10), 8 M urea, 50 mM DTT and 1% SDS. The suspension is incubated at
60°C for 90 minutes with vigorous vortexing at 10-minute interval (Riesenman et al,
1999). The lysis product was centrifuged atl2000 g for 15 minutes. The supernatant
was treated with 0.1 M HC1 and acetonitrile, vortexed for 30 seconds and centrifuged at
1763 g for 5 minutes. The supernatant was washed with the dichloromethane by vertex
mixing for 30 seconds and centrifuged at 1763 g for 5 min. The aqueous layer is used
for drug assay. This is how amount of drug adsorbed onto spore coat was determined.
Amount of drug used(x) and amount of drug adsorbed by 15mg spores (y) data was
plotted as depicted in Figure 2. The data fitted very well into Langmuir adsorption
isotherm: >+

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