NO314387B1 - Zinc-bound somatotropin complex and essentially non-aqueous somatropin preparation with long-term release characteristics for parenteral administration to an animal for growth and productivity growth - Google Patents

Abstract

Metal-bound somatotropin complex suitable for use in a sustained release preparation intended for parenteral administration to an animal by injection or implantation to increase the growth or productivity of the animal, which comprises the reaction product of bovine or porcine somatotropin and ions of a metal selected from the group consisting of zinc, iron, calcium, bismuth, barium, magnesium, manganese, aluminum, copper, cobalt, nickel and cadmium, where the metal ions make up up to 2% by weight of the reaction product and are present in an amount sufficient to prolong the release of the somatotropin from the preparation. Somatotropin preparation with long-term release characteristics after parenteral administration to an animal to increase the growth or productivity of the animal, comprising bovine or porcine somatotropin and a water-soluble compound of a metal ion selected from the group consisting of zinc, iron, calcium, bismuth, barium, magnesium, manganese, aluminum, copper, cobalt, nickel and cadmium, where the metal ions make up up to 2% by weight of the preparation and are present in a sufficient amount to prolong the release of somatotropin from the preparation.

Description

The invention relates to metal-bound somatotropin complex for use in a sustained-release preparation for parenteral administration to an animal by injection or implantation to increase the growth or productivity of the animal. Furthermore, the invention essentially relates to non-aqueous somatotropin preparations with prolonged release characteristics for parenteral administration to an animal to increase the growth or productivity of the animal.

Although prolonged activity of some biologically active (bioactive) polypeptides can be achieved by administering only very small amounts parenterally, others are required in sufficient serum concentrations and/or have such a short serum half-life that a significant amount must be administered (e.g. at least approx. 100 mg) to provide the desired biological effect over a longer period of time such as e.g. 1 week or longer. Somatotropins (growth hormones) are an example of such polypeptides.

To prevent undesired frequent release into an animal's bloodstream, certain polypeptides have been administered parenterally in liquid carriers which may optionally contain agents for reducing hydration (antihydration agents), or in association with metals or metal compounds which further lower their solubility in body fluids. To avoid the need for unacceptably large amounts of such a carrier, it would be advantageous to have significant concentrations of the polypeptide in the carrier. However, most bioactive polypeptides are highly viscous at significant concentrations and thus difficult to inject or otherwise administer at such concentrations. Furthermore, many commonly used antihydrating agents increase the viscosity and can reduce the appropriate injectability of such preparations. For these and other reasons, it has the right combination of (1) a sufficiently rapid release to produce the desired biological effect in the animal, (2) a sufficiently slow release to prolong the action to a sufficient extent, (3) a sufficiently large amount for release at the required rate over the prolonged period of time and (4) a sufficiently small volume and sufficiently low viscosity for convenient administration, have usually been difficult to achieve.

As each polypeptide is different, e.g. in terms of three-dimensional structure and reaction with other substances, the possibility of achieving a long-term effective release with a high content of polypeptide in a suitable carrier is impossible to predict or demonstrate theoretically. Still, in many cases, such preparations with prolonged release must be developed if the biological effect of the polypeptide is to be provided in a usable, economical way.

Methods for achieving long-term release of bioactive substances have long been sought in order to reduce the frequency of treatments and/or minimize damage to the animal being treated. Sustained release has been achieved for a number of such substances by various means. One system is the use of oil solutions that can be injected intramuscularly, subcutaneously or otherwise. In some cases, substances with low oil solubility have been administered in oil suspensions.

E.g. it is described in US patent document no. 2 491 537 release up to 24 hours for penicillin suspended in oil (e.g. vegetable) which is gelled with pectin, a cellulose compound, or a protein such as e.g. gelatin. Prolonged release of insulin and steroid hormones is also suggested. US Patent No. 2,507,193 describes release in rabbits for up to 11 days using 300,000 units/ml of procaine penicillin suspended in peanut oil gelled with 5% aluminum monostearate (A1MS). US Patent No. 3,016,330 describes AlMS-coated penicillin suspended in sesame oil. Chien, Journal of Parenteral Science and Technology, vol. 35 (3), p. 109 (1981) discusses long-term bioavailability of penicillin G procaine suspended in vegetable oil gelled with 2% A1MS, and states that more than 2% A1MS appears to be of only limited benefit in prolonging effective penicillin levels and that suspensions containing more than 2% A1MS are too viscous for practical use.

Slurries in oil have also been used for certain other low molecular weight therapeutic substances than penicillin. For example are described in US Patent No. 3,676,557 long-acting preparations of up to 50% pamoate salts of normorphinones in oil slurries gelled with A1MS. US Patent No. 4,016,273 describes preparations of up to 40% pamoate salts of oxazepines in oils gelled with aluminum stearates which have prolonged release.

Systems for sustained release of certain bioactive polypeptides have also been described. E.g. described in US Patent No. 2,964,448 slurries of relaxin (approx. 2%) in a vegetable oil gelled with A1MS. The patent document states that such a slurry provides relaxation comparable to that obtained with oil without gelling agent (e.g. 5 to 7 days) and describes a more prolonged effect (up to 23 days) by heat treating the slurry containing A1MS .

US Patent No. 3,869,549 describes injectable preparations with prolonged release containing "extremely small amounts", e.g. "parts of a milligram" of a peptide. Although growth hormones are mentioned, the specific examples relate to water-soluble preparations of corticotropin (ACTH) which are active for 7 to 8 days. In particular, preparations are described which contain acid addition salts of ACTH analogues suspended in peanut oil gelled with aluminum distearate (AIDS) or adsorbed on AIDS which is then dispersed in such oil. In both cases, the analogue is present in the injectable preparations in concentrations of only 0.03 to 0.1% and a weight ratio between the peptide and the aluminum salt that is not greater than 0.5.

Preparations for long-term release of analogs of LHRH hormone are described in US Patent No. 4,256,737. These preparations contain salts of the hormone, including multivalent metal salts (e.g. zinc salts), in vegetable oil gelled with the aluminum salt of fatty acids. The LH-RH analogues are administered at concentrations from 0.01 to 1% in the injected preparation.

Others have described aqueous suspensions of metal salts or complexes of polypeptides for prolonged parenteral release. E.g. long-term active aqueous suspensions of a precipitation product of water-soluble gonadotropins and aluminum or zinc hydroxide are described in US Patent No. 3,852,422. As zinc is naturally present in pancreatic insulin, prolongation of insulin release from aqueous slurries due to reaction between insulin and various metals (eg, zinc, nickel, cobalt, and cadmium) has been investigated. See US Patent Nos. 2,143,590, 2,174,862, 2,882,203, 2,290,014 and 3,102,077.

An object of the invention is to provide preparations which are useful for long-term release of a biologically active polypeptide in an animal, and which are composed of components which are biologically compatible with the animal.

Another purpose is to provide such preparations which have a sufficiently rapid release to produce the desired biological effect in the animal.

Another object is to provide such preparations which have a sufficiently slow release to maintain the desired biological effect with an advantageously extended duration.

Another purpose is to provide such preparations which have a sufficiently high content (adequate amount) of the polypeptide to maintain the required release rate over such a long period of time.

Another object is to provide such preparations which have a sufficiently small volume to provide easy parenteral administration. This is particularly important when the amount of polypeptide to be administered is necessarily large.

Another purpose is to provide such preparations where the components and the ratios between them are chosen so that the preparations have sufficiently low viscosity to provide easy injection or other administration. This is also very important when injection of a relatively large amount of the polypeptide is required.

In some embodiments, it is intended that such components and their ratios provide a sufficiently high viscosity to aid in the formation in the animal of depots favoring more sustained release. In many cases this is difficult to achieve together with the recently mentioned low viscosity objective.

Another object of the invention comprises the provision of metal-bound somatotropin complex which is advantageously included in such preparations. These and other purposes of the invention will become clearer from the detailed description below.

It has been found that the above objects can be realized with a substantially non-aqueous somatotropin preparation with sustained release characteristics for parenteral administration to an animal to increase the growth or productivity of the animal, which is characterized by comprising the reaction product of bovine, human or porcine somatotropin and multivalent ions of zinc metal, where the metal constitutes up to 2% by weight of the reaction product. Furthermore, the objects can be realized with a metal-bound somatotropin complex for use in a sustained-release preparation for parenteral administration to an animal by injection or implantation to increase the growth of or productivity of the animal, which is characterized by comprising the reaction product of bovine, human or porcine somatotropin and polyvalent ions of zinc metal, where the metal constitutes up to 2% by weight of the reaction product.

In this description, all percentages in preparations are based on weight and temperatures are in °C unless otherwise stated.

As used in the specification and in the claims, the term "substantially non-aqueous" means substantially anhydrous or containing water in such a small proportion that it does not in an intolerable manner accelerate the release of the polypeptide in the animal. Although this proportion of water varies between the individual preparations according to the invention, it is usually less than approx. 2% (even more typically less than about 1%) in a form that has such an effect on polypeptide release. The term "non-toxic" here refers to ingredients in preparations according to the invention which are reasonably safe and/or harmless when used in appropriate amounts and under suitable conditions in parenteral administration of such preparations.

The term "biologically active" or "bioactive" polypeptide herein means a polypeptide which, after appropriate parenteral administration to an animal, has a detectable effect on a biological process in the animal. The effect can be hormonal, nutritional, therapeutic, prophylactic or something else, and can imitate, supplement or inhibit a naturally occurring biological process. Although there are a large number of different such effects and processes, for example stimulation of growth, milk production, egg or offspring production and/or feeding efficiency in food animals can be mentioned. Other examples include the production of wool, leather or other non-food products from animals. Although lower molecular weight polypeptides (e.g., at least about 300) may be used, these polypeptides typically have a substantial molecular weight, e.g. at least approx. 1000, usually at least approx. 4000, in the most preferred embodiments at least approx. 9000, and in many even more preferred embodiments at least approx. 18,000 and up to approx. 30,000 or higher. Although the polypeptide may be in its active form in preparations according to the invention before administration to an animal, the expression here also includes polypeptides that develop bioactively after such administration.

The somatotropins administered in the preparation according to the invention are only slightly water-soluble (ie at least 100 parts of water at room temperature are needed to dissolve one part of somatotropin). In many desirable embodiments, they are not very soluble in the biocompatible oil used, i.e. not soluble at room temperature in a concentration higher than approx. 2%, and most preferably no higher than approx. 1%. 1 some cases, e.g. preparations containing different somatotropins, the somatotropins are both slightly water-soluble and essentially insoluble in the oil.

As mentioned above, each of the preparations according to the invention contains as a continuous base, a biocompatible oil, i.e. an oil which has no intolerable harmful effect on the somatotropin, the animal or, in the case of animals whose products enter the food chain, the consumers of such products . It is preferred that such oils have a low degree of acidity and are essentially free of rancidity. As used herein, the term "oil" means a fatty oil or fat that is liquid at the body temperature of the animal. Thus, such an oil will melt or at least start to melt during approx. 40 °C and preferably below approx. 35 °C. Oils that are liquid at approx. 25 °C can facilitate injection or other administration of some preparations according to the invention. In some cases, polyunsaturated (eg, partially hydrogenated) oils may be preferred due to greater biocompatibility with the animal or for other reasons.

In a preferred embodiment, the biocompatible oil is composed mainly of triglycerides, i.e. long-chain (usually Cg-C24', preferably Ci2-Ci8) fatty acid esters of glycerol, or mixtures of triglycerides and such fatty acids (preferably in only minor proportions, e.g. less than about 10% free fatty acid). In some embodiments, glycerol may be replaced by other trihydroxy or polyhydroxy compounds. Oils that are particularly preferred include vegetable oils such as e.g. olive oil, sesame oil, peanut oil, sunflower seed oil, soybean oil, cottonseed oil, corn oil, safflower oil, palm oil, rapeseed oil and mixtures of such oils. Sesame and peanut oils are highly preferred for many embodiments. Oils of animal or mineral origin or synthetic oils (including long-chain fatty acid esters of glycerol or propylene glycol) can also be used provided they are sufficiently biocompatible.

In most embodiments, such an oil constitutes a dominant proportion by weight of such preparations. It is desirable that the unbroken phase of biocompatible oil in most cases contains finely divided, small particles of somatotropins relatively uniformly dispersed, e.g. in a slurry. The upper limit of somatotropin content is where the oil ceases to exist in an unbroken phase because there is insufficient oil to completely enclose substantially all of the somatotropin in the preparation.

Surprising and unexpected results have been obtained by using high contents of somatotropin in such preparations even when the viscosity is thereby increased to a significant extent. With such high contents, a reaction between the somatotropin and oil has also been discovered, which in many cases favors a long-term release of somatotropin from a long-lasting depot. As previously mentioned, this reaction is promoted in many cases when the somatotropin is bound to a metal.

Consequently, preparations according to the invention contain a somatotropin at desirable high content levels, e.g. at least 10%. Even higher content of somatotropin, e.g. at least 15%, are often desirable and particularly effective. Content of 20% or more, e.g. at least 30% or even up to 42% or higher, can be advantageously used in parenterally injectable preparations. Such preparations can cause long-term release of the somatotropin (measured in blood

the circulation of cattle or other animals) for periods of up to 30 days or more.

Mainly non-aqueous preparations comprising content levels of somatotropin as high as approx. 10%, as far as is known, has not been proposed before. Within the prior art, oil preparations are limited to very low contents of polypeptides, i.e. no more than approx. 2%. (See US Patent Nos. 2,964,448, 3,869,549 and 4,256,737).

The preparations according to the invention may also include, in addition to the biocompatible oil, an "antihydration agent", a term which here means a substance that reduces the hydration of a specific preparation according to the invention, or the somatotropin and/or biocompatible oil therein, thereby reducing and / or stabilizes the release rate of the somatotropin from the preparation after administration to an animal. A wide variety of non-toxic antihydrating agents are known. For example, there are "gelling" agents which, when dispersed and in some cases heated to dissolve in the oil, give the amount of oil greater viscoelasticity (and therefore greater structural stability) and thereby reduce the rate of penetration of aqueous (e.g. body) fluids through the oil.

The exact mechanism of these agents in the present invention is not fully understood. Thus, it has been observed that certain known "gelling" agents provide the desired antihydration effect even when the oil containing such an agent has not been heated to increase the gelling effect, or when the gelation, once it has occurred, is substantially been eliminated (e.g. by means of shear forces). Furthermore, various antihydrating agents which have no significant ability to gel the oil are suitable for use in connection with the invention. Magnesium stearate is an example.

Exemplary antihydration agents include various salts of organic acids, e.g. fatty acids with from 8 (preferably at least 10) to 22 (preferably up to 20) carbon atoms, e.g. aluminium, zinc, magnesium or calcium salts of lauric acid, palmitic acid, stearic acid and the like. Such salts can be mono-, di- or tri-substituted, depending on the valence of the metal and the degree of oxidation of the metal by that acid. Particularly useful are the aluminum salts of such fatty acids. Aluminum monostearate and aluminum distearate are particularly preferred antihydrating agents. Others which may be used include aluminum tirstearate, calcium mono- and -distearate, magnesium mono- and -distearate and the corresponding palmitates, laurates and the like. In many embodiments, the concentration of such an antihydrating agent, based on the weight of the oil plus the agent, will advantageously be between 1% and 10% (most typically between 2% and 5%), although other concentrations may be suitable in some cases.

Generally, both the somatotropins and the antihydrating agents tend to increase the viscosity of the compositions according to the invention. With many somatotropins, especially those with a relatively high molecular weight and/or complex secondary or tertiary structure, this presents a problem which it has now been discovered can be overcome by using a relatively high weight ratio of somatotropin to antihydrating agent. According to the invention, this ratio is usually at least 1, more typically at least 3, even more typically at least 4, and usually at least 6. Although usually less critically expressed in formulation viscosity, that ratio is usually not greater than 40 and more typically greater than 20 .

By using such conditions, it has now been discovered that even with the higher preparation viscosities that accompany relatively high somatotropin concentrations, advantageously prolonged and effective release of the somatotropin is obtained. Even more surprising is the fact that in some such preparations, the rate of release actually increases as the somatotropin content (and therefore the preparation viscosity) is increased.

The preparations according to the invention can be used for long-term release of somatotropins in animals. Somatotropins can be used to increase lean-to-fat ratio, nutritional efficiency and milk production in various mammalian species including cattle (eg dairy cows), sheep, goats and pigs.

As used herein, the term "somatotropin" means a polypeptide having biological activity and chemical structure substantially similar to the activity and structure of a somatotropin produced in the pituitary gland of an animal. Such somatotropins include naturally occurring somatotropins produced by somatotropic cells from the pituitary gland, and alternatively somatotropins expressed by means of genetically transformed microorganisms such as e.g. E.coli. other bacteria or yeast. Such alternatively produced somatotropins may have an amino acid sequence that is identical to the naturally occurring somatotropin, or may be analogues that have one or more variations in the amino acid sequence that may provide increased biological activity or another benefit. Somatotropins for which the present invention is particularly applicable include bovine and porcine somatotropins, e.g. microbially expressed bovine and porcine somatropins. These somatotropins optionally have a methionine residue at the N-end, e.g. a methionine that transcribes from microbial translation of an ATG initiation signal in a gene for the polypeptide. In some cases, however, it may be desirable that such methionine residues in the polypeptide are no more than approx. 20% (preferably no more than about 10%) formyl methionine, to reduce any tendency in the animal's defense against foreign substances to break down the polypeptide.

Observation of injections of preparations according to the invention indicates that a significant amount of the somatotropin is in some cases released immediately after injections. This is referred to as a "burst" which is believed to be due to an increase in surface area caused by injection or other administration. In some cases, it may be desirable to have a moderate outbreak, e.g. to activate a desired biological effect. A characteristic that is useful in the design of preparations according to the invention is the ratio between the immediate onset level determined by measuring the concentration of somatotropin in the serum of the treated animal shortly after administration, and the long-term release level determined by measuring the concentration of somatotropin in the serum from the animal at a later time. In connection with the present invention, the onset level is the concentration of somatotropin in serum 24 hours after injection, and the prolonged release level is the concentration of somatotropin in serum 14 days after injection. These concentrations are used to calculate an "extended-to-prolonged-release" ratio which is believed to be generally beneficial between approx. 1.2 and approx. 6, e.g. between 1.5 and approx. 3.

Another useful characteristic when evaluating and designing preparations according to the invention is "sprayability", a measure of how well the preparation flows through an injection needle. If particles of the somatotropin are too large or the preparation is too viscous, a disproportionately high pressure may be required to force the preparation through such a cannula. In connection with the present invention, "sprayability" is determined by measuring the time that a volume unit of a preparation according to the invention needs to pass through a cannula of dimension 18 which has an inner diameter of 0.838 mm and a length of 4 cm, when the preparation is applied to a pressure of 1193 kPa in a syringe connected to the needle. It is desirable that "sprayability" for preparations according to the invention is at least 0.03 ml per second. Preferably, such sprayability is higher, e.g. at least approx. 0.1 ml per second or, even more desirable, at least approx. 0.3 ml per second.

Preparations according to the invention can be prepared by adding somatotropin to oil alone or to oil with an antihydration agent suspended or dissolved in the oil. It is often convenient to dissolve an antihydrating agent to provide a gelled oil. When preparations according to the invention are prepared by a method that begins by making a gelled oil, a gelling agent such as e.g. a fatty acid salt of aluminium, is added as a powder to a certain amount of oil with stirring to give a slurry of the powder. It is desirable that the stirred slurry can be heated at least to the melting point of the fatty acid salt (eg at least 155 °C for A1MS) where the salt will dissolve in the oil. Lower temperatures can be used if the gelling agent or other antihydrating agent is sufficiently dissolved. Vigorous and continuous stirring helps to avoid agglomeration of the fatty acid salt and maintains the dispersion. Generally, the heating and stirring should be continued until the slurried salt is completely dissolved. It is often desirable to maintain stirring for an additional time to ensure complete dissolution.

The oil solution of fatty acid salt can then be cooled, e.g. to room temperature, where a reasonably stable gel structure will occur. The gel should be stored under vacuum or in the presence of a desiccant to avoid contamination with water that could adversely affect the gel structure.

The somatotropin can then be added to the oil at this temperature (eg room temperature) where intolerable harmful effects are avoided (eg denaturation). E.g. bovine somatotropin has been added to such an oil at temperatures from approx. 4 to approx. 125 °C without harmful effects on biological activity. This addition of somatotropin is preferably carried out under vacuum to avoid contamination with water or air bubbles. It is desirable that such addition is carried out by slowly adding finely divided somatotropin to the oil which is being mixed with high shear, in order to provide a uniform dispersion of the somatotropin particles. Size reduction of the somatotropin particles is often desirable and can be carried out e.g. by using a ball mill where a slurry of the somatotropin is mixed with a quantity of stainless steel balls with diameters such as is 0.3 to 0.6 cm. This can advantageously be carried out at the same time as such dispersion in the lower part of a container where mixing is carried out with high shear. This is particularly advantageous for highly charged somatotropins that are difficult to reduce in size to particles with an average particle diameter based on volume of no greater than about 15 µm (ie 50% of the volume of the particles have diameters no greater than about 15 µm). The use of somatotropins with a small particle size (e.g. with an average particle diameter not greater than about 10, preferably not greater than about 5 nm) has been found to be desirable in order to increase the sprayability of preparations according to the invention. By using such a ball mill, the somatotropin particles can easily be reduced to such a preferred mean particle diameter which is no larger than approx. 5 µm. The preparation according to the invention can then be recovered from the ball mill by filtration (advantageously under vacuum).

As previously mentioned, the preparations according to the invention are attractively useful for parenteral administration, e.g. by injection intraperitoneally or, usually more desirably, subcutaneously or intramuscularly. The duration of sustained release is the time period during which the somatotropin is delivered at the required rate for the desired biological effect, usually indicated by the concentration of the somatotropin in the animal's bloodstream. Depending on the specific somatotropin and the specific biological effect, it is desirable that the period of prolonged release is at least approx. 7 days. In other cases, it can be at least approx. 15 days, or more desirable for many applications at least approx. 30 days, or even at least approx. 60 days or longer. According to the present invention, it has thus been found that preparations comprising bovine somatotropin associated with zinc give an average concentration of bovine somatotropin for at least 7 days in the serum of a milk-producing cow injected with a 2.5 ml dose, which is at least approx. 12 ng/ml, which is very beneficial when it comes to increasing milk production and/or the efficiency of the conversion from forage to milk in cattle. To provide an effective amount of bovine somatotropin for the treatment of dairy cows, e.g. to increase milk production, it is desirable to use a preparation that contains at least approx. 300 mg of a zinc-bound somatotropin to provide such an increased serum level of active bovine somatotropin for at least approx. 15 days. It is an important attractive feature of the invention that the zinc-bound somatotropin is advantageously present in a sufficiently high concentration (e.g. at least about 15%) to open the possibility of using a conveniently small volume of the preparation (e.g. about 10 ml or less, eg between 1 and about 3 ml) for easy administration.

Production of biologically active somatropins bound to metal

For the production of biologically active somatropins bound to metal, a somatotropin (e.g. bovine) can be dissolved in a number of different buffered solutions. It is preferably dissolved in an aqueous urea solution buffered with tris(hydroxymethyl)aminomethane (TRIS) or another suitable buffering agent. A desirable upper limit for urea concentration is usually approx. 6M, in some cases a urea concentration of approx. 4.5M preferred. Lower urea concentrations, e.g. 3M or as low as 2M or even IM, can be used but with lower solubility for the somatotropin.

The pH of the buffered urea solution is preferably between 7 and 10.5. Between these pH limits, the recovery of somatotropin precipitated from solution is usually at least 60%. Generally, a higher degree of recovery (eg at least 90%) can be achieved with a pH between 9 and 9.5.

The temperature of the solution during precipitation should be sufficiently low to prevent oligomerization of the somatotropin. It is usually desirable that such temperatures are lower than 10 °C and more preferably lower than 5 °C.

To provide a somatotropin bound to a monovalent metal, the solution (depyrogenated and sterilized as previously mentioned) is treated by diafiltration (or dialysis) to exchange the urea with a solution of the metal bicarbonate (eg, a 25 mM NaHCO 3 solution, pH 9.5) or another suitable salt. Several extractions are preferably carried out with bicarbonate solution to ensure complete extraction of urea. The solution is then treated by further diafiltration with water to remove excess NaHCO 3 indicated by the onset of precipitation for MBS. Extraction of sodium somatotropin by freeze-drying gives a powder of the sodium salt of the somatotropin.

To provide a somatotropin bound to a multivalent metal, the solution (depyrogenated and sterilized as previously mentioned) is brought into contact with a multivalent salt (e.g. zinc salt). Use of an IM solution of zinc chloride has been found to give acceptable precipitated zinc somatotropin from 4.5M urea solution of the somatotropin, although higher or lower concentrations of the chloride may be used. The ZnC^ solution is preferably added slowly, e.g. as in titration, while the somatotropin solution is stirred.

As addition of the ZnCl 2 solution is continued, the somatotropin solution first reaches a gray-white, then a pearly white color when the stoichiometric amount of ZnCl 2 is added. E.g. by adding 4 ml IM ZnCl2 to 400 ml of a solution with pH 9.5 containing approx. 20 mg somatotropin per ml, and 0.09M TRIS in 4.5M urea, a uniform, pearly white zinc somatotropin suspension is formed. Additional ZnCl2 (eg, up to about 10 mL of IM ZnCl2 solution) can be added to ensure complete precipitation.

It is often desirable that the slurry is then diluted to reduce the tendency for the precipitate's particle size to increase. Dilution with up to approx. 3.5 parts by volume of water to approx. IM urea has been found satisfactory in causing the zinc somatotropin particles not to agglomerate. Recovery by diafiltration (or multiple centrifugations and washings) to remove urea, TRIS and zinc chloride ions, followed by freeze-drying yields a powder with particle sizes typically smaller than between 10 and 20 µm.

When the somatotropin is bovine, the particles usually contain between 0.3 and 1% zinc (between approx. 1 and 4 molecules of zinc per molecule of somatotropin). If the rate of zinc addition is increased, there will be larger amounts in the precipitate, e.g. up to 4 to 5%. Such large amounts may be due to further binding of zinc to active acid sites on the somatotropin, e.g. in additional aspartic and/or glutamic acid residues or possibly in histidine residues, or in the carboxyl terminus of the polypeptide. It is not intended that this theory for zinc binding should be regarded as limiting the scope of the invention. Generally, precipitation using a substantially minimal amount of zinc is considered advantageous.

The examples below illustrate particular embodiments and aspects of the invention.

Example 1

This example illustrates the preparation of a bovine somatotropin bound to zinc according to the invention.

Bovine somatotropin with N-terminal methionine (MBS) was prepared as described by Seeburg et al. in "Efficient Bacterial Expression of Bovine and Porcine Growth Hormone", 2(1) DNA 37-45 (1983. The somatotropin was recovered in a usable form by lysing the bacterial cells and then separating the somatotropin from bacterial cell debris.

In a method for extracting MBS, the bacteria were killed by treating with 50% sulfuric acid in a sufficient quantity to lower the pH of the culture liquid to 1.7. The culture liquid was neutralized with NaOH and centrifuged, which gave a cell mass that was suspended in urea, homogenized, cooled to approx. 4 °C (this temperature was maintained until the MBS freeze-drying referred to below), centrifuged and washed three times, dissolved in guanidine hydrochloride (7M), centrifuged to remove insolubles, filtered, passed through a column of G25 Sephadex® where the guanidine was exchanged with urea, filtered and then passed through a DE52 ion exchange column. The volume of the flowing fluid was reduced approx. 30 times using ultrafiltration through hollow fibres. The concentrated solution was passed through a chromatography column with G75 Sephadex®, another volume reduction column with hollow fiber and then dialyzed to exchange the urea first with NaHCO 3 solution and then distilled water to precipitate the MBS. The precipitate was freeze-dried to give a white solid (slightly soluble in water) containing a polypeptide (MBS) with the amino acid sequence NH2-met-phe(l)-pro(2)... leu(126 ... phe( 190rCOOH as stated in the previously mentioned publication by Seeburg et al.

The MBS obtained was dissolved in a solution with 4.5 M urea, 0.09 M TRIS with 21.5 mg MBS per ml, 4 °C and pH 9.5. The MBS solution was depyrogenated by mixing with 0.2 g of mixed anionic/cationic ion exchange resin beads (Biorad AG-501X8) for each ml of sterile MBS solution. The mixture was stirred for approx. 10 minutes at 4°C and then filtered with a 1 µm nylon filter to remove the beads containing adsorbed pyrogens.

The depyrogenated MBS was sterilized by passing the solution through a 0.2 umesh radiation sterilized encapsulated plate filter to remove non-sterile foreign bodies such as bacteria or loose contaminants from previous processing.

MBS was converted to a zinc salt (ZnMBS) by adding IM ZnCl 2 while stirring the depyrogenated MBS solution. The precipitated ZnMBS contained approximately 1% zinc. The solution containing ZnMBS in solid form was then diluted with sterile depyrogenated water to a urea concentration of IM.

ZnMBS was recovered by centrifugation at 10,000 x g for 30 min, while the solution was kept at 4 °C. ZnMBS was slurried in sterile depyrogenated water at 50 ml ZnMBS/ml using high shear mixing. ZnMBS was recovered by centrifugation at 10,000 x g for 30 min, resuspended in sterile depyrogenated water at 50 mg ZnMBS/ml using high shear mixing, and then freeze-dried to obtain a white fluffy powder of sterile ZnMBS.

Example IA

This example illustrates an alternative preparation of ZnMBS.

A depyrogenated and sterilized solution of 1 mg MBS per ml in 4.5M urea, 0.05M TRIS, 10 °C and pH 8.8 was recirculated through a holding vessel using a positive pressure pump. IM ZnCl2 was added to the suction side of the pump until the concentration of the solution was 0.01M ZnCl2 which precipitated ZnMBS. Dilution water was added to give a concentration of 10 mg MBS per ml which gave further precipitation of ZnMBS. The resulting slurry of ZnMBS was then circulated at 25 °C through a hollow fiber membrane for diafiltration, with pores that would allow molecules up to molecular weight 100,000 through, until the concentration reached 40 mg MBS per ml, then water was added to adjust the filtration rate through the membrane until substantially all Zn, urea and TRIS had been removed from the slurry. Water addition was stopped so that the concentration was approx. 80 mg MBS per ml. The concentrated slurry was then freeze-dried, whereby a dry, white powder of ZnMBS with particle sizes in the range from 0.5 to 11 µm was obtained.

Example 2

This example illustrates the preparation of a preparation according to the invention which contains a somatotropin bound to zinc.

One volume of sesame oil (Fisher NF Grade) was added to a 3-neck round-necked flask. An antihydration agent (A1MS) was added to 5% of the total amount

A1MS and sesame oil. The flask was placed in an oil bath at 155°C and stirred to disperse the A1MS as quickly as possible. Stirring was continued for 20 minutes during which the A1MS was completely dissolved in the oil. The flask was removed from the bath, kept under vacuum and allowed to cool to 25°C. On cooling, the solution turned into a thick gel. The cooled gel was fed to a ball mill with a high shear agitator in a layer of stainless steel balls with diameters of 0.32, 0.48 and 0.64 cm. Vacuum was created in the mill and ZnMBS powder (prepared as described in Example 1) was slowly added using a screw feeder until the preparation contained 40% ZnMBS (13.3 weight ratio of ZnMBS to AlMS). Stirring was continued for 6 hours during which the mean diameter of the ZnMBS particles was reduced from 20 µm to 5 µm. The resulting, essentially non-aqueous, gelled oil suspension of ZnMBS was separated from the steel balls by filtration.

Example 3

This example illustrates an effective use of a preparation according to the invention in the long-term release of a polypeptide, i.e. a bovine somatotropin, to increase milk production in dairy cows.

A substantially non-aqueous preparation was prepared essentially as follows

example 2 by dissolving 5% A1MS in sesame oil which had been heated to 155 °C. The oil was cooled, whereby a gelled oil was obtained. ZnMBS was dispersed and mixed into the oil until the preparation contained 32% ZnMBS in an unbroken phase of oil (9.4 in weight ratio between ZnMBS and A1MS). Syringes equipped with 3.8 cm long needles of size 18 were filled with 2.54 g (2.5 ml) of the preparation so that a dose containing 805 mg of ZnMBS was obtained. The preparation had a sprayability of 0.136 ml/second. Blanks of 5% A1MS in sesame oil without the polypeptide were also prepared and 2.4 g were loaded into identical syringes.

The preparations were injected into 23 dairy cows of the Holstein type in the second and third trimesters of their second (or subsequent) lactation period. The cattle were randomly divided into 4 groups of 5 or 6. Two groups were injected intramuscularly (IM) in the buttock area, one with the ZnMBS-containing preparation and the other (a control group) with the blank preparation. At the same time, two other groups were injected subcutaneously (SQ) in the area above the shoulder blade with the ZnMBS-containing preparation or the blank preparation.

Cumulative mean values according to the least squares method for average milk production (adjusted for differences in milk yield before treatment so that the same ratio was maintained) as shown in table 1, where milk production is expressed in kilograms of milk per day. As shown in Table 1, a single IM or SQ injection of an appropriately administered preparation of the invention provides a rapid and long-lasting improvement in milk production at very high levels of statistical significance.

Blood samples were analyzed for bovine somatotropin which, without administration in accordance with the invention, is normally present in the bloodstream of cattle.

Representative analyzes by means of "radioimmunoassay" ("RIA") are shown in Table 2 where the concentrations of bovine somatotropin in blood serum are indicated in nanograms per ml (ng/ml).

Example 4

This example illustrates the effectiveness of preparations according to the invention when it comes to providing long-term release of a somatotropin (MBS) in animals by using several different substances in such preparations.

In this example, ZnMBS preparations were formulated essentially as described in Example 3 using combinations of the following ingredients: Biocompatible oil: Sesame seed or peanut oil Antihydrating agent: A1MS at a concentration of 3% or 5% of oil plus

A1MS.

Somatotropin content: ZnMBS in a concentration of 20%, 30% or 40% of

the entire preparation.

A1MS was dispersed in the oil. The dispersion, after being heated to and held at 155°C for 15 minutes, was allowed to cool to 25°C whereby the oil gelled. ZnMBS was added and dispersed using a high shear mixer (Polytron homogenizer) so that a suspension of ZnMBS was formed in the gelled oil. The suspension was filled into tuberculin syringes with needles of size 18.

By means of subcutaneous injection on the dorsal side of the upper scapular area, the preparations indicated in Table 3 were administered to 16 groups of 8 immunologically suppressed female rats of the Sprague-Dawley type (IFS-D).

Blood samples were analyzed by RIA for bovine somatotropin. Analysis results in table 4 are given in ng/ml blood plasma. Such plasma levels are shown in Table 4 for blood samples taken before injection on day 0 (injection day). Some baseline measurements for rats in Examples 4 through 7 are higher than some baseline and released polypeptide measurements for cows in Example 3. This is due in part to species differences in normal somatotropin levels and in part to the fact that the RIA in Example 3 was more accurate).

Example 5

This example illustrates the effectiveness of preparations according to the invention in terms of long-term release of polypeptide (MBS) using other fatty acid salts of aluminum as antihydration agents. In these preparations, aluminum monolaurate (A1ML) and aluminum monopalmitate (A1MP) were used as antihydrating agents together with sesame and peanut oils.

In this example, gelled oils containing a 3% A1ML or A1MP were produced in essentially the same way as in example 4. ZnMBS was suspended in the gelled oils at a concentration of 30% of the entire preparation (14.3 in weight ratio between ZnMBS and A1ML or A1MP). Each preparation was injected into a group of 8 rats by IFS-D in the amounts indicated in Table 5.

Analyzes of blood samples taken from the rats on the indicated days after injection resulted in the concentrations of bovine sematotropin shown in Table 6, where the readings on day 0 are the starting point for the analyses.

Example 6

This example illustrates the effectiveness of preparations according to the invention when it comes to long-term release of a somatotropin (MBS) where olive oil or corn oil is used.

In this example, gelled oils were prepared essentially as in example 4 by using 3% A1MS based on A1MS plus the oil. The slurries of 30% or 40% ZnMBS were injected into two groups of 8 rats of type IFS-D in the quantities indicated in Table 7.

Analyzes of blood samples taken from the rats on the indicated days after injection gave the concentrations of bovine somatotropin shown in table 8, where the readings on day 0 are the starting point for the analyses.

Example 7

This example illustrates preparations according to the invention which comprise approx. 10% of the somatotropins, MBS and ZnMBS in peanut oil. This example further illustrates that prolonged action of the polypeptide can be increased by using the polypeptide bound to a metal and by using an antihydrating agent. Preparations indicated in Table 9 for injection were prepared essentially as in Example 4.

Example 9

Each preparation was injected subcutaneously into a group of 8 rats of IFS-D in an amount of 300 µl. Analyzes of blood samples taken from the rats on the indicated days after injection indicated plasma concentrations as shown in table 10 where the readings on day 0 are the starting point for the analyses.

Comparison of the results for Groups 30 and 31 illustrates enhancement of sustained release of MBS for at least 7 days using a multivalent metal bound to the somatotropin. Comparison of the results for groups 32 and 33 illustrates the increase in sustained release of MBS using an antihydrating agent when MBS is bound to such a multivalent a metal.

Example 8

This example illustrates preparations according to the invention which contained 10% bovine somatotropin without the presence of an antihydrating agent in each of the following oils: sesame, peanut, corn, olive, saffron, cottonseed, palm, rapeseed and soybean oil. Separate amounts of each oil were stored at each of the following temperatures: 4°C, 25°C, 50°C, 75°C, 100°C and 125°C. ZnMBS was dispersed and mixed with each oil as in Example 2 until the concentration reached 10%. Mixing was continued until the somatotropin has a mean particle diameter not greater than 15 (im. Each preparation has a sprayability greater than 0.1 ml/second.

Example 9

This example illustrates preparations according to the invention prepared as in example 8 with the exception that A1MS was dispersed in each oil at a concentration of 5% based on the oil plus A1MS before the addition of the somatotropin. These preparations have a sprayability greater than 0.1 ml/second.

Example 10

This example illustrates preparations according to the invention prepared as in example 8 with the exception that the addition of the somatotropin was continued until the preparations contained 40% bovine somatotropin. Dispersion and mixing were continued until the somatotropin had an average particle diameter no greater than 15 µm. These preparations have a sprayability greater than 0.03 ml/second.

Example 11

This example illustrates preparations according to the invention prepared as in example 10 with the exception that A1MS was dispersed in each oil at a concentration of 5% based on the oil plus A1MS before the addition of the somatotropin. These preparations have a sprayability greater than 0.03 ml/second.

Example 12

This example illustrates preparations according to the invention which contain 10% of a bovine somatotropin in each of the following oils: sesame, corn, olive, saffron, cottonseed, palm, rapeseed and soybean oil. Each oil was heated to 160 °C and stirred to facilitate dissolution of A1MS. Once 1% A1MS was dissolved, each oil was cooled to 25°C. ZnMBS was dispersed and mixed in the cooled oil as in Example 2 until the concentration reached 10% and the average particle diameter was reduced so that it was no more than 15 µm. Each preparation has a sprayability greater than 0.1 ml/second.

Example 13

This example illustrates preparations according to the invention prepared as in example 12 with the exception that the addition of the somatotropin was continued until each preparation contained 40% somatotropin. The preparations were mixed as in example 2 until the somatotropin had an average particle diameter that was not greater than 15 nm. Each preparation has a sprayability greater than 0.03 ml/second.

Example 14

This example illustrates preparations according to the invention which contain 10% of a bovine somatotropin in the following oils where A1MS is dissolved in a concentration of 5% based on the oil plus A1MS: sesame, peanut, corn, saffron, cottonseed, palm -, rapeseed and soybean oil. Each oil was heated to 160 0 C and stirred to facilitate dissolution of A1MS. Once the A1MS was dissolved, each oil was cooled to 25°C. ZnMBS was then dispersed and mixed in the cooled oil as in Example 2 until the concentration was 10%. The dispersion was further mixed until the somatotropin had an average particle diameter not greater than 15 µm. Each preparation has a sprayability greater than 0.1 ml/second.

Example 15

In this example, preparations according to the invention are prepared which contain 42% of a bovine somatotropin by continuing the addition of the somatotropin to preparations according to example 14 until each preparation contains 42% somatotropin. While the oil was still maintained as an unbroken phase, the somatotropin was dispersed and mixed as in Example 2 until it had an average particle diameter no greater than 15 µm. Each preparation has a sprayability greater than 0.03 ml/second.

Example 16

This example illustrates preparations according to the invention which contain 20% of a bovine somatotropin in the oils used in example 9, but where A1MS is replaced with one of the following antihydrating agents: aluminum distearate or -tristearate, aluminum mono-, -di- or -tripalmitate or -laurate, magnesium mono- or -distearate, -laurate or -palmitate, and calcium mono- or -distearate, -laurate or -palmitate. The antihydrating agent was added to the oil before adding the somatotropin. ZnMBS was added as in Example 2 until the concentration was 20%. Dispersion and mixing were continued until the somatotropin had an average particle diameter no greater than 15 µm. Each preparation has a sprayability greater than 0.03 ml/second.

Example 17

This example illustrates preparations according to the invention which contain other concentrations of a bovine somatotropin in oils corresponding to the types of oils used in example 16, with the exception that the addition of the somatotropin was continued until the concentration was 25%, 30% or 75%. Dispersion and mixing were continued until the somatotropin had an average particle diameter no greater than 15 µm. Each preparation has a sprayability greater than 0.03 ml/second.

Example 18

Essentially the same results as in Examples 8 to 17 were obtained when other bovine somatotropins were used, e.g. those having the following amino acid sequences:

Claims (5)

1. Metal bound somatotropin complex for use in a sustained release preparation for parenteral administration to an animal by injection or implantation to increase the growth or productivity of the animal, characterized in that it comprises the reaction product of bovine, human or porcine somatotropin and polyvalent ions of zinc metal, where the metal constitutes up to 2% by weight of the reaction product. 2. Somatotropin complex according to claim 1, characterized in that the molar ratio between metal and somatotropin in the reaction product is 1:1. 3. Somatotropin complex according to claim 1, characterized in that the metal-bound somatotropin complex is essentially free of unreacted metal. 4. Substantially non-aqueous somatotropin preparation with sustained release characteristics for parenteral administration to an animal to increase the growth or productivity of the animal, characterized in that it comprises bovine, human or porcine somatotropin and a water-soluble compound of a polyvalent ion of zinc metal, where the metal constitutes up to 2% by weight of the preparation and is present in a sufficient amount to prolong the release of somatotropin from the preparation, and is dispersed in a biologically compatible oil, wherein said oil is present in a sufficient amount to form one continuous phase. 5. Preparation according to claim 4, characterized in that the molar ratio between the metal and the somatotropin is 1:1.