AU711760B2 - Controlled release vitamin B12 compositions and methods of using them - Google Patents

Description

TO 006126285359 p. 0:3/27 18-DEC-1998 17:46 FROM R.J. PRRK SON TO 0061262853593 P. 03/27 Regulation 3,2

AUSTRALIA

PATENTS ACT, 1990 COMPLETE

SPECIFICATION

FOR A STANDARD

PATENT

ORIGINAL

4 .4 4 4 4 Name of Applicant: Actual Inventor: Address for service in Australia: Invention Title: NEW ZEALAND PASTORAL AGRICULTURE

RESEARCH

INSTITUTE

LIMITED

DANNY H LEWIS, KEVIN S BURKE, NEVILLE

DONOVAN

GRACE

A J PARK SON, Level 11, 60 Marcus Clarke Street, Canberra ACT 2601 CONTROLLED RELEASE VITAMIN

B,.

2

COMPOSITIONS

AND METHODS OF USING THEM The followng statement is a full dcsoriptioft of this invention, including the best rnethad of puifbrmring it known to melus i 18-DEC-1998 17:46 FROM A.J. PRRK SON TO 0061262853593 P.04/27 2 CONTROLLED RELEASE VITAMIN B, COMPOSITIONS

AND

METHODS OF USING THEM This invention relates to controlled release compositions containing vitamin B,.

It also relates to methods of increasing and maintaining serum vitamin B 1 2 concentrations and liver stores of vitamin B 2 in animals, particularly ruminants, and humans.

Cobalt deficiency is a major problem in livestock worldwide. Responses to Co supplementation have been reported in many countries including the UK, USA, South Africa, Brazil, Australia and New Zealand, see for example Journal of Department of Agriculture of Western Australia 13:199-201, 1936, and The New Zealand Veterinary Journal 14:191-196, 1966. The Co requirements of sheep are about double those for cattle (New Zealand Journal ofAgricultural Research 29:443-448, 1986), with the young lamb being the most sensitive to Co deficiency.

Cobalt is required by the rumen microorganisms for the synthesis of vitamin B 1 2 (hydroxocobalamin hydrochloride) (British Journal of Nutrition 24:857-877, 1970).

Vitamin B, 2 is then absorbed and acts as a cofactor for two enzymes: methylmalonyl CoA mutase and methionine synthase (Biological Trace Element Research 22:153-164, 1989, Journal of Nutrition 122:1384-1390, 1992). The conversion of propionic acid to glucose via succinic acid and the tricarboxylic acid cycle requires methylmalonyl CoA mutase, and the transfer of methyl groups from homocysteine to methionine is catalysed by methionine synthase. Co deficiency therefore disrupts the energy and protein metabolism of ruminants.

Increasing the Co intakes by an annual application of 175-350 g CoSO 4 7H7O/ha is an effective method for preventing Co deficiency in grazing animals where fertilisers are applied regularly. However the direct treatment of grazing animals to prevent Co S0. deficiency is more difficult and labour intensive as weekly and fortnightly doses of 7 mg and 28 mg Co respectively are needed, and subcutaneous injections of soluble vitamin

B

1 preparations at a rate of 0.05 to 0.1 mg vitamin B 2 /kg liveweight are only effective for up to 4 to 6 weeks when assessed in terms of liveweight gains and elevated blood vitamin B 12 concentrations.

Accordingly, it would be desirable to have an alternative method useful for the long term prevention of vitamin B, 2 deficiency in animals. It would also be desirable to have a means of elevating vitamin B, levels in a human for an extended period of M time, as a means of treating disorders such as pernicious anaemia, multiple sclerosis and neurological disorders; and to supplement HIV sufferers who have been demonstrated to be deficient in vitamin BI 2 It is an object of the present invention to go some way towards achieving the above desiderata or at least to offer the public a useful choice.

Accordingly, in a first aspect the present invention may broadly be said to consist in a controlled release composition suitable for administration to an animal, comprising biodegradable microparticles comprising micronised vitamin B 12 incorporated in a polymeric matrix, wherein the matrix comprises an aliphatic polyester such as a polylactide, a polyglycolide or a copolymer of lactic and glycolic acids and polycaprolactone and copolymers thereof with lactic or glycolic acids, wherein the amount of vitamin B 12 present in the polymeric matrix and the release rate of vitamin B 12 from the polymeric matrix is such that at least 0.33pg-1.25tg vitamin B 1 2 per kg liveweight per day is released over at least 50 days which is sufficient to provide the animal with its daily vitamin B 1 2 requirements, and also to elevate the blood serum vitamin B 1 2 level of the animal and maintain an elevated vitamin B 1 level for said period of time.

Preferably said composition is formulated ready-for-use, thereby avoiding the need of the user to engage in preparatory steps such as reconstitution.

Preferably said composition is in an injectable form.

'.25 Preferably said composition is adapted to release said vitamin B 12 at a near uniform rate.

Preferably the polymeric matrix comprises poly(lactide-co-glycolide) copolymer, :-30 more preferably a 95:5 molar ratio of poly(lactide-co-glycolide) copolymer, or polylactide polymer alone having an inherent viscosity in chloroform of about 0.70 or 0.80 dl/g at 30 0

C.

Preferably the composition comprises a microcapsule composition wherein vitamin B, 2 is present in an amount of about 5 to about 25% by weight of the 18-DEC-V398 17:47 FROM R.J. PRRf< SON TO 006 1262853533 P. 06/27 18-DEC-1998 17:47 FROM R.J. PRK SON TO 006126853593 P.06/27 4 composition.

More preferably, the vitamin B 12 is present in an amount of about 9 to about by weight, most preferably about 12-13% by weight.

Preferably, the composition comprises sufficient vitamin to maintain a release rate of at least 0.33/g vitamin per kg liveweight of the animal per day for a sufficient length of time to maintain the elevated blood serum vitamin B, 1 levels and increased liver vitamin concentrations for at least about 200 days, most preferably about 360 days.

In a further aspect the present invention may broadly be said to consist in a controlled release composition suitable for administration to an animal, comprising injectable, biodegradable microparticles comprising vitamin Bt incorporated in a polymeric matrix comprising a poly(lactide-co-glycolide) copolymer, and wherein the vitamin is present in an amount of about 5 to 50% by weight of the composition, preferably about 7 to about In a further aspect the present invention may broadly be said to consist in a method of increasing vitamin B 2 blood serum levels and liver vitamin B 1 concentrations in an animal or human comprising administering to the animal or human a controlled release composition comprising injectable, biodegradable microparticles comprising vitamin B 12 S: incorporated in a polymeric matrix, wherein the matrix comprises an aliphatic polyester such as a polylactide, a polyglycolide or a copolymer of lactic and glycolic acids and '"2S polycaprolactone and copolymers thereof with lactic or glycolic acids, wherein the amount of vitamin B 12 present in the composition and the release rate of vitamin B ,,from the composition are sufficient to provide the animal or human with its daily vitamin B,2 requirements as well as elevating the blood serum vitamin level and maintaining an elevated blood serum vitamin B 1 2 level for a predetermined period of time.

3Q Preferably the amount of vitamin B, 1 in the composition and the release rate are sufficient to maintain the elevated blood serum vitamin level for at least 50 days, preferably at least 360 days after treatment.

Preferably, the composition provides a sufficient dose of vitamin B, 2 and a release 4 rate of vitamin from the composition such that at least about lpg vitamin B 1 2 per kg liveweight is released per day for a sufficient length of time to maintain the elevated vitamin B, levels for at least about 50 days, more preferably about 360 days.

18-DEC-1998 17:47 FROM R.J. PRRK SON TO 0061262853593 P.07/27 More preferably, the composition provides a depot of about 0.1 to about 0.4 mg vitamin per kg liveweight, and the release rate of vitamin Bi from the composition is such that from about 0.33 /zg to about 1.25 Ag vitamin B 1 per kg liveweight is released in vivo per day.

In one preferred embodiment of the invention the animals are sheep. In this embodiment, preferably sufficient composition is administered to provide about 6- 8mg vitamin

B

1 per animal, and about 20 4g to about 25 ,g vitamin

B,

2 is released from the composition per animal per day.

Preferably the composition is prepared and packaged ready-to-use for injection by the subcutaneous or intramuscular route.

Preferably said composition is stable for at least 24 months.

Preferably said composition further includes an oil based physiologically acceptable non-aqueous suspension medium. The suspension medium preferably comprises a vehicle for injection that will protect the polymer matrix from breakdown through hydration.

Preferably said oil based suspension medium is peanut oil.

Preferably said oil based suspension medium contains a physiologically acceptable resuspending agent.

"..2S Preferably said resuspending agent is beeswax.

S* More preferably said beeswax is of a suitable pharmaceutical grade.

a. Most preferably the composition comprises a polymeric matrix comprising a 95:5 molar ratio of poly(lactide-co-glycolide) copolymer, or alternatively, solely polylactide polymer having an inherent viscosity in chloroform of about 0.70 or 0.80 dl/g at and a concentration of vitamin of about 12-13% by weight of the composition.

3 In another embodiment the invention may be said broadly to consist in a method of formulating a composition as defined herein above, the method comprising the steps of: 18-DEC-1998 17:48 FROM R.J. PARK a SON TO 006126285:359:3 P.08/27 18-DEC-1998 17:48 FROM R.J. PRRK SON TO 0061262853593 P.08/27 6 dissolving a polymer in a suitable organic solvent, adding micronised hydroxocobalamin HTC1 to said solution and forming a suspension thereof in said solution, emulsifying said suspension in water and removing said organic solvent by extraction or evaporation, and recovering rnicrospheres containing vitamin B 12 therefrom.

The recovered microspheres may then be suspended in a physiologically acceptable suspension medium.

Preferably the microspheres are resuspended in a suspension medium containing a physiologically acceptable resuspending agent ini order to provide a ready-to-use preparation.

Preferably said polymer is either a poly(Iactide-co-glycolide) co-polymer or a polylactide polymner.

Preferably said solvent is ethylacetate.

0: Preferably said suspension is emulsified with 3% polyvinyl alcohol in aqueous solution.

Preferably said suspension remains emulsified in water until said microspheres are hardened.

Preferably said suspension medium is peanut oil.

39 Preferably said resuspension agent is beeswax.

a The invention further provides a product produced by the aforemnentionled method.

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be i: i 18-DEC-1998 17:48 FROM A.J. PARK SON TO 0061262853593 P.09/27 7 incorporated herein as if individually set forth.

The invention consists in the foregoing and also envisages constructions of which the following gives examples.

Preferred forms of the invention will now be described in more detail.

Vitamin is water soluble, stable and has a molecular weight of 1355 Daltons.

The applicants have discovered that vitamin B1, can be incorporated into biodegradable microparticles to provide controlled release compositions. The compositions may be injected subcutaneously or intramuscularly, as a means of increasing blood serum vitamin levels in animals or humans, and also increasing liver vitamin BI, storage.

The compositions and methods of the invention may be used, for example, to prevent or treat cobalt deficiency in grazing animals, such as sheep and cattle. The applicants have discovered that controlled release compositions, which release vitamin B, 2 at a rate such that at least about 0.33 ug vitamin B 12 per kg liveweight per day is provided to the animal, will provide the animal's daily vitamin requirements and also significantly elevate blood serum vitamin Bt 2 levels, and increase liver storage of vitamin Bi2, It is possible, using the preferred composition of the invention, to maintain the elevated vitamin B,2 levels for as long as 12 months, or even longer, with a single dose. Although vitamin B 12 is relatively non-toxic and it is possible to administer to an animal many times the daily requirement of vitamin B, 2 without adverse effects (as the excess is simply excreted), this is wasteful of vitamin B2. Accordingly, it is preferred the composition provides between about lg and about 1.254g vitamin B 1 per kg liveweight per day.

The compositions of the invention and methods of administering the same have been found to be effective in increasing and maintaining vitamin levels in sheep at an elevated level for extended periods of time, up to about 12 months. Previously available soluble vitamin preparations which provide 0.05 to 0.1 mg vitamin BI 2 /kg liveweight are only effective for about 4 to 6 weeks.

Preparation ofmicroparticle compositions: Aliphatic polyesters such as lactide and glycolide polymers and copolymers are well known in the art, as is their use for the delivery of active agents such as growthpromoting steroids. Reference is made in this regard to US Patent Nos. 5,288,496, 4,389,330 and 4,530,840, the contents of which are herein incorporated by reference.

These references teach the preparation of injectable, biodegradable microparticles TO 0061262855593 P. 10/27 1B-DEC-1998 17:49 FROM fl.J. PFRK SON TO 06162853593 P. 10/27 Y50.

*0* 30 a a..

a comprising a polymeric matrix comprising a polylactide, a polyglycolide or a copolymer of lactic and glycolic acids, and containing various active agents that are not water soluble. The methods disclosed in US 5288496, with substitution of vitamin

B

12 as the active agent, are the basis from which the methods used to prepare microparticles for inclusion into the compositions according to the present invention were developed. The processing conditions and formulation parameters were modified to reduce the rate at which the polymer would hydrate upon administration so as to reduce the rate of leaching of the vitamin and thereby extend the duration of release.

In preferred embodiments of the invention, as noted above, the compositions are designed, to provide a release rate such that at least about 0.33Mg vitamin

B

1 is released per kg liveweight per day, more preferably from about 1 to about 1.25 jg vitamin Bt 2 per kg liveweight per day. This is based on experimental observations that the daily net vitamin requirement for young sheep is 0.44 ug vitamin per kg liveweight (British Journal of Nutrition 24:615-633, 1970), and the ability for vitamin to be stored in tissues, especially the liver. Sufficient vitamin Bz 1 is provided in a single dose of the composition to maintain elevated levels of vitamin for at least about 50 days, more preferably 360 days. When the animals are sheep, this translates to a dosage size of about 6-8mg vitamin B, 2 and a release rate of from about 20 ,g to about 25 4g vitamin B, 2 per day per animal.

In the methods according to the present invention farm animals, such as sheep.

cattle, deer, horses, goats and pigs, are injected with the controlled release vitamin B 2 compositions according to the invention. In preferred embodiments, the animals are injected subcutaneously or intramuscularly with a suspension of the microparticle composition. Conveniently, peanut oil may be used to reconstitute the microparticles but other suitable vehicles will be apparent to those skilled in the art. Peanut oil is the preferred vehicle for injection of said microparticles.

It is beneficial to add a resuspending agent to the oil to assist in keeping the microspheres in suspension. A preferred resuspending agent is bees wax.

The following examples describe the preparation of specific controlled release compositions comprising injectable, biodegradable microparticles containing vitamin B,, according to the present invention.

TO 00~1262855593 P. 11/27 18-DEC-1998 17:49 FROM R.J. PARK SON TO 006126253593 P. 11/27 9 nample 1 Preparation of Microaprticle Composition: This example describes the procedure for preparing a microparticle composition according to the present invention in which hydroxocobalamin hydrochloride is present in an amount of 9.4% by weight and the excipient is a 95:5 molar ratio ofpoly(lactideco-glycolide) copolymer having an inherent viscosity in chloroform of 0.70 dlg at The copolymer, 6.76 g (Medisorb) was dissolved in 51,2 g of ethyl acetate (Fisher Scientific) in a 125 ml Erlenmeyer flask. Micronized hydroxocobalarin HCI (Sigma), 0.75 g, was placed in a 50cc centrifuge tube and chilled in an ice bath. About 45 ml of the polymer solution was added to the centrifuge tube containing hydroxocobalamin HC1 and was cooled for 10 minutes. The contents of the centrifuge tube were then sonicated (Techmer TM375,1/4" microtip) for two minutes. The resulting drug suspension was added to the remainder of polymer solution in the Erlenmeyer flask, followed by the addition of 16.2 g of ethyl acetate which was used to rinse the remaining drug from the centrifuge tube. The polymeric drug suspension was cooled in the ice bath for an additional 10 minutes.

To 151.4 g of a 3% PVA aqueous solution (polyvinyl alcohol, Air Products), g of ethyl acetate was added. The aqueous solution was placed in a 350 ml jacketed glass reactor equipped with a 1" teflon turbine impeller driven by an overhead stirrer motor.

The jacket of the reactor was connected to a circulator immersed in an ice bath. The S. impeller stirring speed was adjusted to 411 RPM.

"'The polymeric drug suspension was quickly added to the PVA solution to form an oil-in-water emulsion. Additional 3% PVA, 13.8 g, was used to rinse the flask containing the drug suspension and was also added to the reactor. After approximately 1 minute of stirring the emulsion was drained from the reactor by gravity through an opened stopcock into 3.8 litres of 8*C water in a 4 litre stainless steel beaker while stirring. The microspheres were hardened for about 1 hour- The resultant product was collected on a 25 micron sieve screen and then transferred into 2 litres of 9C water in a stainless steel beaker, while stirring for 3 hours as a final wash. The microspheres, having a size range of 25-212 microns, were collected on sieve screens and were air dried for about 18 hours. The product yield was 93%. The microspheres contained 9.4% hydroxocobalamin HCI by weight. Peanut oil containing 1% beeswax was then added to achieve a vitamin B, concentration in the resulting suspension for 6mg/ml.

P.12/27 18-DEC-199B 17:49 FROM R.J. PARK SON T 016839 TO 0061262953593 P. 12/27 a~imple 2: Preprat"On of 95-4 Micrpartide Composition The preparation techniques described above in Example 1 were used to prepare a micropafticle composition according to the present invention containing hydroxocobalalnrn hydrochlooide in an amount of 15.9% by weight, and the excipient is a 95:5 molar ratio of poly(1atide-co-gycolide) copolymner.

Rx~nmple It Preparation of MAO BIeud CnMpositioit A 50:50 blend of the compositions obtained using the procedures of Examples 1 and 2 was prepared, thereby obtaining a microparticic composition according to the present inventijon containing hydroxocobalamfin hydrochloride in an amount of 12.5% by weight.

Example 4! restmeflt pf Aixmalq wth mlcrnparticlp formltionst The microparticlc formulations were prepared according to Examples 1, 2 and 3 above. The microparticles were reconstituted in peanut oil, and a 0.5 ml dose, sufficient 7 to provide about 6-8 mg of vitamin B 12 was prepared.

Animals From 300 castrated Romney lambs, having a mean liveweight of 20 kg, 70 animals were selected on liveweight and randomly placed into 7 groups of 10 animals, The lambs were identified by eartags, injected with the various vitamin treatments as described below and then grazed as a single group.

Treament 2; Two groups of 10 animals were injected intramuscularly with the doses of compositions of either Examples 1, 2 or 3. The control group were injected only with 0.5 ml peanut oil.

All animals were bled from the jugular vein using a 10 ml vacutainer just prior to 39 the injection and then atdays 4 3 0,50, 8 5, 114, 151, 182, 216, 250, 280 and3l10 after treatment.

After centrifuging at 2500 g for 20 minutes the serum was removed and stored at for vitamin 1 2 determinations. On day 310 all lamnbs were slaughtered, and their 3S ~livers removed, weighed and subsarnpled for vitamin 13,.

Pasture samples were collected at 2-3 monthly intervals for Co determination and the lambs were weighed at 2 monthly intervals.

18-DEC-1998 17:50 FROM A.J. PARK SON TO 0061262853593 P.13/27 11 Analytical method The vitamin B 1 2 concentrations in the serum and liver were determined using the Becton Dickinson radioassay method and pasture Co by graphite furnace atomic absorption spectroscopy New Zealand Veterinary Journal 32:105-108 (1984), New Zealand Journal of Agricultural Research 29:443-448 (1986).

The significant differences between treatments were determined by repeated measures analysis of variance.

Results The mean pasture Co concentration was 0,12 mg/kg DM. There were no significant differences in liveweight gains between treatment groups and the overall mean gain was 112±6.2 g/day over the 310 day study with the fastest gains occurring in late spring.

A significant response in growth rates to vitamin supplementation would only be expected on Co deficient pasture, that is, pastures with Co concentrations of less than 0.08 mg/kg DM.

The effect of the vitamin B 2 injections on the serum vitamin B, concentrations are shown in Table 1. At day 14 only in the case of the Example 3, Group 1 sheep were the serum vitamin B 2 concentrations significantly higher (P<0.05) than the other treated and untreated animals. From days 30 to 250 the serum vitamin B, concentrations of the Example 2, Groups 1 and 2, and Example 3, Group 1 sheep were significantly higher 5: (P<0.01) than the untreated control animals while similar pattern was observed for the Example 3, Group 2 sheep from days 50 to 250. Significantly increased serum vitamin concentrations were found at days 85 and from days 182 to 280 for the Example 1, Group 2 sheep and from days 182 to 216 for the Example 1, Group 1 sheep. No differences were found between any treatment on days 280 and 310.

The influence of vitamin injections on the liver storage of vitamin B 12 at day 310 is shown in Table 2. The Example 3, Groups 1 and 2 sheep had significantly higher (P<0.05) hepatic stores of vitamin when compared to the untreated control lambs, 0 bee '0*0 we.

~0 0 0 0 0** 0 0 *0 Table 1. Effect of injectable vitamin B, 2 microsphere preparations on the mean serum vitamin B. concentrations (pmole/f1) of lambs.

Days after injection 0 14 30 50 85 114 151 182 216 250 280 310 Control 968 1302' 987A 695A 1511A 807A 1255A 867A 1966- 770A 683 440 Example I Group 1 699 1056' 1397A 1297A 1960A 1104A 1541 250 0 b 4700S 1078 A 681 398 Example 1 Group 2 855 1131' 1 1 1 6 A 129 2 A 2 3 00 10 9 7 A 1550 A 2720 B 4950 B 1350P 706 393 Example 2 Group I 840 1781' 2148B 2200 3788" 2522 2855 s 2 43 3 6 5 0 33 B 1322 694 407 Example 2- Group 2 524 1354" 20578 1842B 3985" 2628B 3314" 3546 B 5500° 1757 B 962 485 Example 3 Group 1 1300 2 0 2 8 b 1957B 2014° 3485" 2142" 26008 3128 B 4214 s 1371" 762 440 Example 3 Group 2 681 1642" 1600 1885 B 3657B 2 1 42 2300B 2485b 56140 1374" 778 398 Within a column means with different superscripts are significantly different avb P<0.05 AVB P<0.01.

I i lit! I S S. S S S S S S S S

S

S S S S S. 13 Table 2. Effect of injectable vitamnin B 12 microsphere preparations on the mean (L standard error) liver vitaminm B 12 content (nol/lIkg fresh tissue) of lamnbs after 3 10 days.

C Ub

M

n- Liver vitamin B 1 nmole/kg fresh tissue Control Example 1 Group I Example 1 Group 2 Example 2 Group 1 Example 2 Group 2 Example 3 Group 1 Example 3 Group 2 368±40.94 43 5±39.

419±23.63 496:L80.1la 48&L29.9" 625±4 8 3 b 5 37=15 0. 1 Mean with different superscripts significantly different at P<0,05.

I HV' !i IIr I i

U)

0062.855. I 16/27 18-DEC-1998 17:51 FROM R.J. PARK SON TO 006162853593 P. 16/27 14 Example 5: Treatment of Animals Varying Treatment Parameters This was to determine the effect of the following parameters on serum vitamin B 1 concentrations.

1. The route of administration eg. subcutaneous (SC) v intramuscular

(IM)

injection.

2. The efficacy of formulations eg. 95:5 copolymer (lactide/glycolide) and 100 polymer (lactide).

3. The size of vitamin BI, depot eg. 95:5 (6 and 9 mg) or 100: (9 v 12 mg).

4. The suspending agent, namely beeswax.

Animals Three hundred lambs, aged about 6 months, with a mean liveweight of 30 kg and a mean serum vitamin B 1 2 concentration of 850 pmole/1 were used for the study. On the basis of serum vitamin B,2 concentrations and liveweight 13 groups of 10 lambs/group were randomly selected and eartagged for identification. The lambs were managed and grazed as a single flock.

Treatments The following treatments were administered.

.i Group 1 Control: 1 ml of peanut oil only intramuscular (IM) Group 2 Control: 1 ml of peanut oil containing 1% beeswax (IM) Group 3: 95:5; 6 mg in 1 ml peanut oil (IM) Group 4: 95:5; 9 mg in 1 ml peanut oil (IM) Group 5: 100: 9 mg in 1 ml peanut oil (IM) Group 6: 100: 12 mg in 1 ml peanut oil (IM) Group 7: 95:5; 6 mg in 1 ml peanut oil subcutaneous (SC) Group 8: 95:5; 9 mg in 1 ml peanut oil (SC) Group 9: 100; 12 mg in 1 ml peanut oil (SC) Group 10: 95:5; 6 mg in 1 ml peanut oil with 1% beeswax (IM) Group 11: 95:5; 6 mg in 1 ml peanut oil with 1% beeswax (SC) Group 12: 100; 12 mg in 1 ml peanut oil with 1% beeswax (IM) Group 13: 100; 12 mg in 1 ml peanut oil with 1% beeswax (SC) 18-DEC-1998 17:51 FROM R.J. PARK SON TO 0061262853593 P.17/27 Preparation of Vitamin BI Composition Peanut oil was the vehicle for administration. To ensure that the vitamin Bl,/polymer or the encapsulated vitamin B, 2 remained in suspension. 1% of beeswax was added to the oil peanut to prevent the vitamin B, 1 /polymer from settling to the bottom of the vial thus making it difficult to accurately administer the required dose of vitamin B, 2 To prepare the peanut/beeswax mixture 1g of beeswax was dissolved into 100 ml oil at a temperature of 75'C, then allowed to cool in room temperature before adding the vitamin B/polymer. The 95:5 formulation contained 11.2% vitamin BI 2 and the 100 formulation contained 14.3% vitamin B t, For each group doses of vitamin B 1 2 /polymer were calculated and weighed out to give the appropriate depot size (ie. 6, 9 or 12 mg) for each formulation before being mixed with 10 ml of the 1% beeswax peanut oil mixture. For example to make up doses using the 95:5 formulation containing 11.2% vitamin B to provide a depot of 6 mg vitamin B, 2 /sheep/dose 535.7 mg of the 95:5 formulation was mixed with ml of 1% beeswax peanut oil. A 1ml dose then provided a depot of 6 mg of vitamin B, 2 /sheep/dose.

Treatment and sampling protocols All lambs were bled just prior to the injections with the various vitamin B 12 treatments and then at days 15, 27, 47, 68, 83, 96, 115, 142 and 180 after the injection to collect sera for vitamin B 1 2 determinations. At 96 days lambs from Group 2 (1 ml peanut oil containing 1% beeswax IM) and Group 11 (95:5 6 mg in peanut oil 1% beeswax SC) were slaughtered and their livers removed for vitamin Bn 2 determinations.

Results and Discussion "The mean serum vitamin B, 2 concentration of the control lambs was 638 pmole/l. All of the vitamin treatments increased serum vitamin B 1 2 concentrations to greater than 2600 pmole/l by day 15 after which the concentrations slowly decreased but remained significantly higher (p<0.01) than the untreated controls at day 96 (for example 553 v 962 pmole/1) and at day 180 (281 v 530 pmole/1). This was associated with a significant increase in the vitamin B12 stores of the liver (265 v 478 nmole/kg fresh tissue). The 18-DEC-1998 17:52 FROM fl.J. PRRK SON TO 006126285:3593 P.18/27 18-DEC-1998 1?:52 FROM A.J. PARK SON TO 0061262853593 P.19/27 16 following preliminary conclusions can be made.

1. Beeswax is an effective agent to keep the encapsulated vitamin B, in suspension. It has no effect on the release of vitamin BI, from the injected vitamin B 12 copolymner depot, 2. There is no difference between subcutaneous and intramuscular methods of injection on the rate of release of vitamin B 12 The subcutaneous injection is preferred because many animal remedies and vaccines are administered subcutaneously by farmers and veterinarians.

3. To date the 95:5 (lactide/glycolide) formulation providing a 6 mg depot of vitamin B12 administered subcutaneously is the preferred treatment according to the invention to prevent Co deficiency in grazing livestock.

is This was to determine the effect of injecting ewes during mating with 12mg vitamin B, 2 long acting formulation (95:5 lactide/glycolide containing 11.2% Vitamin B3 12 on: serum Vitamin B 2 concentrations during pregnancy and lactation in ewes.

liver storage of Vitamin B in pregnant ewes just prior to parturition.

liver storage of Vitamin B 2 in foetuses just prior to parturition.

milk Vitamin B 12 concentrations during lactation.

serum Vitamin B1 2 concentrations in suckling lambs.

liver storage of Vitamin B 12 in suckling lambs.

Methods 3QAt the time of mating 2 groups of 28 mixed age ewes, with a mean liveweight of S4 kg, were randomnly selected from 300 animals, Ewes were identified with eartags and run as a single flock up to lambing. At lambing the control untreated and the Vitamin B 12 treated ewe were separated for lamibing so that lambs from untreated and treated ewes could be readily identified. After *3i lamnbing all ewes and lambs will be run as a single flock until weaning.

The control ewes were injected with 1 ml of peanut oil/beeswax I_.1iI ti ,Wii nI 18-DEC-1998 17:52 FROM R.J. PRRK SON TO 00612~2B5- r. 1 r 17 intramuscularly while treated ewes were injected with 1 ml peanut oil/beeswax containing 12 mg Vitamin B 12 encapsulated in 95:5 lactide/glycolide polymer prepared by the method of example 1. Injections were given during mating, days preparturition and 40 days postparturition.

Blood samples were taken just prior to the injections and at 15, 27, 47, 68, 83, 96, 115, 140 and 178 days after the first injection, This covers the period from mating to late lactation. Gestation length in ewes is 145 days. At 115 days 8 ewes from each group were put down using a captive bolt gun and their liver and the livers of their foetuses removed.

Preliminary Results The long acting Vitamin B 12 significantly increased (p<0.01) the serum Vitamin concentrations (1430 v 2460 nmole/l) in the ewes during pregnancy and lactation and the storage of Vitamin B 2 in the livers (813 v 1455 nmole/kg fresh tissue) of the ewes and their foetuses (354 v 873 nmole/kg fresh tissue).

Further, the milk vitamin B, 2 concentrations (3680 v 5320 pmole/1) were increased at day 60 of the lactation, while liver vitamin B 2 stores (192 v 279 nmole/kg fresh tissue) were increased in 30 day old lambs.

Conclusion Increasing the Vitamin Bi 2 status of pregnant ewes increased the Vitamin

B

12 status of their new born lambs.

GENERAL OBSERVATIONS The efficacy of the injectable vitamin formulations was assessed on their ability to significantly increase and maintain serum vitamin Bi, concentrations above those of the untreated controls. The relationship between growth rates and serum Vitamin B, 2 levels in sheep has been well documented (New Zealand Veterinary Journal 33: 1-5, 1985). It will be seen that all of the treatments were effective in elevating vitamin B 12 levels for extended periods of time. The microparticle compositions according to the present invention, having selected depot sizes of 6-12 mg and daily release rates of 20-25.g from the vitamin B 2 incorporated in the microspheres, were effective in meeting daily vitamin B 1 2 requirements (British Journal ofNutrition 24:615-633, 1970) as well as increasing the vitamin B 1 2 status of the sheep for 8-9 months.

18-DEC-1998 17:52 FROM R.J. PARK SON TO 0061262853593 P.20/27 18 71 The appropriate daily release rates of vitamin

B

12 can be calculated from the changes in serum vitamin B This approach is the best way of determining the daily vitamin

B,

1 release rates because the animal biochemical and physiological factors which can and do influence the release of vitamin as reflected by changes in serum vitamin Bt, concentrations, are taken into consideration.

Using the data from the vitamin

B

2 formulations described in Examples 2 and 3 (Table 1) the daily vitamin B 12 release rates were calculated by dividing the depot size or amount of vitamin

B

1 2 injected (6000 gg) by the period over which the serum vitamin

B

12 concentrations were significantly elevated (250 days). The calculated daily release rates of vitamin B 2 was 24 /g.

As presently available injectable vitamin preparations are only effective for 4-6 weeks, the injectable vitamin B 12 preparations according to the present invention, which are effective for much longer periods, and up to 8-9 months in preferred embodiments of the invention, are believed to be a major step forward in the management and prevention of Co deficiency in grazing animals, in situations where applying Co containing fertilisers and supplements is not a practical or economic solution. The 8-9 month injectable vitamin means that ewes and lambs need only be treated about once or twice a year and this operation S: could be fitted in with the timing of a number of husbandry practices such as drenching and vaccinating. The treatment of ewes, grazing Co deficient pastures, 6-8 weeks prior to mating would ensure an increase in the storage of vitamin B,, in the foetal liver while the increase in the vitamin B, 2 content of colostrum and milk together with the increased liver vitamin B2 stores would continue to prevent Co deficiency in the 30 day old suckling lamb. At tailing or at weaning, the treatment of 4 week or 12-16 week old lambs respectively, which are most sensitive to Co deficiency, would then protect for a further 8-9 months.

3 It is envisaged that the vitamin B 12 compositions and methods according to the present invention may also prove useful in medical applications in humans, such as for treatment of pernicious anaemia, multiple sclerosis and neurological disorders.

o

Claims (44)

1. A controlled release composition suitable for administration to an animal, comprising biodegradable microparticles comprising vitamin B 12 incorporated in a polymeric matrix, wherein the matrix comprises an aliphatic polyester such as a polylactide, a polyglycolide or a copolymer of lactic and glycolic acids and polycaprolactone and copolymers thereof with lactic or glycolic acids, wherein the amount of vitamin B 12 present in the composition and the release rate of vitamin B 12 from the composition is such that at least 0.33ug-1.25ug vitamin B 12 per kg liveweight per day is released over at least 50 days which is sufficient to provide the animal with its daily vitamin B12 requirements, and also to elevate the blood serum vitamin Bl 2 level of the animal and maintain an elevated vitamin B 12 level for said period of time.

2. A controlled release composition according to claim 1, which is formulated ready-for-use.

3. A controlled release composition according to claim 1 or 2, which is in an injectable form.

4. A controlled release composition according to any preceding claim which is adapted to release said vitamin B 1 2 at a near uniform rate.

5. A controlled release composition according to any preceding claim wherein :2 5 the polymeric matrix comprises poly(lactide-co-glycolide) copolymer.

6. A controlled release composition according to claim 5 wherein the polymeric matrix comprises a 95:5 molar ratio of poly(lactide-co-glycolide) copolymer, or polylactide polymer alone having an inherent viscosity in chloroform of about 0.70 dl/g at

7. A controlled release composition according to any preceding claim comprising a microcapsule composition wherein vitamin B 12 is present in an amount of about 5 to about 25% by weight of the composition.

8. A controlled release composition according to claim 7, wherein the vitamin B 1 2 is present in an amount of about 9 to about 20% by weight.

9. A controlled release composition according to claim 7 or 8, wherein the vitamin Bl 2 is present in an amount of about 12-13% by weight.

A controlled release composition according to claim 1 comprising sufficient vitamin B 12 to maintain a release rate of at least 0.33/ag vitamin B 1 2 per kg liveweight of the animal per day for a sufficient length of time to maintain the elevated blood serum vitamin B 12 levels and increased liver vitamin B 1 2 concentrations for at least about 200 days.

11. A controlled release composition according to claim 10 comprising sufficient vitamin B 12 to maintain a release rate of at least 0.33p/g vitamin B 12 per kg liveweight of the animal per day for a sufficient length of time to maintain the elevated blood serum vitamin B 12 levels and increased liver vitamin B, 2 concentrations for at least about 360 days.

12. A controlled release composition suitable for administration to an animal, comprising injectable, biodegradable microparticles comprising vitamin B, 2 incorporated in a polymeric matrix comprising a poly(lactide-co-glycolide) copolymer, and wherein the vitamin B 1 2 is present in an amount of about 5 to by weight of the composition.

13. A controlled release composition according to claim 12, wherein the vitamin B 1 2 is present in an amount of about 7 to about S .25

14. A method of increasing vitamin B 12 blood serum levels and liver vitamin B 2 concentrations in an animal or human, comprising administering to the animal or human a controlled release composition comprising injectable, biodegradable microparticles comprising vitamin B 12 incorporated in a polymeric matrix, wherein the matrix comprises an aliphatic polyester such as 30 a polylactide, a polyglycolide or a copolymer of lactic and glycolic acids and :polycaprolactone and copolymers thereof with lactic or glycolic acids, wherein the amount of vitamin B 1 2 present in the composition and the release rate of vitamin B 1 2 from the composition are sufficient to provide the animal or human :.with its daily vitamin B 12 requirements as well as elevating the blood serum 35 vitamin B 12 level and maintaining an elevated blood serum vitamin B 1 2 level for a predetermined period of time.

A method according to claim 14, wherein the amount of vitamin B12 in the composition and the release rate are sufficient to maintain the elevated blood serum vitamin B 1 2 level for at least 50 days.

16. A method according to claim 15, wherein the amount of vitamin B 12 in the composition and the release rate are sufficient to maintain the elevated blood serum vitamin B 12 level for at least 200 days.

17. A method according to claim 15 or 16, wherein the amount of vitamin B 12 in the composition and the release rate are sufficient to maintain the elevated blood serum vitamin B 1 2 level for at least 360 days.

18. A method according to claim 14, wherein the composition provides a sufficient dose of vitamin B 1 2 and a release rate of vitamin B 1 2 from the composition such that at least about 1/_g vitamin B 1 2 per kg liveweight is released per day for a sufficient length of time to maintain the elevated vitamin B 1 2 levels for at least about 50 days.

19. A method according to claim 18, wherein the composition provides a sufficient dose of vitamin B 12 and a release rate of vitamin B 1 2 from the composition such that at least about 1/pg vitamin B 1 2 per kg liveweight is released per day for a sufficient length of time to maintain the elevated vitamin B 12 levels for at least about 200 days.

20. A method according to claim 18 or 19, wherein the composition provides a ":sufficient dose of vitamin B 1 2 and a release rate of vitamin B, 2 from the composition -such that at least about 1/.g vitamin B,2 per kg liveweight is released per day for a sufficient length of time to maintain the elevated vitamin B 12 levels for at least about 360 days.

21. A method according to claim 14, wherein the composition provides a depot of about 0.1 to about 0.4 mg vitamin B 1 2 per kg liveweight, and the release rate of vitamin B 1 2 from the composition is such that from about 0.33 /tg to about 1.25 ,g vitamin B 12 per kg liveweight is released in vivo per day.

22. A method according to claim 14 wherein the animals are sheep or cattle.

23. A method according to claim 22 wherein sufficient composition is administered to provide about 6-8mg vitamin B, 2 per animal, and about 20 /g to about 25 vitamin B 1 2 is released from the composition per day.

24. A method according to any one of claims 14-23, wherein the composition is prepared and packaged ready-to-use for injection by the subcutaneous or intramuscular route.

A method according to claim 24, wherein said composition is stable for at least 24 months.

26. A method according to claim 24 or 25, wherein said composition further includes an oil based physiologically acceptable non-aqueous suspension medium.

27. A method according to claim 26, wherein said oil based suspension medium is peanut oil.

28. A method according to claim 26 or 27, wherein said oil based suspension medium contains a physiologically acceptable resuspending agent.

29. A method according to claim 28, wherein said resuspending agent is beeswax.

"30. A method according to claim 29, wherein said beeswax is of a suitable *5 pharmaceutical grade.

31. A method according to any one of claims 14-30, wherein the composition comprises a polymeric matrix comprising a 95:5 molar ratio of poly(lactide-co- glycolide) copolymer and a concentration of vitamin B 12 of about 12-13% by weight of the composition. *8a*

32. A method according to claim 21 wherein the composition comprises solely polylactide polymer having an inherent viscosity in chloroform of about 0.7 dl/g at 30'C, and a concentration of vitamin B 12 of about 12-13% by weight of the .0.35 composition.

33. A method of formulating a composition as claimed in any one of claims 1 to 13, the method comprising the steps of: 23 dissolving a polymer in a suitable organic solvent, adding micronised hydroxocobalamin HC1 to said solution and forming a suspension thereof in said solution, emulsifying said suspension in water and removing said organic solvent by extraction or evaporation, and recovering microspheres containing vitamin B 1 2 therefrom.

34. A method according to claim 33, further comprising the step ofresuspending the microspheres in a suspension medium containing a physiologically acceptable resuspending agent to provide a ready-to-use preparation.

A method according to claim 33, wherein said polymer is either a poly(lactide-co-glycolide) co-polymer or a polylactide polymer.

36. A method according to any one of claims 33-35, wherein said solvent is ethylacetate.

37. A method according to any one of claims 33-36, wherein said suspension is emulsified with 3% polyvinyl alcohol in aqueous solution.

38. A method according to any one of claims 33-37, wherein said suspension remains emulsified in water until said microspheres are hardened. a -25

39. A method according to any one of claims 33-38 wherein the suspension medium is peanut oil. a.

A method according to any one of claims 33-39, wherein said resuspending agent is beeswax.

41. A composition produced by the method of any one of claims 33-40.

42. A controlled release composition according to claim 1 substantially as herein ".35 described or exemplified.

43. A method according to claim 14 substantially as herein described or exemplified.

44. A method according to claim 33 substantially as herein described or exemplified. A composition according to claim 41 substantially as herein described or exemplified. en. C a. a a S a. C a Ce a. a a a a a p S C *a a a C a. C C a a