Composition comprising solanum glaucophyllum for preventing and/or treating hypocalcaemia and for stabilizing blood calcium levels
09757415 · 2017-09-12
Assignee
Inventors
Cpc classification
A61K36/81
HUMAN NECESSITIES
A61K9/2054
HUMAN NECESSITIES
International classification
A61K36/81
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention provides a pharmaceutical and/or veterinary composition comprising a retard release formulation of a Solarium glaucophyllum preparation. The composition further preferably comprises a calcium source. The composition of the invention is suitable to prevent and/or treat hypocalcaemia. Furthermore, the composition is suitable to prevent milk fever in cows. Advantageously, the composition is administered only once before calving so as to prevent the occurrence of mil fever in cows.
Claims
1. A method for preventing parturient paresis in a gestating ruminant at risk thereof, the method comprising: administering orally to the gestating ruminant 72 to 24 hours before birth a single dose of a pharmaceutical or veterinary composition comprising a slow release formulation and an instant release formulation, each formulation comprising a source of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides that is a Solanum glaucophyllum extract obtained by maceration or percolation of dried leaves of the Solanum glaucophyllum with a solvent, the Solanum glaucophyllum extract being totally free of solanine, chaconine and solasodine, the pharmaceutical or veterinary composition is administered to the gestating ruminant only once and in an amount providing 50 ng to 1.5 μg of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides per kg bodyweight of the gestating ruminant, the composition comprises the slow release formulation and the instant release formulation in a ratio from 1:1 to 4:1 as a weight ratio of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides present in the slow release formulation relative to the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides present in the instant release formulation, the composition further comprising dolomite as the only calcium source in the composition, and the composition has a density of at least 1.2 g/ml, the dolomite is 70-85 wt. % of the slow release formulation, the slow release formulation comprises a binder that is up to 5 wt. % of the slow release formulation and comprises hydroxypropylmethylcellulose (HPMC) and optionally polyvinylpyrrolidone (PVP), the Solanum glaucophyllum extract in the slow release formulation is (i) included in a matrix containing poly(meth)acrylate-methyl/ethyl/2-trimethyl amino ethyl estercopolymerisate or (ii) has a coating comprising poly(meth)acrylate-methyl/ethyl/2-trimethyl amino ethyl estercopolymerisate, and the slow release formulation comprises an amount of the poly(meth)acrylate-methyl/ethyl/2-trimethyl amino ethyl estercopolymerisate that completely releases 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the slow release formulation over a time period that is at least 4 hours as determined in vitro by immersion of the slow release formulation into water at 37° C. and determining the time until all the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the slow release formulation are dissolved in the water, the dolomite is 0-87.5 wt. % of the instant release formulation, and the instant release formulation comprises an excipient which following contact with water and/or a digestive liquid completely releases the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the instant release formulation over a time period that is less than 4 hours as determined in vitro by immersion of the instant release formulation into water at 37° C. and determining the time until all the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the instant release formulation are dissolved in the water, the excipient in the instant release formulation comprises a binder that is up to 10 wt. % of the instant release formulation and comprises HPMC and optionally PVP.
2. The method of claim 1, wherein the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides from the S. glaucophyllum are the only active form of vitamin D3 present in the composition.
3. The method of claim 1, wherein the slow release formulation and the instant release formulation each have a density of at least 1.2 kg/l.
4. The method of claim 1, wherein the composition is in a form selected from the group consisting of a bolus, a capsule, a granular composition, a pellet, a tablet, a gel, a powder, and a compacted powder.
5. The method of claim 1, wherein the coating releases the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides from the slow release formulation by a process selected from the group consisting of (i) diffusion of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides through the coating, (ii) enzymatic decomposition of the coating in specific compartments of the digestive tract, and (iii) a combination thereof.
6. The method of claim 1, wherein the slow release formulation comprises an amount of the poly(meth)acrylate-methyl/ethyl/2-trimethyl amino ethyl estercopolymerisate such that the matrix is eroded completely over the time period that is at least 4 hours.
7. The method of claim 1, wherein the slow release formulation comprises an amount of the poly(meth)acrylate-methyl/ethyl/2-trimethyl amino ethyl estercopolymerisate such that the coating is resistant to the digestive liquid of the stomach but is decomposed when in contact with bile and/or pancreatic juice.
8. The method of claim 1, wherein the excipient in the instant release formulation further comprises at least one disintegrant selected from the group consisting of a sodium salt of carboxymethylcellulose, a sodium salt of carboxymethylated starch, crosslinked PVP, starch and mixtures thereof.
9. The method of claim 8, wherein the sodium salt of carboxymethylcellulose is 1-5 wt. % of the instant release formulation, the sodium salt of carboxymethylated starch is 2-8 wt. % of the instant release formulation, the crosslinked PVP is 0.5-5 wt. % of the instant release formulation, and the starch is 2-10 wt. % of the instant release formulation.
10. The method of claim 1, wherein the excipient in the instant release formulation comprises a filler that is up to 42.5 wt. % of the instant release formulation and selected from the group consisting of lactose, microcrystalline cellulose and a mixture thereof.
11. The method of claim 1, wherein the slow release formulation comprises a filler that is up to 15 wt. % of the slow release formulation and selected from the group consisting of lactose, microcrystalline cellulose and a mixture thereof.
12. The method of claim 1, wherein the Solanum glaucophyllum extract is 10-20 wt. % of the slow release formulation.
13. The method of claim 1, wherein the Solanum glaucophyllum extract is 12.5 wt. % of the instant release formulation.
14. The method of claim 1, wherein the single dose of the composition is administered in an amount providing 350 ng to 600 ng of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides per kg bodyweight of the gestating ruminant.
15. A method for preventing parturient paresis in a gestating ruminant at risk thereof, the method comprising: administering orally to the gestating ruminant 72 to 24 hours before birth a single dose of a pharmaceutical or veterinary composition comprising a slow release formulation and an instant release formulation, each formulation comprising a source of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides that is a Solanum glaucophyllum extract obtained by maceration or percolation of dried leaves of the Solanum glaucophyllum with a solvent, the Solanum glaucophyllum extract being totally free of solanine, chaconine and solasodine, the pharmaceutical or veterinary composition is administered to the gestating ruminant only once and in an amount providing 50 ng to 1.5 μg of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides per kg bodyweight of the gestating ruminant, the composition comprises the slow release formulation and the instant release formulation in a ratio from 1:1 to 4:1 as a weight ratio of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides present in the slow release formulation relative to the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides present in the instant release formulation, the composition further comprising dolomite as the only calcium source in the composition, and the composition has a density of at least 1.2 g/ml, the dolomite is 8.3-25.0 wt. % of the slow release formulation, the slow release formulation comprises a binder that is up to 8.3 wt. % of the slow release formulation and comprises HPMC and optionally PVP, the Solanum glaucophyllum extract in the slow release formulation is (i) included in a matrix containing polymethacrylate-methyl/butyl/2-dimethyl aminoethyl ester-copolymerisate or (ii) has a coating comprising polymethacrylate-methyl/butyl/2-dimethyl aminoethyl ester-copolymerisate, and the slow release formulation comprises an amount of the polymethacrylate-methyl/butyl/2-dimethyl aminoethyl ester-copolymerisate that completely releases 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the slow release formulation over a time period that is at least 4 hours as determined in vitro by immersion of the slow release formulation into water at 37° C. and determining the time until all the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the slow release formulation are dissolved in the water, the dolomite is 0-87.5 wt. % of the instant release formulation, and the instant release formulation comprises an excipient which following contact with water and/or a digestive liquid completely releases the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the instant release formulation over a time period that is less than 4 hours as determined in vitro by immersion of the instant release formulation into water at 37° C. and determining the time until all the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides contained in the instant release formulation are dissolved in the water, the excipient in the instant release formulation comprises a binder that is up to 10 wt. % of the instant release formulation and comprises HPMC and optionally PVP.
16. The method of claim 15, wherein the slow release formulation comprises a filler that is 50-75 wt. % of the slow release formulation and selected from the group consisting of lactose, microcrystalline cellulose and a mixture thereof.
17. The method of claim 15, wherein the Solanum glaucophyllum extract is 16.7 wt. % of the slow release formulation.
18. The method of claim 15, wherein the Solanum glaucophyllum extract is 12.5 wt. % of the instant release formulation.
19. The method of claim 15, wherein the single dose of the composition is administered in an amount providing 350 ng to 600 ng of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides per kg bodyweight of the gestating ruminant.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION
(11) The present invention relates to a composition comprising a S. glaucophyllum preparation.
(12) A “S. glaucophyllum preparation”, for the purpose of the present invention, may be any preparation obtained from the plant Solanum glaucophyllum containing glycosides of 1,25(OH).sub.2 vitamin D.sub.3. Preferably, the preparation is obtained from the leaves of S. glaucophyllum. The 1,25-(OH).sub.2 vitamin D.sub.3-glycosides are the active principle present in S. glaucophyllum. The glycosidic form of 1,25-(OH).sub.2 vitamin D.sub.3 is supposed to require break-up of the glycosidic bond in the gastro-intestinal tract, for example in the rumen of ruminants, before it can be absorbed. The S. glaucophyllum preparation may be referred to herein as the plant preparation.
(13) 1,25-dihydroxyvitamin D.sub.3, also known as calcitriol and 1,25-dihydroxycholecalciferol, is the hormonally active form of vitamin D. Herein below, any one of these designations and also the designation 1,25-(OH).sub.2 vitamin D.sub.3 are used.
(14) According to an embodiment, the S. glaucophyllum preparation is prepared from dried leaves. For example, dried leaves may be ground, comminuted or in any other way transformed into a powder. The powder may be then directly used for the preparation of the composition of the invention.
(15) According to an embodiment, said S. glaucophyllum preparation is in particular selected from the group of comminute and/or powdered dry leaves of S. glaucophyllum, an extract prepared from S. glaucophyllum leaves and from a combination of both of the aforementioned.
(16) In accordance with the above, the S. glaucophyllum preparation may be an extract prepared from S. glaucophyllum, preferably from plant material, such as the whole plant, the aerial plant parts, and/or the leaves. The plant material is preferably dried. An extract may be prepared as is disclosed, for example, in WO 2009/129818. In particular, an extract may be obtained, for example, by maceration or by percolation, for example, using a suitable solvent. As a solvent, a mixture of water and a lower alcohol (preferably ethanol) or acetone at the ratio (v:v) in the range of 80:20 to 25:75 (water: alcohol/acetone) may be used, for example.
(17) In a typical extraction process, the leaves and the solvent solution are mixed at a weight ratio of 1:5 to 1:15 (leaves:solvent), for example, and the active principle is thus extracted by stirring the mixture for about 6 to 24 hours at 25 to 55° C., for example. The process may be repeated following removal of the solvent, for example by filtration and adding new solvent.
(18) According to an embodiment, percolation is used for extracting the active principles. In this case, any suitable commercially available equipment may be used. For example, the plant material may be percolated in a percolator comprising 1 to 10, preferably 4 to 8 cyclically connected tanks, at 35-55° C. for 4 to 8 hours.
(19) One or more antioxidants may be added at 0.05 to 1 wt. %, preferably 0.05 to 0.2 wt. % of dry matter to the plant extract, for example to the liquid phase following percolation. Examples are ascorbinic acid, gallates, BHA and BHT. This may reduce the undesired browning of the liquid phase and of the extract. Browning may also be reduced, alternatively or in addition, by adjusting the pH of the liquid phase to a value of 5.5 to 6.5 using, preferably, a food grade acid, such as citric acid, acetic acid, formic acid, sulfuric acid, hydrochloric acid, phosphoric acid, for example.
(20) The solvent of the liquid phase or phases is preferably reduced and/or at least partially removed, by drying, evaporating and/or under vacuum, for example using a thin-layer evaporator to a concentration of approximately 10-50%, more preferably 30-40%, most preferably about 35% dry matter, to obtain a concentrated liquid extract.
(21) This concentrated liquid extract may be analyzed with respect to the presence of any undesired alkaloids. For example, it may be checked if any one or more of solanine, chaconine and/or solasodine can be detected in the extract. Solanine, such as a-solanine, is a glycoalkaloid poison that may be present in species of the nightshade family (Solanaceae). Alpha-chaconine is a steroidal glycoalcaloid, which has insecticidal and fungicidal properties. Solasodine is also a poisonous glycoalcaloid.
(22) If any one of the aforementioned alkaloids is detected, it may be removed by guiding the concentrated liquid extract over a suitable column, for example. For example, the liquid extract may be chromatographed using a column comprising a strongly or weakly acidic ion exchange resin, which is preferably equilibrated in the H.sup.+ form. This may be a continuous or a batch-wise process.
(23) According to an embodiment, said preparation of S. glaucophyllum is characterised in that glycosides of 1,25-(OH).sub.2 vitamin D.sub.3 are the only active form of vitamin D.sub.3 present in said preparation and/or in that alkaloids occurring sometimes in Solanum species are substantially absent, said absent alkaloids being preferably one, two or all three selected from solanine, chaconine and solasodine. “Substantially absent”, in this regard, means that said alkaloids may be present below the detection level of commercially available detection methods. Preferably, said alkaloids are totally absent in said preparation.
(24) Also the concentration or content of the active principles, 1,25-(OH).sub.2 vitamin D.sub.3-glycosides, in the extract may be determined at this stage. These concentrations may vary between plants and depending on climatic conditions and/or cultivation site and need therefore to be determined so as to control the dose.
(25) The concentrated liquid extract, or, if guided over a column, the eluate of the concentrated liquid extract is then preferably heat-treated, for example by UHT treatment, so as to remove germs.
(26) Before the final drying step, additives, fillers, in particular carbohydrates and derivatives thereof, such as, for example, maltodextrin and the like may be added in suitable amounts. In this way, the concentration of the active principle (1,25-(OH).sub.2 vitamin D.sub.3-glycosides) may be adjusted to a desired value. Additives may be added for improving or adjusting stability, processability and/or plasticity, for example.
(27) Thereafter, the concentrated liquid extract may be dried to a final water content of below 10%, preferably below 7%, even more preferably below 6%, most preferably below 5%. This may be achieved by spray- or vacuum belt drying, for example.
(28) The dried extract above may be directly used in the composition of the invention or may be further processed, for example to obtain one or more formulations exhibiting controlled release characteristics. For example, the extract can be used directly as an instant release formulation. According to another example, the dried extract (or also the liquid concentrated) may be used to prepare specific instant and/or retard release formulations.
(29) As mentioned above, the concentrations of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides in S. glaucophyllum may vary due to different factors, which as such may not all be controlled always. Therefore, it is advantageous to use a S. glaucophyllum sort or variety having generally a high content of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides, in particular in the leaves. According to a preferred embodiment, said S. glaucophyllum is selected from a wild type or cultivated form of S. glaucophyllum, preferably from the plant sort ‘HERVIT’ (according to the Community Plant Variety Certificate EU25473 of Jun. 22, 2009). Of course, other sorts and/or varieties, such as those that are derived from said ‘HERVIT’ sort, may have an ever higher content of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides and may thus be also or preferably used.
(30) According to an embodiment, the composition of the present invention is intended for oral administration.
(31) According to an embodiment, the composition comprises at least one, for example a first formulation, that comprises said S. glaucophyllum preparation, wherein said formulation release the active principle present in the preparation in a retarded or slow release manner.
(32) Retard or slow release preferably means that said formulation has completely released said active principle not before but possibly after a period lasting at least 4 hours, at least 6 hours, preferably at least 8 hours, more preferably at least 10 or at least 12, 14, 16 hours, preferably at least 18 hours, more preferably at least 20 hours, and most preferably not before the expiry of 22, preferably 24 hours following administration of the composition. In accordance with the invention, the active principle may be released constantly but in a retarded manner over time and said release is completed, with respect to the retard release formulation, not before the indicated time.
(33) In other words, the retard release formulation retains and thus prevents release of at least some of the active principle for a period of 4, 6, 8, 10, 12, 18, 20, 22, 24 hours.
(34) For the purpose of the present specification, the expressions “slow release”, “retard release”, “retarded release”, “sustained release” and the like have the same meaning. Generally, the expression “retard release” is used to refer to all of them.
(35) The release duration or the duration of release retention refers in principle to release in the gastrointestinal tract following administration. For determining the release in a controlled experimental setting, an in vitro release text using a dissolution medium may be employed as an approximation. Release of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides may be determined by measuring absorbance in the test recipient at 300 nm, online, for example, or by determining the concentration of the active principle present in the experimental solution in repeated measurements over time (both experiments are conducted in the examples). More specifically, the release time is determined using the current US Pharmacopeia (USP) dissolution test, disclosed in the USP, in particular Chapter Dissolution <711>. The dissolution medium is filtered (0.45 μm porosity filter) and deaerated water (USP deaeration procedure) and has a temperature of 37° C. at the beginning of the test (see: Dissolution Toolkit Procedures for Mechanical Calibration and Performance Verification Test Apparatus 1 and Apparatus 2 Version 2.0, Mar. 22, 2010).
(36) According to an embodiment, said retard release or said first formulation comprises said S. glaucophyllum preparation with at least one excipient, which exhibits retard release characteristics.
(37) According to an embodiment, said retard release characteristics are obtained by including said S. glaucophyllum preparation, for example said extract, in a matrix or by coating said preparation containing said extract with an excipient that releases said active principle in a retarded manner. According to an embodiment, said matrix comprises components that are not or hardly soluble in water and/or wherein said excipient of said coating releases said active principle by diffusion through said coating and/or by way of enzymatic decomposition in specific compartments of the digestive tract. According to an embodiment, release may occur due to erosion in presence of water and/or digestive liquids.
(38) There are many different ways in which the release of the active principle may be retarded in accordance with the invention and the invention is not intended to be limited to a particular retard release formulation. As mentioned, the plant preparation may be embedded in a matrix containing components and/or excipient that prevent, hinder or slow down the dissolution and/or erosion of the formulation once administered. Components that are only partially or not soluble in water may be added in the formulation. An example of an excipient that may be used for preparing the retard release formulation is Eudragit® RS or RL (copolymers of acrylic and methacrylic acid esters with a low content in quaternary ammonium groups). This copolymer may be used as a matrix component, for example in tablets, or used as a coating. Further below, examples of retard release formulations are provided.
(39) According to an embodiment, the coating of said plant preparation and/or of said retard release formulation comprises an excipient, which is resistant to the digestive liquid of the stomach, but which is decomposed when in contact with bile and/or with pancreatic juice. For example, fats and other fatty acid esters may be used in the coating to this end.
(40) According to an embodiment, the composition comprises at least one S. glaucophyllum preparation in the form of an instant release formulation. The expression “instant release formulation” refers to any formulation which does not specifically retard release and to formulations which contain ingredients that speed up release of the active principle contained in the formulation. For the purpose of the present invention, an instant release formulation encompasses normal- and fast release formulations. In an instant release formulation in accordance with the present invention, the active principle is released from the formulation within a time period of 8 hours, preferably within 6 hours, more preferably within 5, 4, 3, 2 hours or even within 1 hour, for example following administration. Also in case of the instant release formulation, the measurement of the complete release duration may be determined by an in vitro experiment as disclosed elsewhere in this specification.
(41) The soluble, dry extract described above is soluble readily in water and may thus be directly used as an instant release formulation for the purpose of the present invention. The instant release may also comprise excipients that increase or accelerate release, such as disintegrants, which may cause rapid swelling and disintegration of the formulation, for example a tablet or a pill, comprising it. Examples of such disintegrants are sodium salts of carboxymethylcellulose and carboxymethylated starch, crosslinked polyvinylpyrrolidone (PVP), or simply starch.
(42) The instant release formulation, and/or the retard release formulation may, independently, further comprise binding materials and fillers as is usual in the art.
(43) Of course, further additives may be added in order to facilitate the production of the particular retard and/or instant release formulation such as the tablet and/or the coating, such as diluents, emollients, plasticizers, detackifiers, anticaking agents, wetting agents and the like.
(44) According to a preferred embodiment, the composition of the invention comprises a first formulation comprising a S. glaucophyllum preparation and a second formulation comprising a S. glaucophyllum preparation, wherein said first formulation is characterized in that it releases at least one active principle present in said preparation more slowly upon administration than said second formulation. Accordingly, said first formulation may be a retard release formulation and said second formulation may be an instant release formulation as discussed elsewhere in this specification.
(45) According to an embodiment, said second formulation comprises said S. glaucophyllum preparation and one or more excipient, which rapidly releases said active principle. For example, rapid release may occur following contact with water and/or digestive liquids, for example in the first stomach or rumen of ruminants or in the stomach of monogastric animals.
(46) According to a preferred embodiment, the composition of the invention comprises a calcium source. Any suitable calcium source comprising preferably a salt of calcium may be used. Calcium sources that have a high bioavailability are preferred. According to a preferred embodiment, the calcium source is dolomite. More preferably, the calcium source is heat-activated dolomite. This form of calcium has a surprisingly high bioavailability with a high or even major part of the ingested calcium being absorbed in the gastrointestinal tract. Dolomite and heat-activated dolomite are commercially available.
(47) Dolomite is particularly advantageous because it provides readily absorbable calcium, and, in addition, magnesium, which is beneficial for the process of muscle contraction during labour at calving.
(48) Furthermore, dolomite is suitable to increase the density of the composition of the invention. According to an embodiment, the composition of the invention, and/or the first formulation and/or the retard release formulation has a density of ≧1.1, preferably ≧1.15 or ≧1.2, or more preferably ≧1.25, even more preferably ≧1.3. The high density is particularly relevant for the retard release formulation, as detailed below.
(49) Accordingly, dolomite may be added to or part of the first formulation, to the second formulation or to both. Alternatively, dolomite may be added to the composition separately.
(50) The advantage of having a density ≧1 applies in particular to ruminants, which may otherwise rehash and ruminate and thereby also physically destroy the retard release formulation by chewing, so that the retard release characteristics are lost. However, if the density is adjusted, at least of the retard release formulation, to the indicated values, the respective formulation will not be rehashed but remains in the rumen or gastrointestinal tract and the active principle is released in a retarded manner as desired.
(51) Dolomite is thus a particularly advantageous additive in that it adds bioavailable calcium, it assist birth labor and increases the density of the formulation containing it.
(52) The composition may be provided in any suitable ingestible form. According to an embodiment, the composition of the invention is provided in the form of any one or more of the group selected from: a bolus, a capsule, a granular composition, a pellet, a tablet, a gel and a powder, for example a compacted powder.
(53) For example, the composition of the invention may be added in the form of a powder to the normal food and taken up with this food.
(54) According to an embodiment, the S. glaucophyllum preparation and/or the instant and/or retard release formulations are provided in the form of a gel.
(55) According to an embodiment, the formulations and/or preparations are homogenously pressed or are compartmented in different layers or are cylindrically arranged. In this latter case, the retard release formulation is preferably provided in the centre of the cylinder, for example in the form of an inner cylinder, the instant release formulation being provided as an outer cylinder encasing the retard release formulation, for example.
(56) If the composition is to be applied to an animal, for example a cow, it may be provided in the form of a bolus. A bolus may be administered using a commercially available bolus applicator. Boluses are routinely used for administrating nutrient supplements and medicaments to cattle.
(57) According to an embodiment, the composition is provided in the form of a layered bolus, pellet or tablet comprising at least two layers, one layer being or comprising the instant release formulation and the other layer being or comprising the retard release formulation.
(58) According to another embodiment, the formulations are provided in the form of tablets. For example, the instant release formulation may be provided in the form of tablets or capsules that are different from the tablets of the retard release formulation. Any one of the two formulations may comprise the calcium source, in particular dolomite, or the calcium source may be provided also in the form of separate tablets. All tablets may, for example, be filled in a bolus capsule, optionally filled with a suitable filler or binder, and administered in this form to the animal.
(59) Tables 1-3 below show exemplary instant release formulations (Table 1) and sustained release formulations (Tables 2 and 3), which are provided in the form of separate tablets containing a dried S. glaucophyllum extract as disclosed in the examples and elsewhere in this specification. Exemplary product names of ingredients that may be used for producing the tablets are also indicated. In some lines of the tables, for example with respect to the disintegrants, different products that may be used in the alternative or in combination are disclosed.
(60) TABLE-US-00001 TABLE 1 Exemplary formulations of instant-release tablets (IR-tablets) Density: 1.2-2 g/ml; diameter: for example about 8 mm weight per Ingredients Tablet function 1. dry extract of 50 mg S. glaucophyllum 2. dolomite CaMg[CO.sub.3].sub.2 q.s. density regulation ca. 0-350 mg Mg and Ca source 3. sodium salt of 8-32 mg disintegrants carboxymethylated starch (Explotab ®, Vivastar ®, Primojel ®); 2-8% crosslinked 2-20 mg polyvinylpyrrolidone (Kollidon CO); 0.5-5% sodium salt of 4-20 mg earboxymethylcellulose (Ac-Di-Sol ®, Vivasol ®); 1-5% starch (Starch 1500); 8-40 mg 2-10% 4. PVP; HPMC 0-40 mg binders (hydroxypropylmethyl- cellulose) 0-10% 5. lactose/ 0-170 mg (q.s.) fillers microcrystalline cellulose Total ca. 400 mg q.s.: as deemed useful
(61) TABLE-US-00002 TABLE 2 Exemplary formulations of retard release tablets (SR- tablets), the matrix only (coating described further below). Exemplary concepts 1 and 3: tablets may have, for example 8 mm diameter and a density of >2 g/ml weight per Ingredients Tablet function 1. dry extract of 40-80 mg S. glaucophyllum 2. dolomite CaMg[CO.sub.3].sub.2 q.s. density regulation ca. 280-340 mg Mg and Ca source 3. PVP; HPMC 0-20 mg binder (hydroxypropylmenthyl- cellulose) 4. lactose/ 0-60 mg (q.s.) filler microcrystalline cellulose Total ca. 400 mg
(62) TABLE-US-00003 TABLE 3 Exemplary formulations of retard release tablets (SR- tablets), the matrix only (coating described further below). Exemplary concept 2: tablets may have, for example 3 mm diameter and a density of about ≧1.2//m1 Masse pro Ingredients Tablette Funktion 1. dry extract of 2 mg S. glaucophyllum 2. dolomite CaMg[CO.sub.3].sub.2 q.s. density regulation, ca. 1-3 mg Mg and Ca source 3. PVP; HPMC 0-1 mg binder (hydroxypropylmethyl- cellulose) 4. lactose/ 6-9 mg (q.s.) filler microcrystalline cellulose Total ca. 12 mg
(63) The tablets described in Tables 2 and 3 are preferably coated so as to retard the release of the 1,25-(OH).sub.2 vitamin D.sub.3-glycosides.
(64) In accordance with concepts 1 and 3 (Table 2), the coating may comprise or be composed of, for example poly(meth)acrylate-methyl/ethyl/2-timethyl amino ethyl ester-copolymerisate (Eudragit® RL, Eudragit® RS) with HPMC (hydroxypropylmethylcellulose), lactose or polyvinyl lacetate for providing pores for the adjustment of the release speed in concepts 1 and 2; as well as detackifiers (for example talc), wetting agent (for example Tween® 80) and plasticizers (examples: diethyl phthalate, triethyl citrate).
(65) In accordance with the concept 2 above (Table 3), the formulation may be processed to tablets or capsules. f processed to tablets, they may be coated, for example, with polymethacrylate-methyl/butyl12-dimethyl aminoethyl ester-copolymerisate (Eudragit® E) with suitable wetting agents (for example sodium lauryl sulfate), pasticizers (stearic acid) and anti-caking agents (for example talc).
(66) If processed to capsules, the coating may comprise and/or be composed of ethyl cellulose (Aquacoat®, Surelease®) with plasticizer (examples: diethyl phthalate, triethyl citrate), wetting agents (for example Tween® 80) and anti-caking agents (for example talc), if necessary.
(67) As mentioned below, the composition of the invention may comprise a retard release formulation and an instant release formulation. referably, the two formulations are contained in the composition at a weight ratio of 1:9 to 9:1, 2:8 to 8:2, 3:7 to 7:3, 4:6 to 6:4 and preferably about 1:1, expressed as weight ratio of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides present in the instant-release formulation with respect to the retard release formulation.
(68) With the goal of keeping the administered dose of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides constant, the following examples using the tablets/capsules of concepts 1 to 3 above are given, in which the total dose is held at 10 g per dried plant extract.
(69) Concept 1: IR: 4 g; SR: 6 g; =>80 IR-tablets and 100 SR-tablets.
(70) Concept 3: IR: 4 g; SR: 6 g; _>80 IR-tablets and 100 SR-tablets.
(71) Concept 2: IR: 6 g; SR: 4 g; =>120 IR-tablets and 2000 SR-tablets.
(72) The above examples illustrate how the skilled person can control the amount of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides that is administered in a retard release and/or in an instant release formulation. It is noted, that the concentration of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides in the dried plant extract is controlled and/or adjusted before the final drying step as disclosed elsewhere in this specification.
(73) The composition and/or formulations disclosed in this specification may be used as medicaments and in methods of prevention and/or treatment of conditions, such as diseases.
(74) The compositions may in particular be used for preventing suboptimal blood calcium levels, for increasing blood calcium levels, for avoiding and/or preventing calcium deficiency, for maintaining healthy, average and/or normal blood calcium levels, in particular in cattle (in particular in cows, goat, and sheep) and also in humans.
(75) The expression “serum calcium levels” and the like preferably refers to total calcium content in blood, including free dissolved as well as protein bound calcium, as may be determined, for example, following collection of total calcium from a blood sample, by flame photometry.
(76) Accordingly, the composition of the present invention may be used to treat and/or prevent hypocalcaemia, in particular in cattle, in particular cows, goat, and sheep. The composition may also be used to treat and/or prevent hypocalcaemia in humans. Hypocalcaemia may have many causes and origins, including, for example, eating disorders, hereditary diseases, vitamin D deficiency, decreased sun exposure, just to mention a few arbitrarily selected causes.
(77) In cattle, hypocalcaemia may concur with calving or may occur shortly before and/or after birth, due to the onset of the mammary milk production. In cows, milk fever may occur, which in severe cases, may lead to death of the cow. Milk fever causes important damages.
(78) Accordingly, the composition and formulations disclosed in this specification may be used to treat and/or prevent milk fever, in particular in milk producing and/or older cows, preferably in the period around and/or after calving.
(79) The presence of a retard release formulation and/or a combination of a retard release and an instant release formulation in the composition is particularly advantageous for the prevention and/or treatment of milk fever. Thanks to the composition of the invention, the composition may be administered only once, in one single treatment, preferably shortly before giving birth. As discussed above, a single administration of calcium or synthetic 1,25-(OH).sub.2 vitamin D.sub.3 could be counterproductive if the moment of administration is not optimal, for example too long time before calving. Thanks to the composition of the invention, the time window within which the composition can be administered becomes larger, making a successful treatment more likely. This applies also and in particular if the composition is only administered once. The longer time window before birth is important, because the exact time of calving can, of course, not always be accurately predicted. The exact length of the gestation period is, of course, subject to a certain variation.
(80) It has been observed that the composition of the invention can be effective if administered for up to 3 days ante partum, preferably up to 4, 5, 6, 7, 8, 9, 10 days ante partum (before birth) or at the first signs of imminent calving. If administered in suitable amounts, a single administration of the composition of the invention may increase blood calcium levels for up to 3-10 days.
(81) According to an embodiment of the method and/or of the composition of the invention, the composition is administered as a single administration dose to a gestating mammal before giving birth, preferably in the period of up to 9 days before birth, 8 days to 0.5 hours, 7 days to 1 hour before giving birth, preferably 96 to 12 hours before birth, most preferably 72 to 24 hours birth or at the first signs of imminent birth. “Before birth” or “before calving” for the purpose of the present specification, refers to the period ending with the onset of the birth process, preferably to the period lasting up to the first contraction/labor pains.
(82) In a gestating cow, the composition is preferably administered as a single dose 2 to 4 days before calving. The indicated time intervals are preferably determined on the basis of an estimation of the average gestation period. If, following administration of the composition 5 or more days lapse without calving taking place, a further application dose (typically a bolus) is preferably administered.
(83) The moment of administration of the single administration dose before birth/calving may be determined by calculation if the day of insemination is known. The gestation period of cows is generally 279-292 days and may be more precisely known for a specific cow breed. Accordingly, the composition of the invention may be administered as a single administration dose 277 to 290 days after insemination, for example 280 days after insemination.
(84) In the prophylactic methods and the methods of treatment of the invention, the composition of the invention is preferably administered to a subject in need thereof. The subject may be a human or one of the above-specified animals. If the method concerns in particular the prevention and/or treatment of milk fever, the subject is selected from cattle, in particular from a cow, sheep or goat, preferably from a gestating animal.
(85) The amount of 1,25-(OH).sub.2 vitamin D.sub.3 to be administered may be determined by the skilled person in dependence of the circumstances and/or the subject in question. As a guideline, 50 ng to 1.5 μg, preferably 100 ng to 1.3 μg, 200 ng to 1.0 μg, more preferably 300 to 800 ng, most preferably 400 to 700 ng, for example 350 to 600 ng 1,25-(OH).sub.2 vitamin D.sub.3-glycosides may be administered per kg bodyweight of the subject. These amounts may apply for cows, for example.
(86) The present invention will now be illustrated by way of examples. These examples do not limit the scope of this invention, which is defined by the appended claims.
EXAMPLES
Examples 1 and 2: Preparation of Extracts from Solanum glaucophyllum
Example 1: Extract Obtained by Maceration
(87) 300 kg dry leaves S. glaucophyllum were grinded to 10-15 mm particle size. The power was added to a solvent mixture and ethanol in a ratio of 7:3 (water:alcohol, v:v) and at a weight ratio of 1:10 (powdered dried leaves:solvent) and stirred for 10 hours at 40° C. for extraction.
(88) The liquid phase is separated by filtration and the process is repeated up to twice using the same leaves.
(89) At the end, 0.5 wt-% of ascorbic acid is added as an antioxidant and pH is adjusted to 6 using acetic acid. The entire liquid phase is evaporated in a thin-layer evaporator to a dry-matter content of approx. 35 wt. %.
(90) The concentrated extract is analyzed and the concentration of 1,25-(OH).sub.2 vitamin D.sub.3-glycosides is determined. The eluate is heat-treated (UHT) for removing germs. Maltodextrin (Ph. Eur. quality) is added to adjust the content of the active principle (total 1,25-(OH)2 vitamin D3) to a content of 50 gg/g (dry matter). The solution is spray-dried to dryness.
Example 2: Extract Obtained by Percolation
(91) The same mixture of dried leaves and solvent mixture as used in Example 1 is percolated in a commercially available percolator for 8 hours at 35-45° C.
(92) The liquid phase (raw extract) is further treated as described in Example I to yield the same final product as in Example 1.
Examples 3 and 4: Instant and Retard Release Formulations Containing the Extract
Example 3: Instant Release Tablet Containing S. glaucophyllum Extract
(93) The ingredients listed in Table 4 below were mixed and pressed to an instant release tablet.
(94) TABLE-US-00004 TABLE 4 Composition of instant release tablet Ingredients (mg/tablet) Dried extract of Example 1 270 Avicel 102 (mikrokristalline Cellulose) 50 Kollidon CL 100 Mg-Stearat veg. 4 Aerosil 200 7 Total weight 431
Example 4: Retard Release Tablet Containing S. glaucophyllum Extract
(95) The ingredients listed in Table 5 below were mixed and pressed to a retard release tablet. The characteristics of the tablet are listed further below in Table 5.
(96) TABLE-US-00005 TABLE 5 Composition of retard release tablet Ingredients (mg/tablet) Dried extract of Example 1 270 HPMC (100M) 400 Eudragit RS 130 Mg-Stearat veg. 8 S02, kolloidal 8 Total weight (target) 830 Average actual weight (mg) 831.2 hardness/tablet (N) ca. 170 dimension (mm) 18 × 8.5 × 6.9 LOD (loss on drying tablet mixture) (%) 2.89 Charge size 435 tablets Yield (acceptable tablets) 290 tablets Yield (%) 66.7
(97) The polymer Eudragit has the property of soaking in contact with the rumen liquid and thereby releasing the soluble extract of Example 1 slowly.
(98) Each tablet contains 50 ng analytically determined 1.25(OH).sub.2 vitamin D.sub.3 per 10 mg tablet.
(99) In other words, a tablet of, for example, 430 mg contains 21.5 μg 1,25-(OH).sub.2 vitamin D.sub.3.
(100) The retard release characteristics were evaluated by a standard USP dissolution test (USP basket, 900 ml water, 300 nm) (for further details, see indications in this specification further above). Dissolution was measured by monitoring the time-dependent increase in absorption at 300 nm in a spectrophotometer. The increase in absorption is considered as the release of the extract into solution. The results are shown in
(101) Furthermore, the concentration of the released active principle (1,25-(OH)2 vitamin D.sub.3-glycosides) was measured at different times following addition of the tablet to the dissolution medium. The result is shown in
Example 5: Blood Levels of Active 1,25-(OH)2 Vitamin D Following Single Administration of Two Different Doses of the Plant Extract
(102) Twenty cows were allocated in two groups of 10 animals. Each cow received a single administration of a capsule containing 10 mg (group 1) or 20 mg (group 2) S. glaucophyllum extract of Example 1 per kg weight of the animal. Blood samples were collected before and at different times following administration and 1,25-(OH).sub.2 vitamin D.sub.3 concentration was determined using an ELISA test kit (Immundiagnistik Bensheim, Germany). Groups of 3 and 4 animals of each treatment were pooled and the results are shown in
Example 6: In Vivo Experiment Using the Composition of the Invention
(103) Twenty-five cows of the races brown, Fleckvieh and Holstein of a weight of approximately 500-600 kg weight and including lactating and non-lactating subjects were allocated to 5 treatment groups as detailed in Table 6 blow. The animals were given a single dose of the test preparation orally per bolus. Blood was taken at time 0 (before application) and at days 1, 2, 3, 4, 5, 7, 9 and 11 following application. From the blood, serum was prepared and calcium (total), phosphate and 1,25-(OH).sub.2 vitamin D.sub.3 was analyzed according to established clinico-chemical methods.
(104) Treatment: all test preparations were applied to the animal as one single bolus in a gelatine capsule (length 7 cm, diameter 2.2 cm and volume 24 mL, Capsula GmbH, Ratingen, Germany) with the aid of a usual bolus applicator. The volume that was not filled with tablets was filled up with pectin as inert excipient. The control group only received pectin.
(105) The boluses contained, in accordance with the treatment group, instant release tablets as described in Example 3 (groups 2 and 4) and retard release tablets as prepared in Example 4 (groups 1 and 4).
(106) TABLE-US-00006 TABLE 6 Treatment groups Group Preparation Dose 1 Retard release form of the 10 mg/kg body weight, Solanum glaucophyllum (corresponds to 500 ng extract 1,25-(OH).sub.2 vitamin D3) 2 Instant release form of the 10 mg/kg body weight Solanum glaucophyllum (corresponds to 500 ng extract. 1,25-(OH).sub.2 vitamin D.sub.3) 3 Synthetic 1,25-(OH).sub.2 vitamin 500 ng 1,25-(OH).sub.2 D.sub.3 (Cerbios Pharma SA vitamin D.sub.3) Barbengo, Switzerland) 4 Combination of retard- and 5 + 5 mg/kg body weight instant release Forms (corresponds to 500 ng 1,25-(OH).sub.2 vitamin D.sub.3) 5 Control Pectin as inert excipient
(107) The results of these experiments are presented in Tables 7-9 below showing the serum calcium, phosphate and 1,25-(OH).sub.2 vitamin D.sub.3 concentrations determined in each group and the outcome of the statistical comparison of the treatments.
(108) TABLE-US-00007 TABLE 7 Serum calcium in mmol/L (mean t standard deviation) at days 0, 1, 2, 3, 4, 5, 7, 9, 11 in treatment grouns (n = 5). Serum calcium (nmol/1) day Group 1 Group 2 Group 3 Group 4 Group 5 0 2.47 ± 0.06 .sup. 2.50 ± 0.06 .sup. 2.40 ± 0.2 .sup. 2.46 ± 0.11 .sup. 2.40 ± 0.06 1 2.75 ± 0.06 cE 2.67 ± 0.03 E 2.64 ± 0.05 E 2.68 ± 0.12 e.sup. 2.44 ± 0.09 2 .sup. 2.89 ± 0.07 bCE 2.74 ± 0.07 E 2.67 ± 0.09 Ed 2.81 ± 0.12 E 2.39 ± 0.02 3 2.82 ± 0.06 cE 2.75 ± 0.11 E 2.72 ± 0.09 E 2.81 ± 0.08 E 2.42 ± 0.03 4 .sup. 2.86 ± 0.03 BCE 2.72 ± 0.06 E 2.72 ± 0.06 E 2.78 ± 0.10 E 2.45 ± 0.05 5 2.81 ± 0.06 CE 2.64 ± 0.16 e.sup. 2.61 ± 0.02 E 2.76 ± 0.11 E 2.46 ± 0.03 7 .sup. 2.77 ± 0.09 bCE 2.60 ± 0.11 .sup. 2.57 ± 0.06 e.sup. 2.71 ± 0.16 e.sup. 2.44 ± 0.06 9 2.64 ± 0.12 E 2.59 ± 0.07 E 2.56 ± 0.09 .sup. 2.57 ± 0.11 .sup. 2.46 ± 0.02 11 2.48 ± 0.12 .sup. 2.44 ± 0.06 .sup. 2.45 ± 0.02 .sup. 2.45 ± 0.05 .sup. 2.40 ± 0.05 .sup.a(p < 0.05) .sup.A(p < 0.01) vs G1 (SR form alone) b (p < 0.05) B(p < 0.01) vs G2 (IR form alone) c (p < 0.05) C(p < 0.01) vs G3 (1,25(OH).sub.2D.sub.3) d (p < 0.05) .sup.D(p < 0.01) vs G4 (combination) e (p < 0.05) E (p < 0.01) vs G5 (control)
(109) TABLE-US-00008 TABLE 8 Serum phosphate in mmol/L (mean t standard deviation) at days 0, 1, 2, 3, 4, 5, 7, 9, 11 in treatment groups (n = 5). Serum phophorus (m/mol/1) day Group 1 Group 2 Group 3 Group 4 Group 5 0 2.33 ± 0.11 2.44 ± 0.06 ce 2.30 ± 0.07 2.35 ± 0.38 .sup. 2.24 ± 0.17 1 2.98 ± 0.18 CE .sup. 2.84 ± 0.18 CE .sup. 2.50 ± 0.12 ed 2.85 ± 0.24 E 2.07 ± 0.30 2 3.30 ± 0.10 CE 3.25 ± 0.21 E .sup. 2.81 ± 0.25 ED 3.41 ± 0.15 E 2.12 ± 0.21 3 3.51 ± 0.17 cE 3.41 ± 0.23 cE .sup. 3.05 ± 0.19 Ed 3.38 ± 0.21 E 2.22 ± 0.18 4 3.47 ± 0.29 E .sup. 3.45 ± 0.53 E .sup. 3.24 ± 0.34 E 3.43 ± 0.12 E 2.38 ± 0.22 5 2.94 ± 0.10 Ed.sup. 2.71 ± 0.41 ed .sup. 2.60 ± 0.32 E 3.17 ± 0.12 E 2.05 ± 0.15 7 2.98 ± 0.17 CE 2.66 ± 0.35 E .sup. 2.43 ± 0.23 e 2.89 ± 0.19 E 2.06 ± 0.10 9 2.63 ± 0.25 bcE 2.10 ± 0.16 d.sup. 2.05 ± 0.44 2.43 ± 0.24 e.sup. 1.97 ± 0.08 11 2.47 ± 0.23 E .sup. 2.26 ± 0.34 .sup. 2.10 ± 0.16 2.43 ± 0.27 .sup. 2.10 ± 0.06 .sup.a(p < 0.05) .sup.A(p < 0.01) vs G1 (SR form alone) b (p < 0.05) .sup.B(p < 0.01) vs G2 (IR form alone) c (p < 0.05) C (p < 0.01) vs G3 (1,25(OH)2D3) d (p < 0.05) D (p < 0.01) vs G4 (combination) e (p < 0.05) E (p < 0.01) vs G5 (control)
(110) TABLE-US-00009 TABLE 9 Serum 1,25-(OH).sub.2 vitamin D.sub.3 in pg/mL (mean of pooled serum samples) at days 0, 1, 2, 3, 4, 5, 7, 9, 11 in treatment groups n = 5. Serum 1,25-dihydroxyvitamin D.sub.3 day Group 1 Group 2 Group 3 Group 4 Group 5 0 58 30 20 47 30 1 364 291 243 243 10 2 216 156 150 148 30 3 96 58 56 110 36 4 67 28 40 37 8 5 35 53 46 16 28 7 36 34 30 20 19 9 41 22 23 4 20 11 40 16 25 1 21
(111) All treatments showed an increase in serum calcium and phosphate within 24 hours and lasted until 9 days. All groups that received the S. glaucophyllum extracts showed a higher concentrations of 1,25-(OH).sub.2 vitamin D.sub.3, calcium and phosphorous in serum than the group with synthetic 1,25-(OH).sub.2 vitamin D.sub.3. In addition, the effect of the retard release form and the combination was stronger than the effect of the instant release form of the extract. The outcome is also presented in
Example 7: In Vivo Experiment in Gestating Cows, Application Before Birth
(112) In this experiment, a single dose of the inventive composition (Slbv) was compared with the state-of-the-art treatment for milk fever prevention (Bc). All animals in this experiment were randomly selected from 5 different farms. All animals were German Fleckvieh, aged between 2 and 8 years and had between 1 and 7 calvings and were randomly attributed to treatment A (Bc) and treatment B (Slbv). A part from the usual reduction of the calcium content in the diet before birth, no prophylaxis for milk fever was done.
(113) The animals were all gestating cows and the treatments were administered when calving was expected to occur within 72 hours (sampling 1). The days until effective calving were determined and finally varied from 1 to 12 days between the cows.
(114) Treatment: Group A (Bc)
(115) Application of 4 boli of a calcium salt represented as Bovikalc® (Boehringer Ingelheim, Germany, 43 g calcium per bolus). According to producers recommendations: a first Bolus 24 hours a.p., a second bolus around partum, a third bolus 12 to 15 hours after calving and a fourth bolus 24 to 30 hours after calving.
(116) Treatment: Group B (Slbv)
(117) Application of a single dose of a preparation containing:
(118) 7 g extract of S. glaucophyllum from instant release tablets (Example 3; Table 4);
(119) 14 g extract of S. glaucophyllum retard release tablets (Example 4; Table 5);
(120) 16 g activated dolomite;
(121) altogether packed in 2 gelatin capsules (cf.
(122) In all animals, serum calcium, phosphate and 1,25-(OH).sub.2 vitamin D.sub.3 was determined from samples taken before birth and at intervals of 2 days after birth for 7 days (samplings 2-5). Analysis was done using typical clinico-chemical methods.
(123) Results: Group A
(124) All 25 animals in this treatment fulfilled the criteria of correct dosing (the administration time ante partum). One animal showed signs of milk fever.
(125) Results: Group B
(126) Of the 31 animals in this treatment, 26 animals fulfilled the criteria of receiving treatment within 72 to 24 hours a.p. None of these animals showed signs of milk fever. One animal showed signs of milk fever and one animal died because of other reasons not related to the present study.
(127) The results of the serum calcium, phosphate and 1,25-(OH).sub.2 vitamin D.sub.3 is seen in
(128) In conclusion, a single administration of the composition of the invention yields as good or better results as the administration of four doses of a commercially available calcium preparation intended for the prevention of milk fever in cows.