TREATMENT OR PREVENTION OF ANAEMIA IN PREGNANT NON-HUMAN MAMMALS

Abstract

The present invention relates to an iron carbohydrate complex for use in a method of increasing the blood haemoglobin concentration in a pregnant non-human mammal, wherein the pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less, is administered one or more doses of iron carbohydrate complex comprising an amount of elemental iron of 1800 mg or more per dose. The method relates to the further effects of decreasing the rate of stillborn offsprings from a pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less, increasing the blood haemoglobin concentration of offspring litters within 3 days from birth and/or weaning, or increasing the litter size in a subsequent parity of a non-human mammal having a blood haemoglobin level of 105 g/L or less.

Claims

1.-78. (canceled)

79. A method of increasing the blood haemoglobin concentration in a pregnant non-human mammal, wherein the pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less is administrated one or more doses of an iron carbohydrate complex in an amount of 1800 mg or more elemental iron per dose.

80. A method of decreasing the rate of stillborn offsprings from a pregnant non-human mammal, wherein the pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less is administered one or more doses of an iron carbohydrate complex in an amount of 1800 mg or more elemental iron per dose.

81. A method for increasing the blood haemoglobin concentration of offspring litters within 3 days from birth and/or at weaning, wherein a pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less is administered of one or more doses of an iron carbohydrate complex in an amount of 1800 mg or more elemental iron per dose.

82. A method for increasing the litter size of a pregnant non-human mammal in a subsequent parity, wherein the pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less is administered one or more doses of an iron carbohydrate complex in an amount of 1800 mg or more elemental iron per dose.

83. A method for increasing the blood haemoglobin concentration of offspring litters within 3 days from birth and/or a weaning, wherein the pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less is administered one or more doses of iron carbohydrate complex comprising an amount of elemental iron of 200 mg or more per dose.

84. A method for increasing the litter size in a subsequent parity of a mammal, wherein the pregnant non-human mammal having a blood haemoglobin level of 105 g/L or less is administered one or more doses of iron carbohydrate complex comprising an amount of elemental iron of 200 mg or more per dose.

85. The method according to any one of the claim 79, wherein the non-human mammal is a pig, a horse, camel, sheep, goat, or a cow.

86. The method according to any one of the claim 79, wherein the mammal is a pig.

87. The method according to any one of the claim 79, wherein two doses are administered, the first dose being administered 15 to 3 weeks prior to expected farrowing and the second dose being administered 8 to 1 weeks prior to expected farrowing.

88. The method according to any one of the claim 79, wherein the dose is 6 mg/kg BW (body weight) or more, such as 7 mg/kg BW, 8 mg/kg BW, 9 mg/kg BW, 10 mg/kg BW, 11 mg/kg BW, 12 mg/kg BW, 13 mg/kg BW, 14 mg/kg BW, 15 mg/kg BW, 16 mg/kg BW, 17 mg/kg BW, 18 mg/kg BW, 19 mg/kg BW, 20 mg/kg BW, 21 mg/kg BW, 22 mg/kg BW, 23 mg/kg BW, 24 mg/kg BW, 25 mg/kg BW, 26 mg/kg BW, 27 mg/kg BW, 28 mg/kg BW, 29 mg/kg BW, 30 mg/kg BW, or more.

89. The method according to any one of the claim 79, wherein in the iron carbohydrate complex is selected from the group comprising iron carboxymaltose, iron polyglucose sorbitol carboxymethyl ether complex, iron mannitol complex, iron dextran, iron hydrogenated dextran, carboxyalkylated reduced oligo- and poly saccharides, iron sucrose, iron gluconate, iron dextrin, iron hydrogenated dextrin, iron polymaltose, iron hydrogenated polymaltose, iron polyisomaltose, iron hydrogenated polyisomaltose, iron saccharide complex, iron pyrophosphate, iron sorbitol, glycoheptanoic acid, oxidised dextrin, oxidised dextran, oxidised oligo and poly-saccharides, or mixtures thereof.

90. The method according to any one of the claim 79, wherein the iron carbohydrate complex is iron hydrogenated dextran.

91. The method according to any one of the claim 79, wherein the weight average molecular weight of the carbohydrate component of the iron carbohydrate complex is 800 to 50,000 Dalton, preferably 800 to 10,000 Dalton.

92. The method according to any of the claim 79, wherein the apparent molecular weight of the iron carbohydrate complex is 500.000 Dalton or less, such as an apparent molecular weight of 400.000 Dalton or less.

93. The method according to any one of the claim 79, wherein the parenteral administration is selected among the group consisting of intramuscular (IM), subcutaneous (SC), and intravenous (IV) administering.

94. The method according to any of the claim 79, wherein the amount of reprotoxic conservatives in the formulation comprising iron carbohydrate complex is 1 g/L or less.

95. The method according to claim 94, wherein the reprotoxic conservative is phenol.

96. The method according to any one of the claim 79, wherein the dose is administered in 2 minutes or less.

97. The method according to any one of the claim 79, wherein the concentration of the iron carbohydrate complex in the liquid veterinary composition is in the range of 5 g/100 mL to 25 g/100 mL.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention relates to an iron carbohydrate complex for use in a method of increasing the blood haemoglobin concentration, for use in a method of decreasing the rate of stillborn piglets, for use in a method of increasing the survival, health and/or growth of offspring litters until weaning, or for use in a method of increasing the litter size.

[0036] The following describes the experimental protocol supporting the invention.

[0037] 1 Materials and Methods

[0038] 1.1 Target and Study Population

[0039] The study is carried out in a commercial sow herd, which is representative of intensive sow herds in Denmark. The study population consists of pregnant sows with low haemoglobin values and their offspring litters until weaning. The pregnant sows are selected for the study between 60-80 days of gestation.

[0040] 1.2 Study Design

[0041] The study is a randomised clinical trial. Approval from the Danish Animal Experiments Inspectorate and from The Danish Medical Agency is obtained before the start of the study. The study is carried out according to the protocol.

[0042] 1.3 Study Unit

[0043] The study unit is sows and their offspring litters.

[0044] 1.4 Herd Selection

[0045] A prevalence study of anaemia in Danish sows is carried out in 5 herds selected in cooperation with specialised veterinary pig practices. A herd with high prevalence of anaemia is selected for the study.

[0046] 1.4.1 Herd Selection Criteria

[0047] 1. Herd size of at least 1000 sows

[0048] 2. Good record keeping of herd data

[0049] 3. Motivated herd owner

[0050] 4. Iron supplementation of piglets by injection of 200 mg iron dextran at day 4 only

[0051] 1.4.2 Herd Exclusion Criteria

[0052] 1. Provision of extra iron to the sows either orally or injectably (except in feed formulations)

[0053] 2. Breeding or multiplying herd

[0054] 3. Parturition induction e.g. with prostaglandin

[0055] 4. Obvious infectious disease or management problems that may affect stillbirths

[0056] 1.5 Sample Size

[0057] The sample size necessary to estimate a prevalence is calculated by the equation:

N=(Z.sub.(1−α/2).sup.2σ.sup.2)/L.sup.2 (J.P.T.M et al., 2001)

[0058] N=the number of samples taken

[0059] α=0.05 (95% level of confidence)=>Z.sub.(1−0.05/2)=Z.sub.0.975=1.96

[0060] =standard deviation

[0061] L=Expected Absolute Error

[0062] In a herd with 50% of sows suffering from anaemia (<100 g Hb per litre) a sample size of 50 animals is sufficient to determine the prevalence with an allowable error of +/−13%.

[0063] 1.6 Identification of Sows for Clinical Trial

[0064] All multiparous sows from a sufficient number of weekly batches corresponding to 200 animals will be identified at day 60-80 day of gestation. First parity sows will be excluded.

[0065] Initial measurement of haemoglobin in the 200 sows will be performed by HaemoCue measurement. Healthy sows with haemoglobin lower than 100 g/l will be included in the study.

[0066] 1.7 Blood Sampling

[0067] The sows are restrained and 5 mL blood is sampled in EDTA and plain vacutainer tubes from the jugular vein at the start of the trial (week eleven of gestation) and one week before expected farrowing. The samples will be analysed by complete haematology, serum iron, TIBC and serum ferritin as described in sections 4.6.3 and 4.8. Transferrin saturation will also be calculated as described in section 4.9.

[0068] 1.8 Haematology of Sows

[0069] All 200 EDTA stabilized blood samples will be tested initially for haemoglobin concentration by HaemoCue. The equipment necessary for this testing will be provided by Pharmacosmos. Selection of 100 anaemic sows will be based on this testing.

[0070] Samples from the 100 selected sows will subjected to complete haematology (baseline measurement) at the University of Copenhagen.

[0071] The blood samples are analysed for erythrocyte count (RBC), leucocyte count (total leucocyte count and differential leucocyte count), platelets, mean platelet volume (MPV), red blood cell distribution width (RDW), haemoglobin concentration (Hb), haemoglobin distribution width (HDW), haematocrit (HCT), mean cell volume (MCV), mean corpuscular haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). Reticulocyte indices will also be analysed, which include reticulocyte count (absolute and relative), reticulocyte haemoglobin content (Chr), mean reticulocyte corpuscular haemoglobin concentration (CHCMr), reticulocyte cellular volume (MCVr), reticulocyte red cell distribution width (RDWr) and reticulocyte haemoglobin distribution width (HDWr).

[0072] The serum samples are stored until study sows for clinical trial are identified.

[0073] 1.9 Recording of Sow Data

[0074] For each sow the following recordings are made: Age of sow, age of first insemination, number of dead born and live born piglets in previous parity, date of insemination, date of farrowing and parity of the sow.

[0075] 2 Sample Size Calculation for Clinical Trial

[0076] The sample size in each group necessary to calculate differences between groups of animals is calculated by the equation (Graat et al., 2001)

N=2×(Z.sub.α+Z.sub.β).sup.2*SD.sup.2/A.sup.2

[0077] In which

[0078] N=Number of sows required in each group

[0079] Z.sub.α, Z.sub.β=Values of Standard Normal distribution at specified levels of confidence and power

[0080] SD=Standard deviation

[0081] Δ=Estimated difference

[0082] The constant 2 implies that the SD is equal in both groups.

[0083] We assume a two sided test with α of 0.05, Z 0.05=1.96 and a power of 80%, Z.sub.0.20=0.84.

[0084] The haemoglobin difference between two groups (A) is assumed to be 10 g/L and the SD is set to 15 g/L based on previous study.

[0085] When assuming a haemoglobin concentration difference of 9 g/L between treatment and control groups, 50 sows are required per group.

[0086] 2.1 Laboratory Analysis of Frozen Samples

[0087] Serum samples obtained from the selected 100 sows (50 in treatment and 50 in control groups) are analysed for serum iron, total iron binding capacity (TIBC) and serum ferritin, if possible. Serum iron and total iron binding capacity are analysed at the Central Laboratory, University of Copenhagen whereas serum ferritin measurement is performed at professor Ritzmanns laboratory, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, München.

[0088] 2.2 Calculation of Transferrin Saturation (TfS)

[0089] Transferrin saturation is calculated for all the blood samples using the formula: TfS (%)=Serum iron/TIBC×100.

[0090] 2.3 Measurement of Back Fat Thickness

[0091] Baseline back fat measurement of the sows is done using ultrasonic device. The measurement is taken at the last floating rib, 7 cm on either side of the midline of the back according to the guidelines of the manufacturer. Back fat measurement will be performed at start of the trial, at farrowing and at immediately before weaning.

[0092] 2.4 Randomization of Sows

[0093] The selected sows are randomly allocated to two groups:

[0094] 1. Control group

[0095] 2. Treatment group

[0096] Randomisation is done using lottery with ear tag numbers of sows.

[0097] 2.5 Treatment of Sows

[0098] Preparations of test and control substance and equipment for injection will be provided by Pharmacosmos.

[0099] First Dose

[0100] Sows in the treatment group will receive 12.5 mL (2,500 mg) iron injection (Uniferon) intramuscularly in the neck region at day 70 of gestation (6 weeks before farrowing). The control group will receive 12.5 mL of isotonic saline intramuscularly in the neck region in the same day. Any reactions due to the iron injection are noted and treated accordingly.

[0101] Second Dose

[0102] Two weeks after the first dose of iron at day 84 (4 weeks before farrowing), the sows will receive second dose of 12.5 mL (2,500 mg) iron injection (Uniferon) intramuscularly in the neck region intramuscularly. Also, the control sows will receive second dose of isotonic saline.

[0103] 2.6 Management of Sows of Piglets

[0104] All sows are managed according to standard procedures at the farm during the entire study period.

[0105] 2.7 Recording and Classification of Stillborn Piglets

[0106] All fully developed dead piglets in a litter are collected at farrowing. The dead piglets are necropsied and lungs are tested. If the lungs sink in water, the piglet is considered as stillborn. Stillborn piglets are classified as follows:

[0107] 1. Non-fresh stillbirths: Showing signs of degeneration, brown skin colour—these piglets probably die more than a week before onset of farrowing (Randall and Penny 1967)

[0108] 2. Pre-partum stillbirths: Showing no external signs of decay but with the same brick-red colour of all their abdominal organs due to haemolysis and autolysis—these pigs die in utero in the days closely preceding farrowing (Bille et al., 1974)

[0109] 3. Intra-partum stillbirths: Normal colour of the abdominal organs but presence of mucus and/or meconium in the trachea indicating piglets dying during farrowing

[0110] Mummified foetus is recorded but not included in the trial. For each stillborn and live born piglets, the sex is recorded and individual bodyweight obtained, if practically feasible.

[0111] 2.8 Haematology of Piglets

[0112] Blood is collected from the Anterior Vena cava from a subset of two live born piglets per litter. These piglets are selected randomly among all the piglets in that particular litter. The blood from two dead born piglets per litter is collected according to the guidelines of Rootwelt et al., 2012.

[0113] The blood is subjected to complete haematology including serum iron, TIBC and serum ferritin as described in sections 1.8 and 2.1. Lactate in each piglet will also be measured.

[0114] 2.9 Adverse Reactions in Sows

[0115] During the study it is expected that 20% of sows are culled at weaning. Among these sows 10 animals are selected by convenience for studies on injection site reactions. The injection site is assessed macroscopically to determine the extent of tissue damage caused due to injection. Samples for histological examination will be fixed in formalin.

[0116] The number of total born piglets in treated animals will be compared to those of controls. The back fat thickness of sows close to weaning will be recorded.

[0117] 3 Statistical Analyses

[0118] SAS 9.3 is employed for the data analysis. The haematology of sows before and after iron injection are compared using general linear model using PROC GLM procedure in SAS. The differences in haematology in sows between control and treatment sows are calculated using general linear model with PROC GLM procedure. Explanatory factors will include baseline Hb values, number of total born piglets and parity of the sow. The probability of piglet being stillborn in each control and treatment sows is calculated using generalised linear models using PROC LOGISTIC procedure. In this procedure, Hb at baseline measurement, number of total born piglets, parity of the sow, sex of the piglet are explanatory variables that are considered for analysis.

[0119] The differences in haematology between live and stillborn piglets are carried out using linear mixed model with PROC MIXED procedure with sow as the random factor. Other explanatory variables will include litter size, parity of the sow and haemoglobin of the sow.

Example 1

[0120] 100 sows were selected from a herd showing a high prevalence of anaemia. For practical reasons the study was performed in two batches. All data was pooled.

[0121] 5 ml blood was sampled in EDTA from the jugular vein at the start of the trial, i.e. week 8 of gestation, about one week before expected farrowing, and 4 week after farrowing. The blood samples were marked Hb_S1, Hb_S2, and Hb_S3, respectively. The samples were analysed and the blood haemoglobin concentration (Hb) was determined.

[0122] The selected sows are randomly allocated to two groups:

[0123] 1. Control group (C)

[0124] 2. Treatment group (T)

[0125] Randomisation is done using lottery with ear tag numbers of sows. Each group included 50 sows.

[0126] Sows in the treatment group was delivered a first dose of 12.5 ml iron dextran (Uniferon, 20%), corresponding to 2500 mg iron, intramuscularly in the neck region at day 70 of gestation (6 weeks before farrowing). The control group received 12.5 mL of isotonic saline intramuscularly in the neck region in the same day. Any reactions due to the iron injection are noted and treated accordingly.

[0127] Two weeks after the first dose of iron at day 84 (4 weeks before farrowing), the sows received a second dose of 12.5 mL (2,500 mg) iron injection (Uniferon) intramuscularly in the neck region. Also, the control sows will received second dose of isotonic saline.

TABLE-US-00001 TABLE 1 Hb in Sows Base- line Δ Δ Hb Number Average Average Average (Hb_S1, (Hb_S1, (g/l) Group of sows Hb_S1 Hb_S2 Hb_S3 Hb_S2) Hb_S3) <105 C 29 99.33 99.60 99.55 0.28 0.22 T 31 99.10 100.51 100.77 1.40 1.66 >105 C 19 111.16 103.36 103.19 −7.80 −7.97 T 19 111.07 102.26 101.66 −8.82 −9.41

[0128] All fully developed dead piglets in a litter are collected at farrowing. The dead piglets are necropsied and lungs are tested. If the lungs sink in water, the piglet is considered as stillborn.

[0129] Mummified foetus is recorded but not included in the trial. For each stillborn and live born piglet, the sex is recorded and individual bodyweight obtained, if practically feasible.

TABLE-US-00002 TABLE 2 Total of stillborn piglets Baseline Number Number Sum of Stillborn Hb (g/l) Group of sows stillborn liveborn rate <105 C 28 44 477 7.9% T 27 32 451 6.6% >105 C 16 20 276 6.6% T 16 18 255 6.4%

[0130] It is noted that the haemoglobin concentration in sows shown in table 1 is increased for the sows having a baseline Hb below 105 g/l. Specifically, the anaemic sows having a low baseline Hb of <105 g/l experience an increase of 1.40 g/l, compared to 0.28 g/l for the control group, from the start of the trial to one week before expected farrowing and an increase of 1.66 g/l, compared to 0.22 for the control group, from the start to 4 weeks after farrowing. The results show that anaemic pregnant sows benefit from the administration of the iron carbohydrate complex. The Δ(Hb_S1, Hb_S2) and Δ(Hb_S1, Hb_S3) for sows having baseline Hb above 105 g/l are negative indicating that these animals are not benefitting from the treatment.

[0131] The stillborn rate in table 2 is decreasing from 7.9% in the control group to 6.6% in the treated group, when the sows having a baseline <105 g/l are treated with the iron carbohydrate complex. The result indicates that the rate of stillborn offspring is decreased by the administration of an iron carbohydrate complex. Conversely, the stillborn rate is decreasing from 6.6% in the control group to 6.4% in the treated group when the baseline Hb is above 105 g/l, indicating that an improvement in the stillborn rate for this group of sows is not obtained.

REFERENCES

[0132] Archibald, R., Hancock, E. E. I., 1939. Iron Deficiency-Stillbirth of Swine. Canadian Journal of Comparative Medicine 3, 134. [0133] Bille, N., Nielsen, N. C., Larsen, J. L., Svendsen, J., 1974. Preweaning mortality in pigs. 2. The perinatal period. Nordisk veterinaermedicin, 26, 294-313. [0134] Graat, E. A. M., Frankena, K., Bos, H., 2001. Principles and methods of sampling in animal disease surveys, In: Noordhuizen, J. P. T. M., Frankena, K., Thrusfield, M. V., Graat, E. A. M. (Eds.) Application of quantitative methods in veterinary epidemiology. Wageningen Pers, Wageningen, The Netherlands, pp. 45-46. [0135] Jensen, A. K., Pedersen, K. S., Nielsen, J. P., 2013. Association between blood haemoglobin concentration in sows and neonatal piglets. In Proceedings of the 5th ESPHM, Edinburgh, UK. [0136] Jensen, A. K., Nielsen, J. P., 2014. Association between stillborn piglets and haemoglobin concentration in sows at farrowing. In Proceedings of the 6th ESPHM, Sorrento, Italy. [0137] Moore, R., Redmond, H., Livingston Jr, C., 1965. Iron deficiency anemia as a cause of stillbirths in swine. Journal of the American Veterinary Medical Association 147, 746. [0138] Morris, C. J., Earl, J. R., Trenam, C. W., Blake, D. R., 1995. Reactive oxygen species and iron—a dangerous partnership in inflammation. The international journal of biochemistry and cell biology 27, 109-122. [0139] Randall, G. C. B., Penny, R. H. C., 1967. Still birth in pigs: the possible role of anoxia. Veterinary Record 81, 359-361. [0140] Rootwelt, V., Reksen, O., Farstad, W., Framstad, T., 2012. Associations between intrapartum death and piglet, placental, and umbilical characteristics. Journal of Animal Science 90, 4289-4296. [0141] Svetina, A., Vrabac, L., Belić, M., Turk, R., 2006. Relation between erythrocyte parameters and stillbirth in piglets. Veterinarski arhiv 76, 297-303. [0142] Thorn, C. 2010. Hematology of the pig. In Schalm's Veterinary Hematology 6th Edition, Wiley-Blackwell, Ames, Iowa, Weiss, D., Wardrop, K., eds. (Iowa, Wiley-Blackwell), p. 848. [0143] Vinther J., 2013. National average productivity in pig production in 2012 (Landsgennemsnit for productivitet i svineproduktionen 2012). Videncenter for svineproduktion [0144] Zaleski, H. M., Hacker, R. R., 1993. Variables related to the progress of parturition and probability of stillbirth in swine. The Canadian Veterinary Journal 34, 109. [0145] Auvigne, V., et al., 2010. Anaemia in the hyperprolific sow: Effect of injectable iron administration and relation with fattering score.