Uniferon Handbook by Pharmacosmos

The Uniferon® Handbook 5th edition

An accurate pen-side diagnostic tool for use in determining an effective dosage of Uniferon® forthe prevention and treatment of sub-clinical and full-scale iron deficiency anaemia.

© 2018 PHARMACOSMOS ALL RIGHTS RESERVED

Table of Contents Introduction and the F.A.C.T Program About this diagnostic tool..............................................................3 About Pharmacosmos, injectable iron and Uniferon®. ...............4-9 What is the F.A.C.T program and its benefits?....................... 10-11 Industry-Accepted Iron Standards Why optimal haemoglobin (Hb) blood levels make a difference.. 12 Benefits of optimal haemoglobin blood levels............................. 13 Are your piglets at risk of entering an iron gap?.......................... 14 Benefits of a 2nd injection........................................................15-17 Iron Deficiency Anaemia Detection and Treatment How do you know if a piglet is suffering from anaemia?........18-19 How iron treatment sources differ................................................20 The benefits of injection and technique..................................21-22 Every drop counts..................................................................23-24 When and how to select piglets for evaluating herd level amaemia..............................................................................25 How to test your selected piglets.................................................26 Pen-side blood testing for diagnosis of iron deficiency anaemia..........................................................27-29 Other Anaemia Contributors and Conditions What if my piglets get infected with a virus?..........................30-31 About vitamin E deficiency..........................................................32 About mycotoxins in swine feed..................................................33 Resources and References Uniferon® benefits........................................................................34 Iron expert board.........................................................................35 Pharmacovigilance associated with the prevention and treatment of iron deficiency anaemia..........................................36 The basics of adverse events associated with the administration of parenteral iron supplementation...................... 37 Resources and references.....................................................38-40 Uniferon® abreviated prescribing information.............................. 41 Contacts......................................................................................42

About this diagnostic tool This accurate pen-side diagnostic tool enables the modern swine production operations to evaluate and treat their piglets with an effective follow-up dosage of Uniferon® (200 mg/ml) that will prevent or treat sub-clinical or full-scale iron deficiency anaemia.

What you will find on the following pages: • I nformation on our F.A.C.T iron deficiency anaemia assessment program • How and why pigs may become subject to sub-clinical or full-scale iron deficiency anaemia • Industry-accepted iron level standards • How to clinically assess a herd • Effective iron treatment program and administration • Best practices

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About Pharmacosmos Pharmacosmos is the leading manufacturer and developer of prevention and treatment of iron deficiency anaemia in humans and animals. Our Uniferon速 Iron(III) Dextran product is the only prevention and treatment of piglet anaemia approved by health authorities in Europe, Asia and by FDA in the U.S. Uniferon速 is manufactured according to highest quality in our manufacturing facilities in Denmark. Pharmacosmos works closely with producers, veterinarians and partners around the world to incorporate best practice solutions into our treatments - and treatment with the proper administration of Uniferon速 is a simple way to realize higher production yields and higher profits.1,3 Learn more about Pharmacosmos, the manufacturer of Uniferon速, which is the leading world-wide prevention and treatment product for sub-clinical and full-scale iron deficiency anaemia in piglets.

Learn more - visit www.pharmacosmos.com

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The history of injectable Iron Traditional Iron Dextran Injectable iron dextran has been used to treat piglets since the 1950’s.12 The first generation were produced with organic solvents, was difficult to produce with more than 10% iron and had some side effects / risk of tissue staining.13 Gleptoferron Injectable iron based on the gleptoferron formulation was developed in 1967. It was produced using organic solvents and cyanide and, therefore, subject to the presence of residual solvents and cyanide impurities in the product. This method of synthesis has only been approved for veterinary use.13 Modern Iron Dextran Uniferon is a modern iron dextran product produced without any organic solvents or cyanide. It’s the only brand of injectable iron that meets both veterinary and human manufacturing standards, and Traditional Iron Dextran 1950s was approved by EMA in Produced with organic solvents 200810 and the FDA in 2011.4

1967

Gleptoferron Produced with organic solvents and cyanide

Modern Iron Dextran Produced without any organic solvents or cyanide

2008

Uniferon, an improved iron hydrogenated dextran, is introduced

5

Iron is not just iron – efficacy and safety The biological activity of nanoparticles depends on particle content and particle properties.14 Differences in any of these parameters can have significant impact on the efficacy and safety profile of the injectable iron product.14 Even products with the same generic name can differ in their safety and efficacy depending on the manufacturing methods, facilities and controls utilized.15

Potential differences between iron complexes: Particle content: Iron Carbohydrate Particle properties: Particle stability Surface properties Carbohydrate Size

Uniferon

The Uniferon carbohydrate end group is hydrogenated, not cyanide-modified and therefore cyanide-free.

Impurities: Dependent upon the manufacturing process

Gleptoferron

Gleptoferron is produced with the use of organic solvents and cyanide. The gleptoferron carbohydrate end group is cyanide-modified to carboxylic end groups.

6

ory

untry

in

nada

gium

many

gium

many

sia

nmark

Iron is not AS just ironCr – Impurities

Molecule

Pb

Arsenic‡

Chromium

Permitted daily exposure¶ 0.3 µg/kg

Permitted daily exposure¶ 22.0 µg/kg

Concentration (mg/ml)

ISU

Lab 2#

ISU

Lead‡

Permitted daily exposure¶ 0.1 µg/kg

Lab 2#

ISU

Lab 2#

µg/200mg dose µg/200mg dose µg/200mg dose The independent FDA-certified laboratory at Iowa State University analyzed 16 widely used injectable iron products for piglets.

Ironhas dextran 100 3.4 2.0 27.0 22.0 < 0.1 < 0.1 In 15 of them, the concentration of arsenic, chromium and/or lead Ironexceeded dextran 100 4.0 1.9 36.2 32.9 0.1 < 0.1 18 the permitted daily exposure limit for <humans. §

§

§

Iron dextran

200

0.2

0.4

36.0

49.5

4.9

5.8

Gleptoferron

200

< 0.1

< 0.1

25.0

24.1

2.0

1.1

Gleptoferron

200

0.9

< 0.1

32.4

29.6

Only one product – Uniferon® – had non-detectable levels of both ® and where chromium Ironarsenic dextran 200 lead. Uniferon 1.7 1.6 was also 12.0 the 35.0only product 0.3 < 0.1 levels did not exceed human permitted daily exposure limits.

exceeded the human daily exposure limit by > 25%

exceeded the human daily exposure limit by ≤ 25%

Gleptoferron 200 2.2 1.2Laboratory ISU = Iowa State University Veterinary Diagnostic

21.0

Lab 2 = independent laboratory

Gleptoferron

1.4

200

0.5

0.6

AS

28.9Arsenic‡ Permitted daily exposure¶ 0.3 µg/kg

18.0

27.0

33.2

28.9

0.5

Cr

< 0.1Chromium < 0.1 Permitted daily exposure¶ 22.0 µg/kg

< 0.1

< 0.1

2.6

0.6

Pb

Lead‡ Permitted daily exposure¶ 0.1 µg/kg

# # ISU Lab 2# Brand Name Manufacturer† Country Molecule Concentration ISU Lab 2 ISU Lab 2 µg/200mg dose§ µg/200mg dose§ µg/200mg dose§

(mg/ml)

Gleptoferron

Product A

200

US

< 0.1

< 0.1

Iron dextran

100

200 < 0.1 Pharmacosmos US < 0.1 Iron dextran

100 0.4

Product C

Product D

IronUniferon dextran® Gleptoferron

Gleptoferron

Product E

IronProduct dextranF Product G

200

na

IronProduct dextranI

na

Iron dextran

Spain

Iron dextran

200

Canada

Iron dextran

200

< 0.1 0.1

< 0.1

25.0 19.0

Belgium Gleptoferron 200

100

US 2.0

Germany Gleptoferron

< 0.1

200

Gleptoferron 200 2.2 19.4

< 0.1

200

29.0

UK

100

1.4 1.8 65.4 Belgium Gleptoferron 200

Uniferon® 200 Pharmacosmos US

Product J

< 0.1

Gleptoferron Product H

200

100

1.8

Gleptoferron 200

Iron dextran ††

NA

200

28.6

3.4

2.0 27.0 22.0

< 0.1 < 0.1

0.74.0 1.9 < 0.136.2 32.9 < 0.1

< 0.1 < 0.1

0.2

36.0

0.4 36.0 49.5

0.4

0.4

1.7 1.6 12.0 35.0

21.9

1.1

< 0.1 < 0.1

1.2

25.0 24.1

29.6 36.70.9 < 0.1 1.2 32.4 1.5 2.2 1.2 21.0 28.9

29.0

3.1

39.8 0.6 33.2 28.9 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1

< 0.1

0.4

0.7

NA††

Product L

2.0 1.1 0.6 0.5 < 0.1 < 0.1 < 0.1 < 0.1 2.6 0.6 < 0.1 < 0.1

Uniferon® – free metal 200 of heavy < 0.1 < 0.1 25.0 impurities 36.0 0.4 0.4 Please see table next page 1.1 1.2

Germany Gleptoferron

figure. Yellow highlighted cells indicate the element was present at ≤ 25% (Adapted from Radke, S.L. et al. Elemental ed highlighted cells exceed the human daily exposure limit by > 25%. impurities0.1 in injectable iron products for swine.

Product K

Russia Gleptoferron 200 < 0.1 19.0 21.9 Journal of Swine Health and Production.

2018;26(3) [accepted for publication], table 1)

the limit of detection for the assay.

0.3 < 0.1

0.3

1.4 0.5 18.0 27.0

NA††

4.9 5.8

US

Iron dextran

100

2.0 2.2 19.4 36.7

1.2 1.5

7

exceeded the human daily exposure limit by > 25%

exceeded the human daily exposure limit by ≤ 25%

AS

Arsenic‡ Permitted daily exposure¶ 0.3 µg/kg

ISU = Iowa State University Veterinary Diagnostic Laboratory Lab 2 = independent laboratory

Cr

Chromium Permitted daily exposure¶ 22.0 µg/kg

Pb

Lead‡ Permitted daily exposure¶ 0.1 µg/kg

# # ISU Lab 2# Brand Name Manufacturer† Country Molecule Concentration ISU Lab 2 ISU Lab 2 µg/200mg dose§ µg/200mg dose§ µg/200mg dose§

(mg/ml)

Product A

US

Iron dextran

100

3.4

2.0 30.2 27.0

< 0.1 < 0.1

Product B

US

Iron dextran

100

4.0 1.9 36.2 32.9

< 0.1 < 0.1

Product C

Spain

Iron dextran

200

0.2

Product D

Canada

Iron dextran

200

Product E

Belgium Gleptoferron 200

Product F

US

Product G

Germany Gleptoferron

Product H

UK

Product I

Belgium Gleptoferron 200

Uniferon® 200 Pharmacosmos US

Gleptoferron 200 200

Gleptoferron 200

0.4 36.0 49.5

1.7 1.6 12.0 35.0 < 0.1 < 0.1

25.0 24.1

0.9 < 0.1

32.4 29.6

4.9 5.8 0.3 < 0.1 2.0 1.1 0.6 0.5

2.2 1.2 21.0 28.9

< 0.1 < 0.1 < 0.1 < 0.1

1.4 0.5

18.0 27.0

< 0.1 < 0.1

33.2 28.9

Iron dextran

200

< 0.1 < 0.1

0.4

200

0.7

2.6 0.6 < 0.1 < 0.1

Product J

Germany Gleptoferron

< 0.1 < 0.1

25.0 36.0

0.4 0.4

Product K

Russia Gleptoferron 200

0.1 < 0.1

19.0 21.9

1.1 1.2

Product L

US

Iron dextran

100

2.0 2.2 19.4 36.7

Product M

Denmark Gleptoferron

200

Product N

China

Iron dextran

100

1.4 1.8 65.4 39.8

0.6 < 0.1

Product O**

China

Iron dextran

100

28.6 NA†† 1.8 NA††

< 0.1 NA††

< 0.1 < 0.1

29.0 29.0

1.2 1.5 3.1 0.3

Adapted from Radke, S.L. et al. Elemental impurities in injectable iron products for swine. Journal of Swine Health and Production.

2018;26(3):142-145 * All values are rounded to the nearest one significant figure. Yellow highlighted cells indicate the element was present at ≤ 25% higher than the daily limits established for humans. Red highlighted cells exceed the human daily exposure limit by > 25%. † Marketing Authorization holder/NADA owner. ‡ Values reported as < 0.1 µg/200 mg dose were below the limit of detection for the assay. § For all 200 mg/ml products the reported elemental concentrations in µg/200 mg dose are equivalent to parts per million.

For 100 mg/ml products, detected concentrations in parts per million were doubled to represent a typical 200 mg dose. ¶ Permitted daily exposure is the published daily exposure limit for an adult human. Values were converted to µg/kg assuming 50 kg as a conservative adult human body weight and using inclusion limits reported in USP < 232 > and ICH Q3D for human pharmaceutical products. ** Sample was not available for testing at both laboratories. †† Not applicable. # 15 of the products were also analysed at a second independent laboratory concurrently.

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About Uniferon® Uniferon® from Pharmacosmos is an injectable iron supplement for piglets, containing 200 mg iron per ml, which promotes better health and faster growth.1,3 It is well known that injectable iron supplementation of piglets via injection of 200 mg iron during processing on days 1 to 4 postfarrowing has become the industry standard for prevention or treatment of sub-clinical or full-scale iron deficiency anaemia.

Uniferon® is the only world-wide brand of injectable iron that is successfully utilised for the prevention and treatment of sub-clinical or full-scale iron deficiency anaemia, whereby: • It boosts and maintains optimal haemoglobin levels1 • It ensures optimal average daily gain (ADG)1 • It promotes more rapid post-weaning growth1,3 • Is a strongly bound iron complex manufactured without the use of organic solvents or cyanide, avoiding trace residues of these components • It has received global approvals from numerous healthcare authorities in Europe, Asia, and is the only 200 mg/ml FDA approved injectable iron product in the United States8,10

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What is the F.A.C.T program?

Our F.A.C.T program has been designed to provide a 360º view of the iron status of piglets on the modern swine production operation. In close cooperation with production managers and swine veterinarians, the F.A.C.T program: • Provides an overall perspective of the production facility and the influencing factors that impact the iron status in piglets • Introduces an accurate and convenient pen-side diagnostic test for sub-clinical or full-scale iron deficiency anaemia in piglets • Provides a timely and accurate diagnosis for potential sub-clinical or full-scale iron deficiency anaemia in individual piglets, which will help to rapidly depict the iron status of the other litters in the herd as well • Eliminates the guesswork of iron status in piglets and reduces the risk of misdiagnosis of sub-clinical and full-scale iron deficiency anaemia • Provides a progressive approach and includes swine industry best practices • Provides the injectable iron knowledge resources for the industry The acronym F.A.C.T stands for

Farm Anaemia Certification and Training

F.A.C.T Enrolling in F.A.C.T is easy by visiting www.uniferon.com

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What are the F.A.C.T benefits?

F.A.C.T has benefits for swine producers and provides access to a large data base of information and the support to begin making a significant difference in your herd. Do not leave your piglets at risk of misdiagnosis. Be certain about your herd’s iron status by implementing the F.A.C.T program today.

Our F.A.C.T program has three key features: Assessment • Accurate and convenient pen-side blood haemoglobin measurements • Haemoglobin blood level assay results within 30 seconds • Tools for getting started Training • Education on role of iron in physiology • Calculating iron requirements of individual piglets • Piglet handling • Iron administration/injection techniques • Field training for staff, advisors and consultants Overview • 360º overview of welfare and health status of piglets • Access to open scientific literature and market research reports • Conducting on-site clinical trials and studies as necessary 11

Why optimal haemoglobin (Hb) blood levels make a difference Anaemia is a condition which results in a lack of iron-carrying haemoglobin in the red blood cells. It typically occurs when the pigletâ&#x20AC;&#x2122;s iron stores are depleted by rapid growth on an all-milk diet or when the exposure to stress conditions either inhibits the build-up of haemoglobin or increases the degradation of haemoglobin in the blood stream of the piglet. Iron deficiency anaemia is either a sub-clinical condition, or a clinical manifest condition known as full-scale iron deficiency anaemia.2 Haemoglobin is a critical iron-rich protein that enables red blood cells to carry oxygen throughout the body. Also, haemoglobin carries carbon dioxide to the lungs for release and exit from the body, which enables the haemoglobin to transport more oxygen throughout the body. Haemoglobin, along with myoglobin, are two proteins that require adequate levels of iron to optimize growth and support immune system development. Clinical benchmarks for optimal haemoglobin levels and the relationship to piglet sub-clinical or full-scale iron deficiency anaemia are delineated below: <90 = Clinical anaemia11 90-110 = Sub-clinical >110 = Optimal For optimal piglet health and performance, haemoglobin levels must be at or above 110 g/l2 12

Benefits of high haemoglobin blood level Elevated haemoglobin levels are important as these are known to further benefit oxygen transport, immune function, vitality and metabolism in piglets. A recent study has shown remarkable associations of the hematological status at weaning and the weight gain post-weaning. Industry experts believe that optimized postweaning growth requires haemoglobin levels at or above 110 g/l.2 It has been demonstrated that increasing blood levels of Hb were positively associated with growth rates of piglets post-weaning (p=0.0003, estimate=1.8 g ADG/gHb/l). Thus, a variation of 10 g/l in haemoglobin at weaning results in a variation of 18 g/day in weight gain 3 weeks after weaning.3

Estimated ADG (g/day)

300 250 200 150 100 50 0 70

80

90

100

110

Hb

120

130

140

150

In other words, when the pre-weaning blood haemoglobin levels are increased by 10 g/l, the post-weaning weight gains are increased by 18 g/day.

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Are your piglets at risk of entering an iron gap?

300

200 mg administration (day 1-4)

250 200 150

Second administration (before day 17)

100 0 -50

-100 -150 -200

50 1, 76 2, 02 2, 28 2, 54 2, 80 3, 06 3, 31 3, 57 3, 83 4, 09 4, 35 4, 61 4, 87 5, 13 5, 39 5, 65 5, 91 6, 17 6, 43 6, 69 6, 94 7, 20 7, 46 7, 72 7, 98 8, 24 8, 50

50 1,

Hypothetical model for Iron Gap5

Fe 3+ reserve (mg)

Improved weight gains on an all-milk ration increase the risk of developing sub-clinical or full-scale iron deficiency anaemia and, hence, may well prevent optimal weight gains as well. This increased productivity can contribute to a potential “iron gap” due to the depletion of iron stores from birth and low levels of iron in the sow’s milk (approximately 1 mg of daily intake), which are known to have diminished to even lower levels due to increased litter sizes.4

Point where iron requirements exceed availability

-250 Iron consumption in relation to growth

“Iron Gap”

Kg body weight (kg)

Calculate piglet iron requirements in order to avoid entry to an iron gap Visit fact.uniferon.com in order to locate a convenient iron calculator to help you with the assessment of optimal iron requirements for your piglets.

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Benefits of a 2nd injection – improved daily gain post weaning A single 200 mg dose of injectable iron for piglets administered on days 1 to 3 post-farrowing accommodates approximately 4 kg of weight gain on an all-milk diet. A follow-up dose of 200 mg of injectable iron on or about day 14 is recommended to help meet further growth demands for piglets on an all-milk diet.5 Recent on-site clinical trial data reveals increased weight gain and haemoglobin blood levels in piglets when treated with a second 200 mg/ml dose over piglets provided only a single dose.1

325

306 2 injections of Uniferon® 1 injection of Uniferon®

Day 34-50 (post weaning)

Haemoglobin (g/l)

Weight gain (g/d)

Effect of second Uniferon® injection

123

113

Day 34 (at weaning)

(Weight gain and haemoglobin results show statistical significance of p=0.01* and p=0.0001*** respectively)

Administration of a second dose of injectable iron prior to weaning has shown benefits in growth rates and overall performance, especially in piglets exceeding 4 kg of weight gain on an all-milk diet post-farrowing.1, 6

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Benefits of a 2nd injection – improved growth until slaughter A pre-weaning follow-up dose of 200 mg of iron will not only improve the daily gain post weaning, but improve growth until slaughter.

In a study from 201816, 4.628 pigs were enrolled into two treatments groups according to birth weight to determine the impact of iron dosage and blood haemoglobin concentration on wean to market weight gain in commercial pigs.

4.628 pigs enrolled into two treatment groups according to birth weight.

Group A: 200 mg Uniferon® day 5-7 Group B: 200 mg Uniferon® within 24 hours of life and 200 mg Uniferon® at day 5-7 Haemoglobin measurement on a subset (n = 100) of each treatment group.

Weight gain analysis on a subset (n = 1.400) of each treatment group.

RESULTS • Group B had significantly higher average Hb concentration • Group B had significantly higher weight gain through 19-WPW • Group B was the only group with a mean Hb in the optimal range (>110 g/l) at weaning • 63% in Group B had optimal Hb at weaning compared to 8% in Group A • Piglets with optimal Hb at weaning weighed 4,45 kg more at 19-WPW compared to piglets with full-scale iron deficiency anaemia

Table 1. Impact of blood haemoglobin concentration at weaning on post weaning weight. Values within columns not connected by the same superscripts differ (p ≤ 0,1 NS).

Pigs per group

Weight in kg

Hb status at weaning

A

B

Full-scale iron deficiency anaemia

26

5

29,03a

98,43a

Sub-clinical anaemia

65

28

30,84b

99,79ab

Optimal

8

63

32,66c 102,51b

8-WPW 19-WPW

Table 2. Effect of Uniferon® dose on growth until slaughter. Values within columns not connected by the same superscripts differ (p ≤ 0,05*).

Weight in kg Birth

Wean

8-WPW

19-WPW

A

1,36a

5,44b

29,03b

97,07a

B

1,36a

5,44a

29,89a

98,43b

Group

Piglets with optimal haemoglobin at weaning weighed 4,45 kg more at 19 weeks post weaning compared to piglets with full-scale iron deficiency anaemia.

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Benefits of a 2nd injection – improved immune functions Administration of a second dose of injectable iron prior to weaning has shown benefits in combating a challenge of enterotoxigenic Escherichia coli (ETEC).17 30 piglets were challenged with enterotoxigenic Escherichia coli (ETEC) to investigate their ability to combat ETEC in regard to haemoglobin levels.

30 piglets were challenged with enterotoxigenic Escherichia coli (ETEC) to investigate there ability to combat ETEC in regard to iron status.

Group A (n = 10): 100 mg Uniferon® on day 3 Group B (n = 10): 200 mg Uniferon® on day 3 Group C (n = 10): 200 mg Uniferon® on day 3 and 14 Group C (Avg. Hb 124 g/l) had 1/3 less piglets with diarrhoea compared to Group A (Avg. Hb 85 g/l).

Table 1. Pigs ability to combat ETEC in regard to iron status.

Avg. Hb g/l 3-weeks of age

Diarrhoea in procent (%)

A (n = 10)

85

60

B (n = 10)

104

50

C (n = 10)

124

40

Group

Piglets with optimal haemoglobin level are more capable to defend against enterotoxigenic E. coli infection compared to piglets with iron deficiency.

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How do you know if a piglet is suffering from iron deficiency anaemia?

Sub-clinical iron deficiency routinely goes unnoticed because it is nearly impossible to diagnose using only clinically observed symptoms; however, full-scale iron deficiency anaemia is detectable using only clinical symptoms, while the piglet health conditions are often quite severe before being noticed. Therefore, testing for blood haemoglobin levels allows practical and early detection of sub-clinical iron deficiency in piglets, allowing swine producers to administer additional supplemental injectable iron in order to treat the sub-clinical iron deficiency, and virtually eliminate the development of full-scale iron deficiency anaemia in piglets.

Pen-side or laboratory evaluation of the haemoglobin level in a blood sample taken from a vein will indicate whether or not the piglets are suffering from sub-clinical or full-scale iron deficiency anaemia.

Continued on next page.

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First signs of anaemia: • Poor growth • Paleness of the mucous membranes Severe signs of anaemia: • Listlessness (piglets are slow to move, appear to be tired and listless and remain lying down) • Rough hair coat and wrinkled skin • Labored breathing (for example, piglets are virtually out of breath during weaning day movement to the transport truck) • Increased heart rate • Increased respiratory rate or spasmodic movement of the diaphragm muscles following minimal exercise, for example, piglets that are gasping for air with minor levels of exercise, which is especially noticeable when walking a rather short distance to a transport truck on weaning day movement to the nursery

Once visual signs of anaemia are detected using only clinically observed symptoms, the immune system status and the growth rates of piglets have already been quite significantly, and adversely affected.

19

How iron treatment sources differ Colostrum and milk from the sow are both good sources of iron; however, the iron concentration levels in these two sources are inadequate to support optimal growth. Therefore, iron supplementation is required.

Parenteral iron supplementation Supplementation with injectable iron is widely recognized as the most practical, cost-effective, accurate dosing and effective method of prevention or treatment of sub-clinical and full-scale iron deficiency anaemia in piglets.4 Oral supplementation The ultimate challenge for success with orally administered iron supplements is the inefficient absorption of the iron from the immature gut of piglets, hence assimilate oral iron as haemoglobin into the bloodstream. Furthermore, with oral iron supplementation only, there is considerable animal to animal variation in the poor absorption and assimilation of the oral iron, which provides minimum to virtually no absorption and utilization of the oral iron supplement.4 Iron from the sow Swabbing the sowâ&#x20AC;&#x2122;s udder with an iron-rich solution is a relatively poor option, because piglets are unable to effectively absorb and it is typically too labor intensive to be a cost effective, or a long-term solution.4 Iron from the environment Piglets in confinement at commercial swine production operations have little, if any, access to natural soil habitat, whereby the innate rooting behavior of the species could provide a potential source of oral iron and other minerals.4

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The benefits of injection Parenteral administration Once again, the most practical, cost-effective, accurate dosing and effective method of providing supplemental iron to piglets is via injectable administration. With proper injection technique, a single 200 mg dose of supplemental iron at 1 to 4 days of age offers the following advantages:4 • Accurate dosing • Optimal efficacy and weight gain • Cost-effective labor inputs with dosing of piglets between 1 and 4 days of age • Minimized risk of sub-clinical or full-scale iron deficiency anaemia • Supports optimum growth rates of piglets on an all-milk diet • Enhanced immunity and improved general health of piglets Administering a single 200 mg dose of injectable iron to all piglets at 1 to 4 days of age can prevent or treat sub-clinical or full-scale iron deficiency anaemia that may result from low levels of iron available at birth.

21

Injection technique For successful prevention or treatment of sub-clinical or full-scale iron deficiency anaemia, it is important to practice correct injection technique. This is necessary in order to ensure that the iron is dosed correctly and that the iron supplementation enters the blood stream effectively via absorption from the injection site tissue.

Otherwise you will likely experience anaemia in the piglets.

Be certain that all involved persons are aware of the proper injection techniques to help ensure optimal iron supplementation.

1. Choose injection site.

Parenteral administration can be either intramuscular or subcutaneous and the approved/recommended site can vary by country:

in surgical spirits. Allow the skin to dry before injection.

3. Use a small needle.

20 gauge 15 mm needle that has been sterilized.

Intramuscular: Hold the pigletâ&#x20AC;&#x2122;s body towards you. Injection should be intramuscular in the neck 2.5 - 5 cm behind the ear.

4. Pull skin slightly forward before inserting the needle.

Subcutaneous: Hold the piglet in its hind legs, so it hangs head down with hits abdomen facing you. Injection should be subcutaneously in the inguinal skin fold.

6. Release the skin, give the injection, and remove the needle.

The skin then springs back in place, covers the hole in the muscle and seals the injected material in place, minimizing the risk of leakage.

2. Check that the injection site is clean and dry.

If the pigletâ&#x20AC;&#x2122;s skin is dirty or wet, wipe it clean with cotton soaked

5. Quickly and firmly insert the needle.

7. Change needles after every ten piglets or after each litter.

22

Every drop counts A high degree of mental concentration and attention-to-detail discipline during the supplemental injectable iron administration determines the degree of success in the delivery of an accurate dosage of iron.

Biological scientists generally consider 1 drop of liquid equivalent to roughly 0.05 ml, which means that 1 ml of liquid is comprised of 20 drops of liquid. With a 200 mg/ml injectable iron product, each 0.05 ml drop contains about 10 mg of iron.

Therefore, if a total of 4 drops of a 1 ml accurately measured dosage of the injectable iron product is inadvertently lost on the ground, or leaks back on the needle track and falls to the ground during the dosing procedure, this corresponds to approximately 40 mg of the desired dosage being lost (a 20% loss), and the success of the dosage is thus reduced to 80%. Meanwhile, an accurate delivery of the entire 200 mg dosage of supplemental iron provides the best assurance for the prevention or treatment of sub-clinical or full-scale iron deficiency for your piglets.

Continued on next page.

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When swine producing operations choose to administer a 100 mg/ml injectable iron supplement product to piglets, there are several challenges that can occur, including the following: • Insufficient amount of iron for piglets fed on an all-milk diet • With the administration of a single 2 ml dosage of the 100 mg/ml product, there is likely to be an increased rate of backflow and leakage from the injection site via the needle tracks • With the administration of two separate 1 ml doses of the 100 mg/ml product, there could be an increased risk of injection site trauma due to the two separate injections. Similarly, the two separate 1 ml injection sites for administration of a 200 mg dosage of iron using a 100 mg/ml supplemental iron product increases the risk of neurological injury from a second injection site, and trauma associated with the extra handling of the piglets during the second injection Meanwhile, • Losing just a single drop of Uniferon® during the dosing procedure means losing about 10 mg of supplemental iron that a piglet will not be able to absorb or assimilate as haemoglobin in the blood stream to be used to prevent or treat sub-clinical or full-scale iron deficiency anaemia

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When and how to select piglets for evaluating herd level anaemia Iron supplementation dosage regimens for piglets are most often very standardized with little focus on variations in birth weight, growth rate or weaning age in individual herds.

When should haemoglobin levels be evaluated in piglets?

The industry standard involves a 200 mg dosage of supplemental iron between 1 to 4 days of age; however, a critical time for the development of sub-clinical or full-scale iron deficiency anaemia is a few days prior to weaning. This situation occurs when the iron stores from the initial 200 mg dosage of supplemental iron sources has been depleted the uptake of iron from feed or other oral sources is still of minor importance.

How should one select piglets for haemoglobin evaluation?

To determine an overall prevalence of sub-clinical or full-scale iron deficiency anaemia within a herd, blood samples should be taken from random piglets in random litters within the oldest litters of piglets in the farrowing unit.

In a herd with 5% of animals suffering from full-scale iron deficiency anaemia (<90 g Hb/l) a sample size of 20 animals should be sufficient to determine the prevalence of the deficiency with an allowable error of Âą 10%.

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How to test your selected piglets To determine the prevalence of sub-clinical or full-scale iron deficiency anaemia within the herd, a hematological examination is needed. Haematological examinations can be done by handheld photometer or a reference laboratory.

Method 1 - pen-side testing using Hb photometer: A single drop of blood is taken from the ear vein with a simple pin prick. The blood is introduced into a disposable cuvette by capillarity and then placed in the Hb photometer. Results for haemoglobin concentration are obtained in less than a minute.

Method 2 - lab testing using vacutainer tube: Approximately 3 ml blood sample is withdrawn from each piglet by puncture of vena cava or the jugular vein using an EDTA stabilized vacutainer tube. You should ice down the blood samples immediately following the sample collections and moved to refrigerated storage for safe keeping until ice-packed and shipped out to the testing laboratory. Laboratory testing should be conducted as soon as possible, but in any event completed within a 24-hour time frame.

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Pen-side blood test for diagnosing sub-clinical and full-scale iron deficiency anaemia Measuring the haemoglobin with the handheld device HemoCue 201+ Hb photometer is a fast and reliable way to determine the prevalence of sub-clinical and full-scale iron deficiency anaemia within the herd.9 Blood sampling for pen-side testing using HemoCue 201+: The HemoCue 201+ Hb photometer is technically and economically well suited for pen-side haemoglobin examination.

1. A single drop of blood is taken from the piglets to be tested by puncturing the ear vein.

2. The blood samples are introduced one-at-a-time into the disposable cuvettes.

3. The cuvettes are placed one-at-a-time into the HemoCue 201+ Hb photometer.

4. The individual assay results for the piglets being tested for blood haemoglobin concentration are each successively obtained in less than a minute after the blood samples are collected.

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Assay laboratory testing for diagnosing sub-clinical or full-scale iron deficiency anaemia If blood samples are drawn by puncture of the vena cava or the jugular vein, some additional and important assay parameters may be obtained.

Collect a 3 ml sample of whole blood from each of the piglets to be tested in EDTA stabilized and plain vacutainer tubes that will need to be iced down immediately after collection, and moved to refrigerated storage for safe keeping until ice-packed and shipped out for assay by an independent testing laboratory. The testing should be conducted as soon as possible, but in any event should be completed within a 24-hour time frame following collection of the blood samples.

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Tests

Significance

Notes

Blood haemoglobin assay results for piglets from birth to weaning can be utilised to determine a strategy ÂŽ Assay Values between 90 to 110 for dosing of Uniferon that will prevent or treat g/l are indicative of sub-clinical sub-clinical or full-scale IDA. IDA. Assay Values >110 g/l are indicative of optimal blood haemoglobin levels.

Blood Measurement of Hb in blood haemoglobin Assay Values <90 g/l are indicative of full-scale Iron Deficiency Anaemia (IDA).

Neither blood haemoglobin or hematocrit assay values Measurement of packed cell vol- should be used as ume requires the determination single diagnosis test of the volume occupied by RBC parameters; however after whole blood is centrifuged when these two test for approximately 6 minutes. parameters are used in A normal value is 0.45 l/l. conjunction, they are powerful tools for use in Values within a descending diagnosis of sub-clinical range of 0.41 to 0.26 l/l are and full-scale IDA. indicative of sub-clinical or full-scale IDA, respectively.

Hematocrit

Measures volume % of RBC in blood.

TIBC

Measures bloodâ&#x20AC;&#x2122;s capacity to bind iron with transferrin.

Indicates % of iron bound.

Values within the range of 104-188 mmol/l are indicative of IDA.

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What if my piglets get infected with a virus?

In many herds, the presence of a viral infection such as porcine reproductive and respiratory syndrome (PRRS) or porcine circovirus type 2 (PCV2) could represent overall health conditions that are conducive to the development of sub-clinical and/or full-scale IDA due to immunosuppression, in spite of the piglets having received appropriate injectable iron supplementation.

Trial results demonstrate increased weight gains and blood haemoglobin levels in viremic piglets treated with two 200 mg doses, versus the placebo piglets treated with only one 200 mg dose.6 Effect of a second UniferonÂŽ injection in immunosuppressed pigs

Weight gain (g/d)

473

2nd UniferonÂŽ injection Saline injection

Day 12-32

6.38

Haemoglobin (mmol/l)

6.62

523

Day 12-32

(Weight gain and blood haemoglobin results show statistical significance p=0.01*)

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• Sub-clinical and full scale IDA in piglets are conditions that are exacerbated due to viral infections • Adequate iron supplementation will likely require initial and follow-up injectable dosages that will support subsequent increases in blood haemoglobin levels in piglets, which will be quite helpful to the overall health conditions of the piglets in the presence of a viral infection • It appears that the follow-up doses of injectable iron enable the piglets to develop some measure of damage control when challenged with the immunosuppressed conditions encountered during viral infections

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What about vitamin E deficiency in the sows and their piglets?

As the nutritional status of the sow is the determinant for the vitamin E blood level for the piglets, management factors that optimize the vitamin E blood level for the sows are of significant importance and will help prevent the development of sub-clinical or full-scale IDA. The sow’s ability to absorb and transfer vitamin E to the milk varies somewhat from animal to animal. Variation in vitamin E uptake is also seen among piglets, and may even vary within the same litter of piglets.

I f the sows are vitamin E deficient this approach is recommended:

• Analyze and control the feed content of vitamin E

• Make sure that the gestation feed mixture contains a minimum of 80 mg Vitamin E / FUs and that the lactation feed mixture contains a minimum of 92 mg Vitamin E / FUs, where FUs is defined as Feeding Unit for swine

• Up to 250 mg/sow/day of the synthetic vitamin E (acetate bound) may be added to the gestation and lactation rations, but in those instances should be the only source of vitamin E supplementation

If the piglets are vitamin E deficient this approach is recommended:

• Provide natural vitamin E (peroral administration) to each piglet. It is recommended to use natural vitamin E for improved bioavailability 32

What about mycotoxins in swine feed?

There are various conditions which can cause iron absorption in pigs to be reduced. One of them is mycotoxins induced “sub-clinical or full-scale iron deficiency” anaemia.

Mycotoxins in feed Presence of Mycotoxins in swine feed is a well-known, world-wide problem, and it is much more prevalent in humid climates. For example, Fusarium toxins that develop in crops standing in the wet fields prior to harvest are transferred to the swine feeds. These toxins are not easily detoxified when consumed in swine rations, thus, swine production is decreased.

The reason for the reduced swine production is that the mycotoxins weaken the immune system, interfere with the operation of vital organs and inhibit the bone marrow production of RBCs, which leads to sub-clinical or full-scale IDA. The presence of mycotoxins in swine feeds not only weakens the immune system of the sows, but it may also prevent the uptake and assimilation of vitamin E in the feed. As vitamin E is essential for the uptake and assimilation of iron in both sows and piglets, the mycotoxins induced development of sub-clinical or full-scale IDA is enhanced by the reduced absorption of vitamin E in the sow’s ration.

In order to solve the mycotoxins problem, we recommend tracing the sources of the mycotoxins to particular feed components, the use of a mycotoxins mitigating substance to help prevent and/or offset the negative effects of molds and mycotoxins and additionally improve gut health.

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Uniferon® benefits Natural formula: Uniferon® is a strongly bound iron complex manufactured without the use of organic solvents or cyanide, avoiding trace residues of these components Global approval: Uniferon® is the only injectable iron for piglets approved by the medical authorities in Europe, Asia and by the Food and Drug Administration (FDA) in the US8,10 Faster growth: Uniferon® is research-proven to boost haemoglobin levels and average daily gain1,2 Complete absorption: Uniferon® contains high quality iron dextran that is completely absorbed by the piglet7 Human Standards: Uniferon® has a good safety profile and is manufactured to the same standards as those required for human medicine7 Uniferon® is the #1 iron supplement in the world

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Iron Expert Board Pharmacosmos has established the Iron Expert Board (IEB) with a dedicated panel of experts that consists of international Key Opinion Leaders, which represent some of the most widespread and up-to-date swine production experiences and the best intellectual aptitudes in the field of piglet health and iron supplementation.

The goal of the IEB is: • To ensure adequate iron supplementation for all piglets, thus, avoiding the “iron gap” • To widen the full understanding of sub-clinical and full-scale IDA, and the direct relationship between the optimization of blood haemoglobin and the optimization of growth and weight gains in piglets • To avoid inappropriate use of iron and toxic conditions encountered during “iron overload” • To make up-to-date recommendations for injectable iron supplementation for piglets in order to prevent or treat sub-clinical and full-scale IDA

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Pharmacovigilance Each national health care system has a pharmacovigilance system associated with the prescription and administration of medicines in humans and animals, which ensures a satisfactory handling of adverse events in compliance with local laws and guidelines.

Pharmacosmos has the overall responsibility for maintaining a highly compliant and skilled safety surveillance for Uniferon®.

Pharmacosmos has organized worldwide reporting of adverse events via Uniferon® partners that operate within compliance of local health care regulations. In parallel, Pharmacosmos systematically reviews the worldwide scientific literature for the Uniferon® product and is responsible for signal detection.

Comparison over time of the ratio of animals reported for adverse events/ lack of efficacy (ABON)7 Period Ratio (number of animals/number of doses) Period Ratio (number of animals/number of doses)

01.10.2010 – 01.05.2011 – 01.12.2011 – 01.06.2012 – 30.04.2011 30.11.2011 0 31.05.2012 30.11.2012 0.0000005

0.0000003

0.0000005

0.0000002

01.12.2012 – 01.12.2013 27.10.2010 – 30.11.2013 – 30.11.2014 27.02.2015 0.0000002

0.0000004

0.0000005

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The basics of adverse events associated with the administration of parenteral iron supplementation Intramuscular injections of parenteral iron products, in general, may cause transient discoloration/calcifications at the injection site, or hypersensitivity reactions normally considered non-serious adverse events.10 Intramuscular injections of parenteral iron products can very rarely cause a serious adverse event (deaths).

The most likely reasons for a serious adverse events following the administration of parenteral iron products are listed below:10 • Exposure to an intravenous injection • Iron shock associated with genetic factors • Vitamin E and/or selenium deficiencies • Contamination with Clostridia bacteria due to improper handling/ storage conditions The Active Pharmaceutical Ingredient (API) manufactured by Pharmacosmos is a 200 mg/ml solution of iron dextran.

On the rare occasion of an adverse event, the elimination of any likelihood of production related failure is of great concern to Pharmacosmos, whereby all possible scenarios are thoroughly evaluated in conjunction with any adverse event. Maximum care and caution have been exercised in Uniferon® manufacturing to achieve European MRP10 approval and FDA approval in the U.S.8 37

Resources To learn more about UniferonÂŽ and Iron Deficiency Anaemia please visit: uniferon.com Determine the potential mean iron need within a litter with our online Iron/Weight Gain Optimizer (I/WGO): uniferon.com/iwgo Determine the investment advantage of iron supplementation for your operation with our online F.A.C.T Calculator: fact.uniferon.com

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References 1 H augegaard, J. et al., 2008: ”Effect of supplementing fast-growing, late-weaned piglets twice with 200 mg iron dextran intra-musculary” The Pig Journal (2008) 61, 69 – 73 2 N ielsen, J.P. et al., 2013: “Herd diagnosis of iron deficiency in piglets”.

In: Proc. 5th ESPHM Cong., May 2013 3 N ielsen, J.P. & S. Bhattarai 2014: “Association of haematological status at weaning and weight gain post-weaning in piglets.”

In: Proc. 23rd IPVS Cong., June 2014 4 G addy, H.J. et al., 2012: “A review of recent supplemental iron industry practices and current usage of Uniferon® (iron dextran complex injection, 200 mg/ml) in piglets”. In: Proc. 43rd AASV Cong., March 2012 5 V an Gorp, S. et al., 2012: “Preventing iron deficiency by avoiding an iron gap in modern pig production”. In: Proc. 43rd AASV Cong., March 2012 6 B ach, J. et al., 2006: ”Effect of an additional iron injection in a PMWS positive herd in respect to hematocrit, haemoglobin and growth rate”.

In: Proc. 19th IPVS Cong., June 2006 7 P eriodic Safety Update Report (PSUR), MRP renewal.

Product: Veterinary iron dextran, solution for injection Period of reporting: 27.10.2010 – 27.02.2015 8 N ADA 134-708: Uniferon® 200 (Iron Dextran Complex, Injection, 200 mg/ml x 100 ml product) 9 http://www.hemocue.com/ 10 Procedure No. DK/V/0114/01/MR 11 T horn CE. Haematology of the pig. In: Weiss DJ, Wardrop JK, eds.

Schalm’s Veterinary Hematology. 6th ed. Ames, Iowa: Wiley-Blackwell; 2010:843 39

References 12 M artin, L.E., et al. “The pharmacology of an Iron-dextran intramuscular haematinic”. British Journal of Pharmacology and Chemotherapy, 1955; 10:375–382 13 U nited States Patent Office 3,536,696. Patented Oct. 27, 1970: Ferric hydroxide dextran and dextrin heptonic acids 14 R eflection paper on the data requirements for intravenous iron-based nano-colloidal products developed with reference to an innovator product EMA/CHMP/ SWP/620008/2012 15 C rommelin, Daan J.A. et al. ”Non-biological complex drugs – the science and the regulatory landscape”, Springer 2015 16 O lsen, C. et al.,2018: “Evaluation of the impact of iron dosage on post-weaning weight gain, and mortality” In: Proc. 49th AASV Cong., March 2018 17 F riendship, R. et al., 2018: “Are pigs with iron deficiency less able to defend against enterotoxigenic E. coli infection compared to pigs with adequate iron status?” In: Proc. 10th ESPHM. May 2018 18 R adke, S.L. et al. Elemental impurities in injectable iron products for swine. Journal of Swine Health and Production. 2018;26(3):142-145

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Uniferon® abbreviated prescribing information Before prescribing Uniferon® please refer to full local approved data sheet.

Presentation: Solution for injection. Each ml contains 200mg Iron(III) as iron(III) dextran complex and 5mg phenol as a preservative.

Indications: For the prevention and treatment of iron deficiency anaemia in piglets.

Contraindications: Do not use in piglets suspected to suffer from deficiency of vitamin E and/or selenium, in case of hypersensitive to the active substance, or in older pigs as meat staining may occur in pigs of >4 weeks of age.

Special warnings: None.

Special precautions for use: Normal aseptic injection techniques should be practiced. Self-injection should be avoided.

Adverse reactions: Very rarely deaths have occurred in piglets following the administration, which has been associated with genetic factors or deficiency of vitamin E and/or selenium or with an increased susceptibility to infection due to temporary blocking of the reticuloendothelial system. Hypersensitive reactions can occur and injection may cause transient discoloration and calcification at the injection site.

Interactions: May reduce the absorption of concomitantly administered oral iron.

Administration: Intramuscular (recommended) or subcutaneous route as 200mg of iron dextran per piglet. Prevention: a single injection at 1-4 day of age (UK/IE: deep intramuscular injection as a single dose (1ml) into the hind limb at 3-4 days of age). Treatment: a single injection. Uniferon® should not be mixed with other medicines or substances.

Withdrawal period(s): 0 days (UK/IE: for meat 28 days).

Pack sizes: 5 x 100ml, 12 x 100ml, 20 x 100ml, or 12 x 200ml (not all pack sizes may be marketed).

Date of preparation: 12/04/2016. Pharmacosmos A/S, Roervangsvej 30, DK-4300 Holbaek, Denmark.

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Contacts Pharmacosmos Corporate Headquarters: Pharmacosmos A/S Roervangsvej 30 DK-4300 Holbaek Denmark T: +45 59 48 59 59 F: +45 59 48 59 60 E: uniferon@pharmacosmos.com If you have any questions, or want to share your thoughts or advice, we would like to hear from you.

For a complete global UniferonÂŽ partners please visit: uniferon.com

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T: +45 59 48 59 59 E: uniferon@pharmacosmos.com

V2015-03 v 5.0

#1 iron supplement in the world

T: +45 59 48 59 59 E: uniferon@pharmacosmos.com