CALCIUM COMPOSITION

Abstract

The present invention pertains to a composition comprising a calcium source, a TRP agonist, water and optionally an excipient, wherein the amount of the calcium source is at least 5 wt %, based on the total weight of the composition.

Claims

1. Composition comprising a calcium source, a TRP agonist, water and optionally an excipient, wherein the amount of the calcium source is at least 5 wt %, based on the total weight of the composition.

2. Composition according to claim 1 comprising 5 to 85 wt % calcium source and 0.01 to 10 wt % TRP agonist, 0.1 to 90 wt % water and 0 to 50 wt % excipient, based on the total weight of the composition.

3. Composition according to claim 1, wherein the excipient is a binder.

4. Composition according to claim 1, wherein the TRP agonist is dissolved in a feed-suitable solvent.

5. Pre-formed solid preparation comprising the composition according to claim 1.

6. Pre-formed solid preparation according to claim 5, wherein the pre-formed solid preparation is coated with a water-soluble coating composition.

7. Use of the composition according to claim 1 to treat hypocalcaemia in ruminants, preferably cows.

8. Use of the pre-formed solid preparation according to claim 5 to treat hypocalcaemia in ruminants, preferably cows.

Description

EXAMPLES

Examples 1 and 2 and Comparative Example A

[0027] Measurement of Ca-Flux Across Ruminal Epithelium in the Ussing-Chamber

[0028] Ruminal epithelium of sheep was mounted in a conventional Ussing chamber. Chambers contained 16 ml of serosal or mucosal buffer, which was circulated and gassed by a gas lift system (5% CO.sub.2/95% O.sub.2).

[0029] Groups

[0030] In this experiment 24 chambers were divided into three groups. Group 1 (mucosal buffer containing 1.8 mM CaCl.sub.2), group 2 (mucosal buffer containing 3.6 mM CaCl.sub.2) and group 3 (containing 7.2 mM CaCl.sub.2). The same buffer was present on the serosal side of all chambers (Table 1). Each group (1,2,3) was again divided into two parts. One was defined as control group one as EO group. It is noted that the amounts correspond to a normal Ca.sup.2+ concentration in the rumen (1.8 mM) (referred to as Comparative Example A) and to treated rumen corresponding to the administration of about 13 g Ca (3.6 mM) and 26 g Ca (7.2 mM) (referred to as Examples 1 and 2, respectively).

[0031] Procedure

[0032] After an equilibrating time of around 30 minutes .sup.45Ca.sup.2+ (40 kBq per 16 ml chamber) was added to either to the mucosal or serosal “hot” side.

[0033] Depending on the group 16 μl ethanol (Control) or 16 μl essential oil (EO) were added to the mucosal side of the chamber. The essential oil used is menthol, and the actual menthol concentration obtained is 50 μM.

[0034] After 70 minutes the first sample from the “cold” side was taken, followed by five samples of each 900 μl which were taken in an interval of 30 minutes. The sample volume was replaced by an aliquot of the respective buffer. Radioactivity was measured using a β-counter (LKB Wallace-Perkin-Elmer, Überlingen, Germany).

[0035] Epithelia were paired after sorting for transepithelial conductance (Gt) and the net calcium flux was calculated from the difference between mucosal to serosal flux and serosal to mucosal flux for paired epithelia.

TABLE-US-00001 TABLE 1 Chemical composition of the mucosal and serosal buffer solutions used in the experiment. C.E.A Ex.1 Ex. 2 mucosal mucosal mucosal Serosal Chemical mM mM mM mM CaCl.sub.2 × 2H.sub.20 1.8 3.6 7.2 1.8 MgCl.sub.2 1.0 1.0 1.0 1.0 MES free acid 10.0 10.0 10.0 0 Hepes free acid 0 0 0 10.0 NaCl 70.0 70.0 70.0 70.0 HCl 17.0 17.0 17.0 0 Na-acetate 25.0 25.0 25.0 0.0 Na-propionate 10.0 10.0 10.0 0.0 Na-butyrate 5.0 5.0 5.0 0.0 Na-gluconate 0.0 0.0 0.0 40.0 NMDG free base 2.0 2.0 2.0 5.0 KH.sub.2PO.sub.4 0.4 0.4 0.4 0.4 K.sub.2HPO.sub.4 2.4 2.4 2.4 2.4 NaHCO.sub.3 25.0 25.0 25.0 25.0 Enrofloxacine 0.0278 0.0278 0.0278 0.0278 Mannitol 19.0 19.0 19.0 10.0 Glucose 0.0 0.0 0.0 10.0 pH 6.1 6.1 6.1 7.4

[0036] The net transport of calcium across the isolated ruminal epithelium was calculated by subtracting the calcium secretion (serosal to mucosal transport) from the calcium absorption (mucosal to serosal transport) reflecting the real absorption of calcium from the rumen to the blood side. For statistical analysis linear mixed effect models for repeated measures were calculated using mucosal calcium concentration and the addition of essential oils as fixed effects and animal as random effect (SAS System, method MIXED PROC). The effects of calcium concentration and the addition of essential oils was calculated as well as their interaction. Furthermore, the effect of sampling time was included to the model. Effects were evaluated for the repeated measures of the consecutive flux periods.

[0037] Results

[0038] The addition of EO increases Calcium net flux as it was already shown in former studies. Surprisingly it is also possible to increase calcium net flux even under high calcium conditions.

TABLE-US-00002 TABLE 2 Shown are the mean and SEM of measured Ca flux across ruminal epithelium depending on mucosal calcium concentration and the presence or absence of EO. Control EO n = 13 Mean Mean Difference Example Ca (nmol/cm.sup.2/h) SEM (nmol/cm.sup.2/h) SEM (nmol/cm.sup.2/h) P-value A 1.8 22.18 2.22 28.24 2.28 +6.06 <0.05 1 3.6 43.25 2.17 45.77 2.26 +2.52 n.s. 2 7.2 67.00 2.13 75.22 2.39 +8.22 <0.05

[0039] The net calcium absorption significantly increased with increasing mucosal calcium concentrations. Furthermore, the addition of EO increased calcium absorption by 6.06, 2.52 and 8.22 nmol/cm.sup.2/h for the increasing mucosal calcium concentration. There was no significant interaction between the factors calcium concentration and EO addition.

[0040] The calcium flux is increased at all three Ca concentrations when menthol is present compared to the control. The relative flux (i.e. the percentage measured against the control flux) is above 100% for all menthol-containing preparations, with a minimum at a Ca concentration of 3.6 mM.

Examples 3 and 4 and Comparative Example B

[0041] Effect of EO on Cellular Barrier Integrity of Ruminal Epithelium in Ussing-Chamber Depending on Mucosal Calcium Concentration

[0042] Ruminal epithelium of sheep was mounted in a conventional Ussing chamber. Chambers contained 16 ml of serosal or mucosal buffer, which was circulated and gassed by a gas lift system (5% CO.sub.2/95% O.sub.2).

[0043] Groups

[0044] In this experiment 24 chambers were divided into three groups. Group 1 (mucosal buffer containing 1.8 mM CaCl.sub.2), group 2 (mucosal buffer containing 3.6 mM CaCl.sub.2) and group 3 (containing 7.2 mM CaCl.sub.2). The same buffer was present on the serosal side of all chambers (Table 1). Each group (1,2,3) was again divided into two parts. One was defined as control group one as EO group. It is noted that the amounts correspond to a normal Ca.sup.2+ concentration in the rumen (1.8 mM) (referred to as Comparative Example B) and to treated rumen corresponding to the administration of about 13 g Ca (3.6 mM) and 26 g Ca (7.2 mM) (referred to as Examples 3 and 4, respectively).

[0045] Procedure

[0046] Fluorescein is a well-known marker for paracellular permeability. To measure net fluorescein flux rates (J.sub.net-fluor=J.sub.ms-fluor−J.sub.sm-fluor), 16 μl sodium fluorescein was added to a final concentration of 0.1 mM to the buffer solution on the “hot” side of each tissue after the equilibration period of 30 min. Epithelia were paired after sorting for transepithelial conductance (Gt) and the fluorescein flux was measured from the mucosal to serosal side. To determine the specific fluorescence intensity, 50-μL samples were taken from the “hot” side of each tissue immediately after adding. Depending on the group CON (16 μl ethanol) or EO (16 μl EO) were added to the mucosal side of the chamber.

[0047] After 70 minutes the first sample from the “cold” side was taken, followed by five samples of each 900 μl which were taken in an interval of 30 minutes. The sample volume was replaced by an aliquot of the respective buffer. Fluorescence was measured in a plate reader at 490 nm excitation and 525 nm emission (EnSpire Multimode Plate Reader, Perkin Elmer, Waltham, Mass.).

TABLE-US-00003 TABLE 3 composition of the mucosal and serosal buffer solutions used in the experiment. C.E. B Ex. 3 Ex. 4 mucosal mucosal mucosal Serosal Chemical mM mM mM mM CaCl.sub.2 × 2H.sub.20 1.8 3.6 7.2 1.8 MgCl.sub.2 1.0 1.0 1.0 1.0 MES free acid 10.0 10.0 10.0 0 Hepes free acid 0 0 0 10.0 NaCl 70.0 70.0 70.0 70.0 HCl 17.0 17.0 17.0 0 Na-acetate 25.0 25.0 25.0 0.0 Na-propionate 10.0 10.0 10.0 0.0 Na-butyrate 5.0 5.0 5.0 0.0 Na-gluconate 0.0 0.0 0.0 40.0 NMDG free base 2.0 2.0 2.0 5.0 KH.sub.2PO.sub.4 0.4 0.4 0.4 0.4 K.sub.2HPO.sub.4 2.4 2.4 2.4 2.4 NaHCO.sub.3 25.0 25.0 25.0 25.0 Enrofloxacine 0.0278 0.0278 0.0278 0.0278 Mannitol 19.0 19.0 19.0 10.0 Glucose 0.0 0.0 0.0 10.0 pH 6.1 6.1 6.1 7.4

[0048] Statistics:

[0049] All results are given as mean±SEM. P values of <0.05 were considered to be significant.

[0050] N refers to the number of animals, n refers to the number of individual measurements of tissues. Multiple comparisons were performed using one-way (repeated measures) ANOVA using Sigma Plot program for Windows.

[0051] Results

[0052] The paracellular flux across ruminal epithelium under three different calcium concentrations were measured. The results are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Shown are the mean and SEM of measured paracellular fluxrates across ruminal epithelium depending on mucosal calcium concentration and the presence or absence of EO. Fluorescein flux Ca-conc. nmol/h/cm.sup.2 Rel. Example n = 13 mM Mean SEM flux % P-value Con 1.8 0.072 0.027 100 B EO 1.8 0.068 0.025 93.5 n.s. Con 3.6 0.082 0.030 100 3 EO 3.6 0.051 0.025 91.8 n.s. Con 7.2 0.005 0.031 100 4 EO 7.2 −0.045 0.041 75.9 <0.05

[0053] The paracellular flux is considerably reduced with the addition of menthol at high Ca concentration (Example 4) which is according to the invention. Under such conditions (i.e. high Ca concentrations), paracellular barrier integrity of the rumen mucosa is significantly increased, whereby the rumen mucosa is likely less affected by the calcium preparation of the invention. The paracellular flux observed for Example 3 and Comparative Example B are not significantly different from the control.