WO2008107746A2

WO2008107746A2 - Tablet comprising microorganisms and a combination of excipients and the manufacture process thereof

Info

Publication number: WO2008107746A2
Application number: PCT/IB2008/000289
Authority: WO
Inventors: Gian Paolo Strozzi; Luca Mogna
Original assignee: Anidral SRL
Current assignee: Anidral SRL
Priority date: 2007-03-07
Filing date: 2008-01-29
Publication date: 2008-09-12
Anticipated expiration: 2009-09-07
Also published as: WO2008107746A3; ITMI20070452A1

Abstract

The present invention relates to a tablet comprising microorganisms and a combination of technological tableting excipients, and to the manufacturing process thereof. The combination of excipients comprises: 40% to 80% w/w of microcrystalline cellulose, 8% to 35% w/w of anhydrous calcium hydrogenophosphate and 8% to 25% w/w of sodium carboxymethylcellulose, percentages being expressed with respect to the total weight of the combination of excipients. Preferably, the microorganisms are probiotic microorganisms, used singularly or as a mixture made up also of different genus or species.

Description

DESCRIPTION

"Tablet comprising microorganisms and a combination of excipients and the manufacture process thereof"

* * * * * The present invention relates to a tablet comprising microorganisms and a combination of technological ta- bleting excipients including microcrystalline cellulose, anhydrous calcium hydrogenophosphate and sodium carboxymethylcellulose . Tablets comprising microorganisms, in particular pro- biotic microorganisms, are known at the state of the art, which tablets are characterized by a parameter of effectiveness and validity related to the number and vitality of present microorganisms. For manufacturing such tablets tableting methods that are well known at the state of the art can be used, which methods can show various problems related to the fact that tablets to be manufactured contain microorganisms. In particular, the tableting step can lead to a reduction in the number and vitality of the population of microorganisms present in the end tablet. Moreover, there is often a lower stability of the end product, since stressing conditions of the tableting step, beyond reducing more or less considerably the bacterial charge, induce such stress and distress to the cell structures as to cause a rapid decay of the microbial population after the tablet has been manufactured. Therefore, the dosage of microorganisms declared on the end package can differ from the actual dosage due to the reduction in the number of microorganisms during the tableting step and during the storage of the product as tablets (reduction in the so- called product shelf-life) . The compression force applied during manufacturing is estimated by quantifying by means of a durometer the force required to break a tablet. Said force is measured in Kp (Kilopond) (1 Kp = 9.807 Newton (N)). The negative effect of compression on the population of microorganisms is evident both when applied forces are high, e.g. for tablets having a hardness of 10-12 Kp, and when said forces are low, such as for instance for tablets having a hardness of 5-6 Kp. The drawback resulting from the use of high forces is a higher reduction in the number of microorganisms, whereas low forces generally lead to a lower cohesion of the tablet.

The Applicant has surprisingly found that such problems can be solved by using in the manufacture of the tablet a combination of technological tableting ex- cipients comprising: 40% to 80% w/w of microcrystal- line cellulose, 8% to 35% w/w of anhydrous calcium hy- drogenophosphate and 8% to 25% w/w of sodium carboxy- methylcellulose, percentages being expressed with respect to the total weight of the combination of ex- cipients. Said combination of technological tableting excipients according to the invention reduces to a very considerable extent the mortality of microorganisms during the tableting step and, inducing a quantitatively far lower cell stress, also enables to keep the vitality of microorganisms unchanged, thus ensuring a higher effectiveness of the products thus obtained.

Moreover, the combination of excipients according to the invention enables to use lower compression forces while keeping the desired integrity of the tablets thus obtained.

According to a first aspect, the present invention therefore relates to a tablet comprising microorganisms, characterized in that it further comprises a combination of excipients including: 40% to 80% w/w of microcrystalline cellulose, 8% to 35% w/w of anhydrous calcium hydrogenophosphate and 8% to 25% w/w of sodium carboxymethylcellulose, percentages being expressed with respect to the total weight of the combination of excipients. The tablets according to the invention can be used for various types of administration, preferably for oral administration. The tablets according to the invention can be ready to use as such or they can undergo another coating step with one or more poly- meric materials known at the state of the art.

Said combination of excipients is present in the tablets according to the invention preferably in a total amount of 20 to 50% w/w, more preferably 30 to 45% w/w, and still more preferably 35 to 40% w/w, with re- spect to the total weight of the tablet.

Preferably, the combination of technological tableting excipients comprises: 50% to 70% w/w of microcrystal- line cellulose, 15% to 30% w/w of anhydrous calcium hydrogenophosphate and 10% to 20% w/w of sodium car- boxymethylcellulose, percentages being expressed with respect to the total weight of the combination of excipients .

According to a preferred embodiment of the invention, the combination of excipients further comprises 0.8% to 5% w/w, preferably 1% to 3.5% w/w, of magnesium stearate, percentages being expressed with respect to the total weight of the combination of excipients. According to a preferred embodiment of the invention, the combination of excipients further comprises 0.5% to 2% w/w, preferably 0.8% to 1.5% w/w, of silicon di- oxide, percentages being expressed with respect to the total weight of the combination of excipients. The present invention further relates to a process for manufacturing tablets comprising microorganisms, in- eluding: mixing the microorganisms with a combination of excipients comprising: 40% to 80% w/w of microcrystal- line cellulose, 8% to 35% w/w of anhydrous calcium hy- drogenophosphate and 8% to 25% w/w of sodium carboxy- methylcellulose, percentages being expressed with respect to the total weight of the combination of excipients; tableting the mixture thus obtained so as to obtain the tablet. The present invention further relates to the use of a combination of excipients comprising: 40% to 80% w/w of microcrystalline cellulose, 8% to 35% w/w of anhydrous calcium hydrogenophosphate and 8% to 25% w/w of sodium carboxymethylcellulose, percentages being ex- pressed with respect to the total weight of the combination of excipients, for the manufacture of tablets comprising microorganisms.

As far as the microbial population of the formulations according to the present invention is concerned, it is advantageous to use probiotic microorganisms, employed singularly or as a mixture made up also of different genus or species.

Probiotic microorganisms are those microorganisms, mainly bacteria of human origin, which, if regularly taken in a sufficient amount and for an adequate lapse of time, can induce beneficial effects. In a preferred embodiment, the probiotic microorganisms according to the invention comprise at least one microbial strain (or a mixture of several microbial strains) chosen from the group including the genus: Lactobacillus, Bifidobacterium, Lactococcus, Leu- conostoc, Streptococcus, Bacillus, Propionibacterium, Saccharomyces, Enterococcus. Preferably, the following species of the genus Lacto- bacillus are used: L. pentosus, L. plantarum, L. ca- sei, L. easel ssp. paracasei, L. rhamnosus, L. acidophilus, L. delbrueckii, L. delbrueckii ssp. bulgari- cus, L. fermentum, L. gasseri, L. reuteri. Preferably, the following species of the genus Bifido- bacterium are used: B. longum, B. bifidum, B. breve, B. animalis ssp. lactis, B. adolescentis, B. pseudo- catenulatum, B. catenulatum, B. infantis. Preferably, the following species of the genus Lactococcus are used: L. lactis ssp. cremoris and L. lactis ssp. lactis. Preferably the species S. thermophilics of the genus Streptococcus has been used.

Among the bacterial species mentioned above, the bacterial strains listed in the following Table 1 have proved to be particularly preferred.

Table 1

Streptococcus

56 thermo-

DSM 18618 13.09.2006 MOFIN S. R. L. philus

57 Streptococcus thermo-

DSM 18619 13.09.2006 MOFIN S.R. L. philus

58 Streptococcus thermo-

DSM 18620 13.09.2006 MOFIN S. R. L. philus

Streptococcus thermo-

59 DSM 18621 13.09.2006 MOFIN S. R. L. philus

60 Streptococcus thermo-

DSM 18622 13.09.2006 MOFIN S. R. L. philus

Streptococcus theriao-

61 DSM 18623 13.09.2006 MOFIN S. R. L. philus

62 Streptococcus thermo-

DSM 18624 13.09.2006 MOFIN S. R. L. philus

Streptococcus

63 thermo-

DSM 18625 13.09.2006 MOFIN S. R. L. philus

Among these L. rhamnosus LR04 (number of deposit DSM 16605) is one of the particularly preferred probiotic strains of the invention.

In the tablets according to the invention, the micro- organisms, preferably used as freeze-dried culture with a vital charge generally of 100 to 200 billions of colony forming units (UFCs) /gram, are preferably present in an amount of 1 to 20% w/w, preferably of 3 to 10% w/w, still more preferably of 5 to 8% w/w, with respect to the total weight of the tablet.

In one of. the preferred embodiments, in order to enhance the probiotic effectiveness of the formulations according to the present invention, specific prebiotic components are introduced into the powder mixture, thus obtaining a symbiotic composition. The prebiotic component is as a rule a non-digestible material of saccharidic nature, at least partially soluble in water or in aqueous solution, which stimulates the growth and/or activity of one or more strains of pro- biotic strains as described above. Among these pre- biotic agents, dietary fibers are preferred. Preferably, said prebiotic fiber is chosen from the group comprising: fructo-oligosaccharides (FOS) , galacto- oligosaccharides (GOS) , trans-galacto-oligosaccharides (TOS), xylo-oligosaccharides (XOS), chitosan- oligosaccharides (COS) , α-galactosides (such as raffi- nose, stachiose and so on) , pectins, gums, partially hydrolyzed gums, inulin, psyllium, arabinogalactans, acacia fiber, carruba fiber, rye fiber, bamboo fiber, citrus fibers and in general fibers containing a soluble and an insoluble portion in a variable ratio one to the other. Preferably, the prebiotic component is present in the tablet in an amount up to 60% w/w, preferably of 3 to 15% w/w, still more preferably of 4 to 7% w/w, with respect to the total weight of the tablet. In a preferred embodiment, the tablet according to the invention can contain other active components, e.g. vitamins, mineral salts, vegetable extracts or other compounds having a synergic effect or a complementary effect to the one of the population of microorganisms present in the formulations according to the invention. Preferably, said other active components are present in an amount of 10 to 75% w/w. Preferably of 30 to 60% w/w, still more preferably of 40 to 55% w/w, with respect to the total weight of the tablet. The combination of technological tableting excipients according to the present invention ensures an adequate cohesion of the tablet powders and result in the desired release kinetics of the active substances after intake. More particularly, it is believed that microcrystal- line cellulose has disaggregating functions, whereas anhydrous calcium hydrogenophosphate affects tablet friableness, and sodium carboxymethylcellulose acts as a binding agent. It is believed that the ratio of the amounts of micro- crystalline cellulose to those of sodium carboxymethylcellulose is the main parameter determining the speed of disaggregation of the tablet and, therefore, of release of the active components. The lower is the ratio between the two cellulose types, the higher will be the aggregation of the powders making up the formulation, i.e. the force required to disaggregate the tablet, the compression force exerted when manufacturing the tablet being the same. Beyond the functions described above, the Applicant has surprisingly found that the ratio of microcrystal- line cellulose to anhydrous calcium hydrogenophos- phate, as well as the relative amounts of the two substances in the total formulation, play a fundamental role in the safeguard of the vitality of probiotic cells.

According to a preferred embodiment of the invention, conversely, magnesium stearate and silicon dioxide are used, the first one to reduce the adhesion of the powders to the working surfaces of the tableting machine, the second one to improve the flowing properties of said powder.

The various components, generally in powder form, can be mixed in any sequence of addition of the components, preferably keeping in mind to add the culture of microorganisms, preferably in freeze-dried form, as last ingredient, so as to prevent too long a mixing time from inducing a mechanical shock to the wall of the bacterial cells, leading to distress and lower stability of the end product. Formulations produced according to the method of the present invention are characterized by excellent flowing properties, a parameter that helps a uniform loading of the cavities (i.e. the space for housing the specific amount of powder to be tableted) in tableting machines. Said tableting machines typically comprise a device for loading the cavities (generally represented by a feeder for transporting the powder mixture) and metallic disc with an adequate radius, in whose thickness said cavities are obtained, which are present in a variable number depending on the machine, in which cavities the powder is tableted thanks to the simultaneous and synergic movement of two moving parts of the machine, the so-called punches. During the tableting step, the lower punch defines with its position the volume of the cavity (and thus the amount of powder loaded each time) , whereas the upper punch, placed outside the cavity when the latter is loaded with the powder mixture, during the tableting step can lower and get in contact with the powder in the cavity, imposing thanks to the exerted force a more or less evident mechanical deformation to the particles making up the powder. At the end of the tableting step, the upper punch is lifted and thus gets away from the cavity, and then the lower punch is also lifted, thus letting out of the cavity the newly formed tablet.

Said tableting machines further comprise various control devices for the main operating parameters (position of lower punch at beginning of tableting cycle, lowest position reached by upper punch during tablet- ing cycle, speed of rotation of disc containing cavities and so on) .

There are reciprocating and rotary tableting machines, depending on the global operating modes of the machine and as a consequence on the number of tablets the machine can produce in the same time unit. Tablets can therefore be manufactured using tableting machines known at the state of the art and using punches of various shapes, preferably oval and round with double radius .

Formulations prepared according to the present invention enable to obtain tablets containing probiotic microorganisms that are effective and stable in time, also at non-refrigerated temperature. In manufacturing tests for tablets containing L. rham- nosus LR04, the Applicant has used formulations prepared according to the method of the present invention and has obtained highly positive results both in terms of survival and in terms of subsequent stability. In particular, a mortality of 4 to 20% has been found when the compression force applied has been such to obtain tablets with a hardness of 5 to 6 Kp, and a mortality of 15 to 40% has been found when the compression force applied has been such to obtain tablets with a hardness of 10 to 12 Kp. Another advantage of the method according to the invention consists in that it allows the production of sufficiently cohesive tablets also with low compression forces, e.g. tablets with a hardness of 5-6 Kp.

Example 1 - Formulation according to the invention, formulation according to the state of the art and comparison of data of mortality due to tableting and of stability obtained in tablets manufactured with the above mentioned formulations.

Table 2 shows the composition of a tableting mixture according to the invention.

Table 2

Table 3 shows the composition of a tableting mixture having technological excipients according to the current state of the art. Table 3

Mixture components mg / tablet

Probiotic component (e.g. L. rhamnosus LR04 with a titer of 200 •lOVgram) 40.50

Insoluble dietary fiber 32.26

Active substances (vitamins, mineral salts, vegetable ex- tracts, etc.) 413.24

Calcium carbonate 250.00

Hydroxypropylmethylcellulose 43.00

Magnesium stearate 7.00

Silicon dioxide 4.00

TOTAL (1 tablet) 790.00

Table 4 shows the comparison of data of mortality of probiotic bacteria as a function of the various pressures exerted and of tablet stability (expressed as half-life of bacterial charge) after 2 years of storage of said tablets at 25 °C. The tablets obtained with the formulation according to the invention and with the formulation according to the state of the art had a diameter of about 9.3 mm and a length of about 11.5 mm, with a thickness of about 5.7 mm for high hardness tablets and of about 6.2 mm for low hardness tablets.

The above data were obtained by estimating the number of probiotic bacteria present in the biobulk powder and in said powder after tableting using the method of plate vital count, in accordance with the methods known to the skilled technician.

For biobulk powder, an amount of 1 to 4 grams of sample is re-suspended in an adequate volume of a sterile liquid medium, generally 0.85% physiological solution of sodium chloride, added with 1 g/1 of bacteriologic peptone.

After powder dissolution and following homogenization with a suitable piston-equipped tool, the number of cells/ml is reduced by means of subsequent 10-base serial dilutions. From a practical point of view, each time 1 ml of the most concentrated dilution is transferred with a sterile pipette into 9 ml of diluting agent; the operation is carried out for a sufficient number of times so as to bring the amount of microorganisms present per ml of diluting agents to a number of 10 to 300, so that after being transferred to a Petri dish and added with a suitable agarized culture medium, they form separate, and therefore countable, colonies. The protocol complied with for the tablets consists first in breaking three to five tablets and then in carrying out the analysis as described above for biobulk powder. Data show a lower mortality both for high hardness tablets (26% of initial population) and for low hardness tablets (7% of initial population) . Another advantageous aspect of the formulation according to the present invention is the better stability in time of the probiotic component of said formulation with re- spect to the typical stability of a formulation at the state of the art. This is due to the use of a peculiar combination of technological tableting excipients that can strongly limit mechanical damages caused by the tableting step to the probiotic bacterial cells. Stability is expressed as the half-life of the probi- otic component of the formulation, i.e. the time elapsed before the initial charge of vital cells is halved.

The Applicant has found that the half-lives of the probiotic component in the biobulk powder and in the tablets with different hardness values, produced with a formulation according to the invention, are very similar to one another, thus showing that the technological components used are able to minimize mechani- cal damages caused by the tableting step.

Claims

1. A tablet comprising microorganisms, characterized in that it further comprises a combination of excipi- ents including: 40% to 80% w/w of microcrystalline cellulose, 8% to 35% w/w of anhydrous calcium hydro- genophosphate and 8% to 25% w/w of sodium carboxy- methylcellulose, percentages being expressed with respect to the total weight of the combination of ex- cipients .

2. The tablet according to claim 1, wherein said combination of excipients is present in a total amount of 20 to 50% w/w, more preferably 30 to 45% w/w, and still more preferably 35 to 40% w/w, with respect to the total weight of the tablet.

3. The tablet according to claim 1 or 2, wherein the combination of excipients comprises: 50% to 70% w/w of microcrystalline cellulose, 15% to 30% w/w of anhydrous calcium hydrogenophosphate and 10% to 20% w/w of sodium carboxymethylcellulose, percentages being ex- pressed with respect to the total weight of the combination of excipients.

4. The tablet according to any of the preceding claims, wherein the combination of excipients further comprises 0.8% to 5% w/w, preferably 1% to 3.5% w/w, of magnesium stearate, percentages being expressed with respect to the total weight of the combination of excipients .

5. The tablet according to any of the preceding claims, wherein the combination of excipients further comprises 0.5% to 2% w/w, preferably 0.8% to 1.5% w/w, of silicon dioxide, percentages being expressed with respect to the total weight of the combination of excipients .

6. The tablet according to any of the preceding claims, wherein the microorganisms are probiotic microorganisms .

7. The tablet according to claim 6, wherein the pre- biotic microorganisms comprise at least one microbial strain chosen from the group including the genus: Lac- tobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Streptococcus, Bacillus, Propionibacterium, Saccharo- myces, Enterococcus.

8. The tablet according to any of the preceding claims, wherein the microorganisms are present in an amount of 1 to 20% w/w, preferably of 3 to 10% w/w, still more preferably of 5 to 8% w/w, with respect to the total weight of the tablet.

9. The tablet according to any of the preceding claims, further comprising at least one prebiotic com- ponent.

10. The tablet according to claim 9, wherein the pre- biotic component is a non-digestible material of sac- charidic nature, at least partially soluble in water or in aqueous solution.

11. The tablet according to claim 10, wherein the prebiotic component is a dietary fiber.

12. The tablet according to any of the claims 9 to

11, wherein the prebiotic component is present in an amount up to 60% w/w, preferably of 3 to 15% w/w, still more preferably of 4 to 7% w/w, with respect to the total weight of the tablet.

13. The tablet according to any of the preceding claims, having a hardness of 10 to 12 Kp.

14. The tablet according to any of the claims 1 to 12, having a hardness of 5 to 6 Kp.

15. A process for manufacturing tablets comprising microorganisms according to any one of the preceding claims, including: mixing the microorganisms with a combination of ex- cipients comprising: 40% to 80% w/w of microcrystal- line cellulose, 8% to 35% w/w of anhydrous calcium hy- drogenophosphate and 8% to 25% w/w of sodium carboxy- methylcellulose, percentages being expressed with respect to the total weight of the combination of ex- cipients; tableting the mixture thus obtained so as to obtain the tablet.

16. The process according to claim 15, wherein the microorganisms are mixed as freeze-dried culture.

17. The process according to claim 15 or 16, wherein the microorganisms are added to the combination of already mixed excipients.

18. The process according to any one of the claims 15 to 17, wherein during the tableting step a compression force such as to obtain tablets having a hardness of 10 to 12 Kp is applied.

19. The process according to any of the claims 15 to

17, wherein during the tableting step a compression force such as to obtain tablets having a hardness of 5 to 6 Kp is applied.

20. Use of a combination of excipients comprising: 40% to 80% w/w of microcrystalline cellulose, 8% to 35% w/w of anhydrous calcium hydrogenophosphate and 8% to 25% w/w of sodium carboxymethylcellulose, percent- ages being expressed with respect to the total weight of the combination of excipients, for manufacturing tablets comprising microorganisms.

21. Use of a combination of excipients comprising: 40% to 80% w/w of microcrystalline cellulose, 8% to 35% w/w of anhydrous calcium hydrogenophosphate and 8% to 25% w/w of sodium carboxymethylcellulose, percentages being expressed with respect to the total weight of the combination of excipients, for reducing the mortality of microorganisms in the production of tab- lets comprising said microorganisms.