GB2332374A - Controlled release vitamin B12 compositions - Google Patents

Abstract

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 are sufficient to provide the animal with its daily vitamin B 12 requirements, and also to elevate the blood serum vitamin B 12 level of the animal and maintain an elevated vitamin B 12 level of a predetermined period of time.

Description

1 2332374 CONTROLLED RELEASE VITAMIN B12 CONWOSITIONS AND METHODS OF USING

THEM 1 This invention relates to controlled release compositions containing vitamin B 2, It also relates to methods of increasing and maintaining scrum vitamin BUconcentrations and liver stores of vitamin B,, 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, Brazi.4 Australia and New Zealandsee for example Journal ofDepartment of Agriculture of Western Australia 13:199-201, 1936, and 7he New Zealand Veterinary Journal 14:191-196, 1966. The Co requirements of sheep are about double those for cattle (New ZealandJournal 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 V'tarnin B,2 (hydroxocobalamin hydrochloride) (British Journal of Nutrition 24:857-877, 1970). Vitamin B12 is then absorbed and acts as a cofactor for two enzymes: methyImalonyl CoA mutase and methionine synthase (Biological Trace Element Research 22:153-164, 1989, Journal ofNutrition 122:1384-1390, 1992). The conversion of propionic acid to glucose via succinic acid and the tricarboxylic acid cycle requires methyImalonyl CoA mutase, and the transfer of methyl go4s from homocysteine to methionine is catalysed by methionine syntbase. Co deficiency therefore disrupts the energy and protein metabolism of ruminants.

Increasing the Co intakes by an annual application of 175-350 g CoSO. 7HOllia is an effective method for preventing Co deficiency in grazing animals where fertilisers are applied regularly. However the di=t treatment of grazing animals to prevent Co 0 deficiency is more difficult and labour intensive as weekly and fortnightly doses of 7 mg 1 and 28 mg Co respectively are needed, and subcutaneous injections of soluble vitamin F3,2preparations at a rate of 0.05 to 0.1 mg vitamin B,, /kg liveweight are only effective for up to 4 to 6 weeks when assessed in terms of liveweight gains and elevated blood vitamin B,2 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 1) time, as a means of treating disorders such as pernicious anaemia, multiple sclerosis (MS), and neurological disorders; and to supplement HIV sufferers who have been demonstrated to be deficient in vitamin B U.

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,, incorporated in a polymeric matrix, wherein the matrix comprises an aliphatic polyester such as a polylactide, a poly-lycolide or a copolymer of lactic and glycolic acids and 0 polycaprolactone and copolymers thereof with lactic or glycolic acids, wherein the amount of vitamin B,, present in the polymeric matrix and the release rate of vitamin B12 from the polymeric matrix are sufficient to provide the animal with its daily vitamin B,2 requirements, and also to elevate the blood serum vitamin 131, level of the animal and maintain an elevated vitamin B,, level for a predetermined 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.

Preferably the amount of vitamin B12 in the composition and the release rate of vitamin 13,2from the composition are such that at least about 0. 33gg vitamin B,2 per kg liveweight per day is released in vivo, more preferably about I to about 1.25gg vitamin B,2per kg liveweight per day.

preferably said composition is adapted to release said vitamin B12at a near uniform rate.

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

Preferably the composition comprises a microcapsule composition wherein vitamin B,, is present in an amount of about 5 to about 25% by weig t of the gh i i 1 i t 3 composition.

More preferably, the vitamin BU is present in an amount of about 9 to about 20% by weight, most preferably about 12-13% by weight.

Preferably, the composition comprises sufficient vitainin B,,, to maintain a release rate of at least 0.33ig vitamin]3,2 per kg liveweight of the animal per day for a sufficient length of time to maintain the elevated blood serum vitamin B,., levels and increased liver vitamin B I, 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 B12 incorporated in a polymeric matrix comprising a poly(lactideco-glycohde) copolymer, and wherein the vitamin BU is present in an amount of about 5 to 50% by weight of the composition, preferably about 7 to about 20%.

In a Ruther aspect the present invention may broadly be said to consist in a method of increasing vitamin B 12 blood serum levels and liver vitamin B 2 concentrations in an animal or hurnan comprising administering to the animal or human a controlled release composition comprising injectable, biodegradable microparticles comprising vitamin B12 incorporated in a polymeric matrix, w&rein 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 I, present in the composition and the release rate of vitamin B Jrom 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 B12 level and maintaining an elevated blood serurn vitamin B12 level for a predetermined period of time.

Preferably the amount of vitamin B12 in the composition and the release rate are sufficient to maintain the elevated blood serum vitamin B,2 level for at least 50 days, preferably at least 360 days after treatment.

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

4 More preferably, the composition provides a depot of about 0. 1 to about 0.4 mg vitamin B,, per kg liveweight, and the release rate of vitamin B,, from the composition is such that from about 0.33,ug to about 1.25,ug vitamin B,, 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 6Smg vitamin B,, per animal, and about 20,ug to about 25,ug 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 suspeniion medium contains a physiologically acceptable resuspending agent.

Preferably said resuspending agent is beeswax.

More preferably said beeswax is of a suitable pharmaceutical grade.

Most preferably the composition comprises a polymeric matrix comprising a 95:5 molar ratio of polyoactide-co-glycolide) copolymer, or alternatively, solely polylactide polymer having an inherent viscosity in chloroform of about 0.70 or 0.80 dllg at 3CC, and a concentration of vitamin B,2 of about 12-13% by weight of the composition.

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:

1:

i dissolving a polymer in a suitable organic solvent, adding micronised hydroxocobalamin HCI 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 B12 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 in order to provide a ready-to-use preparation.

Preferably said polymer is either a poly(lactide-co-glycolide) copolyrner or a polylactide polymer.

Preferably said solvent is ethylacetate.

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.

Preferably said resuspension agent is beeswax.

The invention flirther provides a product produced by the aforementioned 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

6 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 B,2 is water soluble, stable and has a molecular weight of 1355 Daltons. The applicants have discovered that vitamin B12 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 sennn vitamin B,2 levels in animals or humans, and also increasing liver vitamin B,2 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,2 per kg liveweight per day is provided to the animal, will provide the anirnal's daily vitamin B,, requirements and also significantly elevate blood serum vitamin B12 levels, and increase liver storage of vitamin B,,. It is possible, using the preferred composition of the invention, to maintain the elevated vitamin B12 levels for as long as 12 months, or even longer, with a single dose. Although vitamin B,, is relatively non-toxic and it is possible to administer to an animal many times the daily requirement of vitamin B,, without adverse effects (as the excess is simply excreted), this is wasteful of vitamin BQ. Accordingly, it is preferred the composition provides between about 14g and about 1.25kig vitamin B,, per kg liveweight per day- The compositions of the invention and methods of administering the sarne have been found to be effective in increasing and maintaining vitamin B,, levels in sheep at an elevated level for extended periods of time, up to about 12 months. Previously available soluble vitamin B 12 Preparations which provide 0.05 to 0. 1 ing vitamin B 12/kg liveweight are only effective for about 4 to 6 weeks.

Preparation of microparticle 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 i 1 i 7 A comprising a polymeric matrix comprising a po lylactide, a polyglycolide or a copolymer of lactic---andglycolic acids, and containing various active agents that are not water soluble. The methods disclosed in US 5288496, with substitution of vitamin B,, 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 fonnulation 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 vitaminl 2and 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 0.33Azg vitamin B,is released per kg liveweight per day, more preferably from about I to about 1.25 ug vitamin B, per kg liveweigght per day. This is based on experimental observations that the daily net vitamin BQ requirement for young sheep is 0.44 1.ig vitamin,13 per kg liveweight (British Journal of Nutrition 24:615-633, 1970), and the ability for vitamin B1, to be stored in tissues, especially the liver. Sufficient vitamin B Q is provided in a single dose of the composition to maintain elevated levels of vitamin B,2 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 B12, and a release rate of from about 20 1.ig to about 25 4g vitamin B, 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 vitan-iin B,2 compositions according to the invention. In preferred embodiments, the animals are injected subcutaneously or int=uscularly 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 microparlicles containing vitamin B,, according to the present invention.

8 Example 1 1 Preparation of MierQpqrtiele enmpositiono 7Wis example describes the procedure for preparing a microparticle composition according to the present invention in which hydro xocobalamin hydrochloride is present in an amount of 9.4% by weight and the excipient is a 95.5 molar ratio of poly(lactideco-glycolide) copolymer having an inherent viscosity in chloroform of 0.70 dl/g at WC, The copolymer, 6.76 g (Medisorb) was dissolved in 51.2 g of ethyl acetate (Fisher Scientific) in a 125 ml ErIenmeyer flask. Micronized hydroxocobalamin 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 oliydroxocobalamin HCI 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 a of a 3% PVA aqueous solution (polyvinyl alcohol, Air Products), 4,5 ZID g of ethyl acetate was added. The aqueous solution was placed in a 350 mIjacketed glass reactor equipped with a I " 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 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 rmse 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 VC 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 9 C water in a stainless steel bea-ker, 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 HCl by weight. Peanut oil containing 1 % beeswax was then added to achieve a vitamin B,2 concentration in the resulting suspension for 6mg/ml.

i 9 ExaMple 2 Preparation of 95w5 Micropartiele COMpo, ition Th! preparation techniques described above in Example 1 were used to prepare a microparticle composition according to the present invention containing hydroxocobalamin hydrochloride in an amount of 15.9% by weight, and the excipient is a 95:5 molar ratio of poly(lactide-co-glycolide) copolymer.

ExampIC 3. lereparatinn of 5050 Blend Conillnsition A 50:50 blend of the compositions obtained using the procedures of Examples 1 and 2 was prepared, thereby obtaining a microparticle composition according to the present invention containing hydroxocobalamin hydrochloride in an amount of 12.5% by weight.

F,x2mjple 4: Ire2tment of Animals mdth miernpartiele formulatinus The micropaiticle formulations were prepared according to Examples 1, 2 and 3 above. The microparticles wore reconstituted in peanut oi4 and a 0, 5 mi dose, sufficient to provide about 6-8 mg of vitamin B,,, was prepared.

Aninnals 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 B,, treatments as described below and then grazed as a single group.

Traalfflr,nt.-, Two groups of 10 animals were injected inn-dmuscularly 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 thejugular vein using a 10 rril vacutainerjust prior to the injection and then at days 14, 30, 50, 85, 114, 151, 182, 216, 250, 280 and 3 10 after treatment.

After centrifuging at 2500 g for 20 minutes the scrum was removed and stored at ACC for vitamin BU determinations. On day 310 all lambs were slaughtered, and their livers removed, weighed and subsampled for vitamin B 12.

Pasture samples wore collected at 2-3 monthly intervals for Co determination and the lambs were weighed at 2 monthly intervals- AnalZical metho Tbd vitamin 1312 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 AgricuIturalResearch 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--'62 g/day over the 3 10 day study wth the fastest gains occurring in late spring.

A significant response in growth rates to vitamin B12 SUPPleMentation would only be expected on Co deficient pasture, ffiat is, pastures with Co concentrations of less than 0.08 mg/kg DIA.

The effect of the vitamin B12 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,, concentrations significantly higher (P<0.05) tharl 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 (P<O. 0 1) 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 B,, concentrations were found at days 85 and firom 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 2 80 and 3 10.

The influence of vitamin BU injections on the liver storage of vitamin BU at day 3 10 Is shown in Table 2. The Example 3, Groups 1 and 2 sheep had significantly higher (P<0.05) hepatic stores of vitamin B 2 when compared to the untreated control lambs.

1:

i 11 Table 1. Fffect of injectable vitamin B,, microsphere preparations on the mean scrum vitamin B,, concentrations (pmotell) of lambs.

Days after injection 0 14 30 so 85 114 151 182 216 250 280 310 Control 968 1302' 987 A 695^ 15 1 laA 807 A 1255' 8 67 1966 A 770A 683 440 Example 1 Group 1 699 1056 1397A 1297 A 1960A 1104' 1541A 2500h 47008 1078A 681 398 Example 1 - Group 2 855 1131' 11 16' - 1292A 23W 1097^ 1550, 2720' 49509 13501' 706 393 Example 2 - Group 1 840 1781' 21481' 220OE' 37881 2522' 28559 24331 50330 13228 694 407 Example 2 - Group 2 524 1354' 2057 184f' 398515 26280 3314 35460 5500,' 1757 962 485 Example 3 - Group 1 1300 2028h 1957B 2010 34BY' 2142" 2600" 31281' 4210 1371" 762 440 Example 3 - Group 2 681 1642' 1600A 1885a 3657B 2142' 2300B 24856 56140 1374B 778 398 Within a column means with diffireni superscripts are significantly different avb P<0.05 AVB P<0.0 1 - 12 Table 2. Effect of injectable vitamin BR microsphere, preparations on the mean (L standard error) liver vitamin 13,2 content (nmol/ 1 kg fresh tissue) of lambs after 3 10 days.

Liver vitarnin 13,2 nmolelkg fresh tissue Control Example 1 - Group 1 Example 1 - Group 2 Example 2 - Group 1 Example 2 - Group 2 Example 3 Group 1 Example 3 - Group 2 36&140.4' 435 39.2 419 23.6' 496 80.1' 486 29.9' 625 48.3h 537 50.]h Mean with different superscripts significantly different at P<0.05.

13 Example 5: Irgatment of Animals - MaryLig Ti:e;itment Parameters This was to determine the effect of the following parameters on scrum vitamin B,. concentrations.

2.

3.

4.

1. The route of administration eg. subcutaneous (SQ v intramuscular (IM) injection. The efficacy of formulations eg. 95:5 copolymer (lactide/glycolide) and 100 polymer (lactide). The size of vitamin B, 2depot eg. 95:5 (6 and 9 mg) or 100: (9 v 12 mg). The suspending agent, namely beeswax.

Animals Three hundred lambs, aged about 6 months, with a mean liveweight of 30 kg and a mean scrum vitamin B12concentration of 850 pmolell were used for the study. On the basis of serum vitan-dn B,, 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.

Group I Control: I ml of peanut oil only intramuscular (IM) Group 2 Control: I ml of peanut oil containing I% beeswax (IM) Group 3: 95:5; 6 mg in 1 ml peanut oil (IM) Group 4: 95:5; 9 mg in I ml peanut oil (IM) Group 5: 100: 9 mg in I ml peanut oil (IM) Group 6: 100: 12 mg in I ml peanut oil (IM) Group 7: 95:5; 6 mg in I ml peanut oil subcutaneous (SC) Group 8: 95:5; 9 mg in I ml peanut oil (SC) Group 9: 100; 12 mg in 1 ml peanut oil (SC) Group 10: 95:5; 6 mg in I 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 I ml peanut oil with I % beeswax (IM) Group 13: 100; 12 mg in 1 ml peanut oil with I% beeswax (SC) 14 Preparation of Vitamin B,, Composition 1 Peanut oil was the vehicle for administration. To ensure that the vitamin 13,,/polymer or the encapsulated vitamin B,, remained in suspension. 1 % 1 1 fr of beeswax was added to the o 1 peanut to prevent the vitamin B12/PolyMer OM settling to the bottom of the vial thus making it difficult to accurately administer the required dose of vitamin 13,2.

To prepare the peanut/beeswax mixture I g 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 B12/Polymer...The 95:5 formulation contained 11.2% vitamin 13,2and the 100 formulation contained 14.3 % vitamin B 1. For each group 10 doses of vitamin 1312/polymer were calculated and weighed out to give the appropriate depot size (ic. 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 10 doses using the 95:5 formulation containing 11.2% vitamin 13,2 toprovide a depot of 6 mg vitamin B,2/sheep/dose 535.7 mg of the 95:5 formulation was mixed with 10 ml of I% beeswax peanut oil. A I ml dose then provided a depot of 6 mg of vitamin B,,/sheep/dose.

Treatment and sampling protocols All lambs were bledjust priot 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 B12 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 SQ were slaughtered and their livers removed for vitamin B,, determinations.

Results and Discussion The mean serum vitamin B,, concentration of the control lambs was 638 pmole/1. All of the vitamin B,., treatments increased serum vitamin B12 concentrations to greater than 2600 pmolell 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/I). This was associated with a significant increase in the vitamin B 12 stores of the liver (265 v 478 nmole/kg fresh tissue). The 1!i is following preliminary conclusions can be made.

Beeswax is an effective agent to keep the encapsulated vitamin B, in suspension. It has no effect on the release of vitamin B,2 fTorn the injected vitamin B,2 copolymer depot.

2. There is no difference between subcutaneous and intramuscular methods of injection on the rate of release of vitamin B,2. 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.

Example 6 Treatment of Pregnant Ewes This was to determine the effect of injecting ewes during mating with 12mg vitamin B,, long acting formulation (95:5 lactide/glycolide containing 11.2% Vitamin B I.) on: (a) scrum Vitamin B,2 concentrations during pregnancy and lactation in

ewes. (b) liver storage of Vitamin B,2. in pregnant ewes just prior to parturition.

(c) liver storage of Vitamin B 12 in foetuses just prior to parturition. (d) milk Vitamin B,, concentrations during lactation.

(e) scrum Vitamin B,2concentrations in suckling lambs. (f) liver storage of Vitamin B I, in suckling lambs.

Methods At the time of mating 2 groups of 28 mixed age ewes, with a mean liveweight of 54 kg, were randomly 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 lambing so that lambs fom untreated and treated ewes could be readily identified. After lambing all ewes and lambs will be run as a single flock until weaning.

The control ewes were injected with 1 nil of peanut oil/beeswax 16 intramuscularly while treated ewes were injected with I m.1 peanut oil/beeswax containing 12 mg Vitamin B,, encapsulated in 95:5 lactide/glycolide polymer prepared by the method of example 1. Injections were given during mating, 40 days preparturition and 40 days postpartuntion.

1 B lood samples were taken J ust prior to the injections and at 15, 27, 47, 6 8, 83, 96, 115, 140 and 1.78 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,, significantly increased (p<0.01) the serum Vitamin B,2 concentrations (1430 v 2460 nmole/l) in the ewes during pregnancy and lactation and the storage of Vitamin B,, in the livers (813 v 1455 nmole/kg fresh tissue) of the ewes and their foetuses (354 v 873 mnole/kg fresh tissue). Further, the milk vitamin B,, concentrations (3680 v 5320 pmole/1) were increased at day 60 of the lactation, while liver vitamin B,, stores (192 v 279 nmole/kg fresh tissue) were increased in 30 day old lambs.

Conclusion

Increasing the Vitamin 1312 status of pregnant ewes increased the Vitamin B,, status of their new b om lambs. ' GENERAL OBSERVATIONS The efficacy of the injectable vitamin B,, formulations was assessed on their ability to significantly increase and maintain serum vitamin B12 concentrations above those of the untreated controls. The relationship between growth rates and serum Vitamin 13,2 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,, 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,ug from the vitamin B,2 incorporated in the microspheres, were effective in meeting daily vitamin B12 requirements (British Journal ofNutrition 24:615-633, 1970) as well as increasing the vitamin B,, status of the sheep for 8-9 months.

1 17 The appropriate daily release rates of vitamin B12 can be calculated from the changes in serum vitarnin B,2. This approach is the best way of determining the daily vitamin B,, release rates because the animal biochemical and physiological factors which can and do influence the release of vitamin B12, as reflected by changes in serum vitamin B 2 concentrations, are taken into consideration.

Using the data from the vitamin B 12 formulations described in Examples 2 and 3 (Table 1) the daily vitamin B12 release rates were calculated by dividing the depot size or amount of vitamin B 2 injected (6000 ig) by the period over which the serum vitamin B 2 concentrations were significantly elevated (250 days). The calculated daily release rates of vitamin 13,2 was 24 ig.

As presently available injectable vitamin B 12 preparations are only effective for 4-6 weeks, the injectable vitamin B12 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 B 2means that ewes and lambs need only be treated about once or twice a year and this operation 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 enure an increase in the storage of vitamin B12 in the foetal liver while the increase in the vitamin B I, content of colostrum and milk together with the increased liver vitamin BUstores 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.

It is envisaged that the vitamin B12compositions 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 (MS), and neurological disorders.

18

Claims (48)

YMAT WE CLAIM IS:

1 - A controlled release composition suitable for administration to an animal, comprising biodegradable microparticles comprising vitamin B,2 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,2present in the composition and the release rate of vitamin B,2 from the composition are sufficient to provide tile animal with its daily vitamin B,2 requirements, and also to elevate the blood serum vitamin B12 level of the animal and maintain an elev4ted vitamin B, level for a predetermined 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, wherein the amount of vitamin B,, in the composition and the release rate of vitamin B12 from the composition are such that at least about 0.33,ug Vitamin B1, per kg liveweight per day is released in vivo.

5. A controlled release composition according to claim 4, wherein about I to about 1.2 5 gg vitamin B, per kg liveweight per day is released in vivo.

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

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

8. A controlled release composition according to claim 7 wherein the polymeric matrix comprises a 95:5 molar ratio of poly(lactide-coglycolide) copolymer, or polylactide polymer alone having an inherent viscosity in chloroform of about 0.70 dl/g at 30'C.

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9. A controlled release composition according to any preceding claim comprising a microcapsule composition wherein vitamin B12 is present in an amount of about 5 to about 25% by weight of the composition.

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

A controlled release composition according to claim 9 or 10, wherein the vitamin B 12 is present in an amount of about 12-13% by weight.

12. A controlled release composition according to any preceding claim comprising sufficient vitamin B,2 to maintain a release rate of at least 0.33ig vitamin BU per kg liveweight of the animal per day for a sufficient length of time to maintain the elevated blood scrum vitamin B,, levels and increased liver vitamin B12 concentrations for at least about 50 days.

13. A controlled release composition according to claim 12 comprising sufficient vitamin B,2 to maintain a release rate of at least 0.331Ag vitamin B 2 per kg liveweight of the animal per day for a sufficient length of time to maintain the elevated blood scrum vitamin B 2 levels and increased liver vitamin B12 concentrations for at least about 200 days.

14. A controlled release composidon according to claim 12 or 13 comprising sufficient vitamin B,, to maintain a release rate of at least 0.33ig 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,, levels and increased liver vitamin B, 2 concentrations for at least about 360 days.

15. A controlled release composition suitable for administration to an animal, comprising injectable, biodegradable microparticles comprising vitamin B U incorporated in a polymeric matrix comprising a poly(lactide-co- glycolide) copolymer, and wherein the vitamin B,, is present in an amount of about 5 to 50% by weight of the composition.

16. A controlled release composition according to claim 15, wherein the vitamin B,,, is present in an amount of about 7 to about 20%.

J

17. A method of increasing vitamin B 2 blood serum levels and liver vitamin B,, concentrations in an animal or human, comprising administering to the animal or human a controlled release composition comprising injectable, biodegradable microparticles comprising vitamin B, 2 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,, 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,, requirements as well as elevating the blood serum vitamin BR level and maintaining an elpyated blood serum vitamin B 12 level for a predetermined period of time.

18. A method according to claim 17, wherein the amount of vitamin B,2 M the composition and the release rate are sufficient to maintain the elevated blood serum vitamin B,, level for at least 50 days.

19. A method according to claim 18, wherein the amount of vitamin B 2 in the composition and the release rate are sufficient to maintain the elevated blood serum vitamin B,, level for at least 200 days.

20. A method according to claim 18 or 19, wherein the amount of vitamin B, 2 in the composition and the release rate are sufficient to maintain the elevated blood serum vitamin BU level for at least 360 days.

21. A method according to claim 17, wherein the composition provides a sufficient dose of vitamin B 12 and a release rate of vitamin B 12 from the composition such that at least about lig vitamin B,, 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 50 days.

22. A method according to claim 21, wherein the composition provides a sufficient dose of vitamin B 2 and a release rate of vitamin B, 2 from the composition such that at least about 1,ug vitamin B12 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.

i:

i:

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23. A method according to claim 21 or 22, wherein the composition provides a sufficient dose of vitamin B,.2 and a release rate of vitamin B 12 from the composition such that at least about lig vitamin B,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 3 60 days.

24. A method according to claim 17, wherein the composition provides a depot of about 0. 1 to about 0.4 mg vitamin B,2per kg liveweight, and the release rate of vitamin B12from the composition is such that from about 0. 33 ag to about 1.25,ug Vitarnin B,2 per kg liveweight is released in vivo per day.

25. A method according to claim 17 wherein the animals are sheep or cattle.

26. A method according to claim 25 wherein sufficient composition is administered to provide about 6-8mg vitarnin B,2per animal, and about 20, Ug to about 25 ig vitamin B 2 is released from the composition per day.

27. A method according to any one of claims 17-26, wherein the composition is prepared and packaged ready-to-use for injection by the subcutaneous or intramuscular route.

28. A method according to claim 27, wherein said composition is stable for at least 24 months.

29. A method according to claim 27 or 28, wherein said composition further includes an oil based physiologically acceptable non-aqueous suspension medium.

30. A method according to claim 29, wherein said oil based suspension medium is peanut oil.

3 1. A method according to claim 29 or 30, wherein said oil based suspension medium contains a physiologically acceptable resuspending agent.

32. A method according to claim 31, wherein said resuspending agent is beeswax.

33. A method according to claim 32, wherein said beeswax is of a suitable pharmaceutical grade.

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34. A method according to any one of claims 17-33, wherein the composition comprises a polymeric matrix comprising a 95:5 molar ratio of poly(lactide-coglycolide) copolymer and a concentration of vitamin B,2 of about 12-13% by weight of the composition.

35, A method according to claim 24 wherein the composition comprises solely polylactide polymer having an inherent viscosity in chloroform of about 0.7 dl/g at 3CC, and a concentration of vitamin B,, of about 12-13% by weight of the composition.

36. A method of formulating a composition as claimed in any one of claims 1 to 16, the method comprising the steps of.

dissolving a polymer in a suitable organic solvent, adding micronised hydroxocobalamin HG to said solution and forming a suspension thereof in said solution, emulsifyring said suspension in water and removing said organic solvent by extraction or evaporation, and recovering microspheres containing vitamin B 2 therefrom.

37. A method according to claim 36, further comprising the step of resuspending the MIcrospheres in a suspension m6dium containing a physiologically acceptable resuspending agent to provide a ready-to-use preparation.

38. A method according to claim 36, wherein said polymer is either a poly(lactide-co-glycolide) co-polymer or a polylactide polymer.

39. A method according to any one of claims 36-38, wherein said solvent is ethylacetate.

40. A method according to any one of claims 36-39, wherein said suspension is emulsified with 3% polyvinyl alcohol in aqueous solution.

41. A method according to any one of claims 36-40, wherein said suspension remains emulsified in water until said microspheres are hardened.

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42. A method according to any one of claims 37-41 wherein the suspension medium is peanut oil.

43. A method according to any one of claims 37-42, wherein said resuspending agent is beeswax.

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44. A composition produced by the method of anyone of claims 36-43.

45. A controlled release composition according to claim 1 substantially as herein described or exemplified.

46. A method according to claim 17 substantially as herein described or exemplified.

47. A method according to claim 36 substantially as herein described or exemplified.

48. A composition according to claim 44 substantially as herein described or exemplified.

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