Abstract:
The invention relates to sustained release compositions of growth hormones and/or related compounds and multiple water-in oil-in-water emulsions. The invention also relates to methods for increasing and maintaining increased levels of growth hormones and/or related compounds in the blood of treated animals for extended periods of time, increasing weight gains in animals and increasing milk production of lactating animals by the administration of a composition of the invention.

Description:
BACKGROUND OF THE INVENTION 
     Advances in the fields of biotechnology and genetic engineering have resulted in the availability of sufficient quantities of biologically active macromolecules such as growth hormones and/or related compounds to make the administration of these agents on a commercial scale economically feasible. Administration of growth hormones and/or related compounds to animals has been reported to provide beneficial effects such as increasing weight gains, increasing milk production in lactating animals, increasing growth rate, increasing feed efficiency, increasing muscle size, decreasing body fat and improving the lean meat to fat ratio. 
     The above beneficial effects may be accomplished by daily injection or periodic injection of sustained release or prolonged release compositions. Pending Application for United States Letters Patent by S. Cady, R. Fishbein, U. Schroder, H. Erickson, and B. Probasco, Ser. No. 830,158, filed Mar. 20, 1986, and Application for United States Letters Patent of W. Steber, R. Fishbein and S. Cady, Ser. No. 895,608, filed Aug. 11, 1986 and now abandoned, described sustained release compositions utilizing water dispersible carbohydrate polymer-aqueous systems and solid fat and/or wax-oil systems respectively. Prolonged release nonaqueous compositions of polypeptides, preferably associated with metals or metal compounds, and which may additionally contain antihydration agents dispersed in biocompatible oils, are described in European Patent Application No. 85870135.2, published Apr. 4, 1986. 
     Multiple water-in oil-in water emulsions, represented as W/O/W emulsions, are described as suitable vehicles for the administration of chemotherapeutic agents by L. A. Elson, et al., in Rev. Europ. Etudes Clin. Et Biol., 1970, XV, 87-90 and by J. Benoy et al., in Proceedings of the British Pharmacological Society, Mar. 28 and 29, 1972, 135-136. The use of multiple W/O/W emulsions for oral administration of insulin has been reported by M. Schichiri et al., in Diabetes, Vol. 24, No. 11, 971-976 (1975), and Diabetologia, 10,317-321 (1974). 
     U.S. Pat. No. 4,083,798 describes pourable multiple W/O/W emulsion compositions which are stabilized by the presence of 1% to 4% on a weight basis of a water soluble protein and 1 to 4% on a weight basis of a gelling polysaccharide in the external aqueous phase. S. Matsumoto et al., Journal of Colloid and Interface Science, Vol. 77, No. 2, 555-563 (1980), describe the effects of osmotic pressure gradients on the water permeability of oil layers in W/O/W multiple emulsions; and A. Abd-Elbary, et al., Pharm. Ind., No. 9, 964-969 (1984) describe the efficacy of different emulsifying agents for preparing multiple emulsions. 
     It is an object of this invention to provide injectable sustained release compositions of a growth hormone and/or a related compound, wherein the internal aqueous phase contains the growth hormone and/or related compound emulsified in an oil phase which in turn is emulsified in an aqueous phase. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to novel sustained release multiple water-in oil-in water (W 1  /O/W 2 ) emulsions comprising an internal aqueous phase (W 1 ) containing a growth hormone, growth factor, somatomedin, or biologically active fragment or derivative thereof: dispersed in a water immisciable liquid or oil phase (O); dispersed in an external aqueous phase (W 2 ). The invention is also directed to methods for elevating and maintaining elevated blood levels of a biologically active growth hormone, growth factor somatomedin, or a biologically active fragment or derivative thereof for the purpose of increasing weight gains, growth rate, milk production, or muscle size, improving feed efficiency, and/or decreasing body fat and improving lean meat to fat ratio in an animal. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The compositions of the invention comprise on a weight basis an internal aqueous phase (W 1 ) of about 55% to 99.7% water, 0.2% to 5% salts and/or buffers, 0.1% to 40% of growth hormone, growth factor, somatomedin or a biologically active fragment or derivatives thereof, 0% to 40% polyol, glycol or sugar, and 0% to 2% preservatives and/or stabilizers, dispersed in an oil phase (O) of about 65% to 98% pharmaceutically and pharmacologically acceptable oil or water immiscible liquid, 2% to 40% non-ionic surfactant(s), 0% to 15% thickening agent, gelling agent or a mixture thereof, dispersed in a second aqueous phase (W 2 ) of about 38% to 98% water, 0.2% to 5% salts and/or buffers, 2% to 20% non-ionic surfactant(s), 0% to 15% thickening agent, gelling agent, or a mixture thereof, 0% to 2% perservatives and/or stabilizer, 0% to 60% polyol, glycol or a sugar. Preferred compositions of the invention comprise a W 1  /O/W 2  emulsion on a weight ratio basis of from 1/1/1 to 1/3/8 of the various phases as described above. 
     Stabilizers, preservatives, surfactants, glycols, polyols, sugars, thickening agents, gelling agents, salts, buffers and mixtures thereof which are employed in the compositions of the invention normally comprise on a weight basis from 10% to 25% and preferably 14% to 25% of the total composition. These excipients provide maximum stability of the multiple emulsion, adjust the viscosity of the final composition and control the rate of release of the biologically active agent from the inner aqueous phase by providing the appropriate concentration gradient between the inner aqueous phase (W 1 ) and the outer aqueous phase (W 2 ). 
     Preferred salts and buffers employed in the aqueous phases of the invention are those which are normally used in the preparation of phosphate buffered saline (PBS), containing NaH 2  PO 4 .H 2  O (0.025 mol), Na 2  HPO 4  (0.025 mol), and NaCl (0.15 mol), adjusted to pH 7.1; carbonate buffered saline (CBS), containing Na 2  CO 3  (0.025 mol), HaHCO 3  (0.025 mol), and NaCl (0.15 mol), adjusted to pH 9.4; and saline. 
     Preferred stabilizers employed in the compositions of the invention include dehydroacetic acid and salts thereof, preferably the sodium salt; salicylanilide; sorbic acid, boric acid, benzoic acid and salts thereof; sodium nitrite and sodium nitrate. 
     Preferred non-ionic surfactants for use in the compositions of the invention include the sorbitan oleates and stearates, polyethoxylated sorbitan oleates, and block copolymers of ethylene oxide and propylene oxide; with total amounts of from 2% to 20% on a weight basis being distributed between the oil phase (O) and the outer aqueous phase being preferred. 
     A preferred embodiment of this invention is the incorporating of 1% to 10% of sorbitan monooleate, sorbitan trioleate, sorbitan sesquioleate, ethoxylated (5) soya sterol or sorbitan monostearate in the oil phase (O); in conjunction with the incorporation of 1.0% to 10% of polyoxyethylene (20) sorbitan monooleate or a block copolymer of ethylene oxide and propylene oxide in the outer aqueous phase (W 2 ). 
     Thickening agents, gelling agents and sugars useful in the compositions of the invention may be naturally occurring or synthetic in origin. Thickening agents, gelling agents, suspending agents, bulking substances, tonicity modifiers, or sugars with aluminum monostearate, aluminum distearate, aluminum tristearate, gelatin, polyvinyl pyrrolidone, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, polyethylene glycol, sorbitol, mannitol, glycerol, and lactose are preferred. 
     Pharmaceutically and pharmacologically acceptable water immiscible liquids suitable for use as the oil phase of the invention include oils, liquid fats, water immiscible alcohols and glycols or mixtures thereof. 
     Preferred water immisciable liquids for use as the oil phase (O) in the compositions of the invention include fatty acid glycerides and blends thereof which are liquid at ambient temperatures. Representative examples are synthetic oils, light mineral oils, heavy mineral oils, vegetable oils, such as olive, sesame seed, peanut, sunflower seed, soybean, cottonseed, corn, safflower, palm, rapeseed and coconut; animal oils such as fish oils, fish liver oils, sperm oils; or fractions derived therefrom; and mixtures thereof. 
     Biologically active agents suitable for administration in the compositions of the invention include growth hormones, somatomedins, growth factors, and other biological active fragments and derivatives thereof. Preferred agents include bovine, ovine, equine, porcine, avian, and human growth hormones. The term growth hormones encompasses those which are of natural, synthetic, recombinant or biosynthetic origin. 
     The invention is further illustrated by the following non-limiting examples. 
    
    
     EXAMPLE 1 
     Preparation of sustained release growth hormone multiple emulsions compositions 
     Procedures 
     A. Emulsification by Syringe Technique 
     Lyophilized recombinant bovine growth hormone is dissolved in the primary aqueous phase (W 1 ) and then taken up in a 10 mL all glass syringe. The oil phase is taken up into a second syringe. All air is expelled from both syringes and they are connected via a three way stopcock with Luer-Lok fittings (Pharmaseal K75). The two phases are mixed by passing them from one syringe to another for a specific number of exchanges. All of the sample (W 1  /O primary emulsion) is then pushed into one syringe and the secondary aqueous phase (W 2 ) taken up into the second syringe. Multiple emulsification (W 1  /O/W 2 ) is then accomplished by once again passing the contents of the syringe back and forth. Sufficient multiple emulsion is prepared to provide dosage for testing. The emulsions are remixed prior to each injection to insure that a homogeneous dispersion of the primary emulsion is being administered. 
     B. Emulsification by Homogenization 
     Lyophilized recombinant bovine growth hormone is dissolved in the primary aqueous phase (W 1 ) in a beaker and oil phases added to the beaker with continuous homogenization by a Tissumizer (Tekmar, model SDT-1810) at low speed (20-40 V). The W 1  /O primary emulsion formed is then added with homogenization to the beaker containing the external aqueous phase (W 2 ). The multiple emulsion formed is checked by brightfield light microscopy. 
     Utilizing the above procedures with the materials listed in Table I below yields the multiple (W 1  /O/W 2 ) emulsion growth hormone compositions listed in Table II below. 
     
                       TABLE I______________________________________Abbreviation       Material______________________________________K. Alg      Potassium AlginateHVO         Hydrogenated Vegetable OilLMO         Light Mineral OilHMO         Heavy Mineral OilCBS         Carbonate Buffered SalineCB          Carbonate BufferGel         Gelatin Type A, 150 BloomCorn        Corn OilCot         Cotton Seed OilSes         Sesame OilLect        Lecithin UF--HAMS         Aluminum MonostearateDextrin     Carbohydrate (Nadex 772)BW          BeeswaxSq          SqualeneCO          Castor Oil (Trylox-CO5, Emery)CMC         Carboxymethyl cellulosePG          Propylene GlycolSTO         Sorbitan trioleateSMO         Sorbitan monooleateSSO         Sorbitan SesquioleateMMO         Mannide monooleatePSMS        Polyoxyethylene (20) sorbitan       monostearatePSMO        Polyoxyethylene (20) sorbitan       monooleateSMS         Sorbitan monosteatatePSML        Polyoxyethylene (20) sorbitan       monolauratePSE         PoIyoxyethylene (2) stearyl       etherPOE         Polyoxyethylene (2) oleyl etherSLI         Sodium lauriminodipropionateBCP.sub.1   Block copolymer of ethylene-       oxide and propylene oxide       Average molecular weight - 8,350BCP.sub.2   Block copolymer of ethylene-       oxide and propylene oxide       Average molecular weight - 5,000BCP.sub.3   Block copolymer of ethylene-       oxide and propylene oxide       Average molecular weight - 7,700BCP.sub.4   Block copolymer of ethylene-       oxide and propylene oxide       Average molecular weight - 10,800BCP.sub.5   Block copolymer of ethylene-       oxide and propylene oxide       Average molecular weight - 12,500Sorb        Sorbitol aqueous solution USP       (70% w/w)EPS         Ethoxylated (5) Phytosterol______________________________________ 
    
     
                                           TABLE II__________________________________________________________________________Multiple Emulsion Growth Hormone CompositionsCompo-Phase W.sub.1 containing growth hormone                  Phase O            Phase W.sub.2  W.sub.1 /O/W.sub.                                                    2sitionComponents (% w/w)                  Components (% w/w) Components (%                                                    ratio__________________________________________________________________________1    CBS(100)          LMO(90),STO(10)    CBS(93), Sorb(5),PSMO(2)                                                    1/1/1.332    CBS(100)          HMO(96),SMO(10),AMS(2),PSMO(2)                                     CBS(93),PSMO(2),Sorb(5)                                                    1/1/1.333    CBS(100)          HMO(92.3),SMS(7.7) CBS(97),BCP.sub.1 (3)                                                    1/1/1.334    CBS(100)          HMO(89),EPS(11)    CBS(97),BCP.sub.1 (3)                                                    1/1/1.335    CBS(100)          HMO(90),MMO(10)    CBS(93),PSMO(2),Sorb(5)                                                    1/1/26    CBS(100)          HMO(82),Lect.(13),PSMO(5)                                     CBS(91),SMO(2),PSMO(7)                                                    1/1/27    CBS(100)          LMO(88),AMS(1),MNO(10),PSMO(1)                                     CBS(97.8),Gel(0.2),PSMO(2)                                                    1/1/18    CBS(100)          LMO(76),AMS(2),MMO(20),PSMO(2)                                     CBS(97),BCP.sub.1 (3)                                                    1/1/29    CBS(100)          LMO(89),AMS(1),STO(10)                                     CBS(93),Sorb(5),PSMO(2)                                                    1/1/210   K.Alg(0.36),Sorb.(5),PSMO(2),CBS                  HVO(67),MMO(33),   CBS(93),Sorb(5),PSMO(2)                                                    1/1/111   Dextrin(3),CBS(90)                  HMO(87.2),SSO(10.5),PSMS(2.3)                                     CBS(96),Gel(2),PSMO(2)                                                    1/3/212   CBS(100)          LMO(89),AMS(10),STO(1)                                     CBS(96),Gel(2),PSMO(2)                                                    1/3/813   CBS(100)          LMO(89),AMS(1),STO(10)                                     CBS(93),Sorb(5),PSMO(2)                                                    1/1/1.3314   CBS(100)          LMO(89),AMS(1),STO(10)                                     CBS(93).Sorb(5),PSMO(2)                                                    1/1/1.3315   CBS(73),Sorb(25),BCP.sub.1 (2)                  BCP.sub.2 (12.5),Sq(50),BW(37.5)                                     CBS(18.75),Sorb(67.5),                                                    1/1/2                                     PSMO(13.75)16   CBS(67),Sorb(33)  Corn(83.4),CO(16.6)                                     CBS(95.15),CMC(2),PSMO(1),                                                    1/2/2                                     PSML(1),NaCl(0/85)17   CBS(67),Sorb(33)  Cot(83.75),PSE(11.25),POE(5)                                     CBS(83.3)CO(16.6)                                                    1/2/218   CBS (100),        Ses(95),SMO(5),    CBS(90),BCP.sub.3 (5),BCP.sub.4                                     (5)            1.5/2.5__________________________________________________________________________ 
    
     EXAMPLE 2 
     Effectiveness of injectable compositions of the invention 
     The efficacy of injectable compositions of this invention is demonstrated utilizing a hypophysectomized (hypox) rat assay. The hypophysectomized rat does not produce its own growth hormone and is sensitive to injected bovine growth hormone. The response measured is growth over a period of time such as ten days. 
     Each of the hypox albino rats (Taconic Farms, Sprague Dawley derived) is injected with a sufficient quantity of representative compositions prepared in Example 1 to provide a dose of 2400 micrograms of bovine growth hormone in 0.2 mL of W 1  /O/W 1  multiple emulsion. 
     Test Procedure 
     Prior to the test, the animlas are weighed and the animals to be used for the test are selected based on body weight. Only those animals whose body weights are one standard deviation from the mean body weight of the group are selected. The resulting group is then randomly divided into treatment groups consisting of eight rats/group by a computer generated randomization procedure. The test animals are then transferred to a clean cage and housed four rats/cage. On the initial day of the study the test animals are weighed and any animals with excessive weight gain or loss (±grams) are replaced. The animals are then assigned to test groups and treated. 
     At the end of the ten-day test period, total weight gain for each animal is recorded and the average weight gain per rat for each treatment determined. The results of these experiments, which are summarized in Table III below, demonstrate the effectiveness of injectable compositions of this invention. 
     
                                           TABLE III__________________________________________________________________________Efficacy of sustained release compositions of the invention forincreasing weight gains in hypox ratsAverage body weight (g)/animal                Average weight gain (g)/animalCompo-Day   Day      Day         Day Day                Days                    Days                       Days                           Days                              Dayssition0  2  4  7   10 0-2 2-4                       4-7 7-10                              0-10__________________________________________________________________________1    90.3   93.4      98.9         103.4             105.6                3.1 5.4                       4.6 2.1                              15.22    90.0   94.4      98.1         100.3             102.6                4.4 3.8                       2.1 2.4                              12.73    84.8   89.5      92.0         92.5             93.0                4.8 2.5                       0.5 0.5                              6.34    86.4   89.6      93.4         97.4             95.5                3.3 3.8                       4.0 -1.9                              9.15    90.8   93.0      96.8         96.1             98.1                2.3 3.8                       -0.6                           2.0                              7.56    86.0   94.1      95.4         95.4             97.4                6.1 1.3                       0.0 2.0                              9.47    93.8   104.7      105.5         108.3             110.0                10.8                    0.8                       2.8 1.7                              16.18    86.9   91.7      91.7         93.1             95.1                4.9 0.0                       1.4 2.0                              7.39    89.3   92.1      94.8         99.6             102.6                2.9 2.6                       4.9 3.0                              13.410   92.9   95.9      99.3         100.1             101.6                3.0 3.4                       0.9 1.5                              9.811   94.3   103.1      102.3         100.8             100.4                8.9 -0.9                       -1.5                           -0.4                              6.112   91.1   94.0      94.6         98.4             99.1                2.9 0.6                       3.8 0.8                              8.113   94.3   98.0      100.5         105.5             103.6                3.8 2.5                       5.0 -1.9                              9.414   94.6   97.3      103.8         106.4             104.8                2.6 6.5                       2.6 -1.6                              10.115   92.3   94.3      96.9         98.6             98.9                2.0 2.6                       1.8 0.3                              6.716.sup.190.9   91.0      92.7         96.4             94.9                0.1 1.7                       3.7 -1.6                              3.917.sup.189.3   89.6      92.4         92.5             92.0                0.4 2.8                       0.1 -0.5                              2.818   91.9   97.9      97.6         102.4             104.3                6.0 -0.3                       4.8 1.9                              12.4__________________________________________________________________________ .sup.1 Bovine growth hormone dose 1200 micrograms. 
    
     EXAMPLE 4 
     Effectiveness of compositions of the invention for increasing and maintaining increased levels of growth hormone in blood 
     Groups of three wether lambs weighing approximately 35 kg each are treated with the compositions described in Table IV below. 
     Prior to injecting the formulation, one pretreatment blood sample is obtained from each animal at 24 hours before treatment. These animals are acclimated to the facilities and fed daily at 8:00 a.m. Care is taken so as not to excite the sheep any more than necessary, as this may stimulate a natural release of growth hormone. 
     On the day of treatment, blood samples are taken just prior to injection. Each sheep then receives a single injection of the formulation. Blood samples are collected at 0, 2, 4, 6, 24, 48, 72, 96 hours and periodically thereafter. 
     The serum is separated from the clot by centrifugation and the serum frozen and delivered to the Analytical Laboratory for growth hormone by radioimmunoassay procedures. 
     The results of these experiments which are summarized in Table V below demonstrate the effectiveness of the compositions of the invention for increasing and maintaining increased blood levels of growth hormones. Comparable results are obtained with other compositions of the invention. 
     
                       TABLE IV______________________________________               % w/w of  % ofComposition         Phase     Total______________________________________A.      W.sub.1 Phase   Recombinant bovine   growth hormone  12.5      3.75   CBS             87.5      26.3   O Phase   LMO             89.0      27.1   AMS             1.0       0.03   STO             10.0      3.0   W.sub.2 Phase   CBS             93.0      37.1   PSMO            2.0       0.8   Sorb(70%)       5.0       2.0B.      W.sub.1 Phase   Recombinant bovine   growth hormone  7.3       2.8   Gel             13.3      5.1   Water           79.4      30.4   O Phase   SES             91.9      24.9   CO              1.8       0.5   SSO             7.3       1.97   W.sub.2 Phase   Gel             1.0       0.65   Water           79.0      26.9   BCP.sub.5       20.0      6.74C.      W.sub.1 Phase   Recombinant bovine   growth hormone  13.25     2.65   CB              86.75     17.35   O Phase   SES             95.0      28.5   SMO             5.0       1.5   W.sub.2 Phase   BCP.sub.3       5.0       2.5   BCP.sub.4       5.0       2.5   Water           90.0      45.0______________________________________ 
    
     
                       TABLE V______________________________________Bovine growth hormone blood levels in sheep    (ng/mL)    Sheep #Time       1        2         3     Average______________________________________Composition A (2 mL)-24     hrs    7.1      8.0     6.6   ---23    hrs     5.1      4.0     4.8   4.6-22    hrs     5.4      4.5     3.8   4.60      hr      9.6      6.0     7.5   7.72      hrs     222.0    816.0   979.0 672.34      hrs     177.0    505.0   689.0 457.06      hrs     166.0    368.0   468.0 334.01      day     146.0    49.1    77.3  90.82      days    27.9     19.8    21.5  23.13      days    27.9     33.7    28.0  23.24      days    21.7     11.0    22.0  11.66      days    6.6      6.6     7.4   6.98      days    7.8      6.9     9.3   8.010     days    10.1     7.5     7.7   8.413     days    4.7      5.3     7.1   5.715     days    6.5      4.7     4.7   5.317     days    5.3      3.9     8.3   5.820     days    6.4      6.6     5.8   6.322     days    5.7      6.1     7.1   6.324     days    2.8      4.5     6.0   4.4Composition B (5 mL)-24    hrs     2.6      4.0     2.3   3.00      hr      1.5      4.0     2.9   2.81      hr      13.8     10.3    10.4  11.5          11.1     8.3     8.0   9.22      hrs     376.0    66.4    38.5  160.3          20.7     24.6    20.9  22.14      hrs     109.8    102.6   53.3  88.66      hrs     171.8    119.8   156.1 149.21      day     65.1     176.4   319.4 187.02      days    17.1     38.9    67.8  41.33      days    9.9      22.0    31.7  21.24      days    9.6      14.6    38.9  21.05      days    5.1      9.4     28.5  14.36      days    2.2      7.7     48.2  19.48      days    2.0      18.3    98.1  39.510     days    1.5      13.7    80.9  63.413     days    1.7      11.7    81.0  31.515     days    1.8      15.1    73.7  30.217     days    4.0      13.7    73.1  30.3Composition C (2.5 mL)-24    hrs     2.7      2.4     1.8   2.30      hr      2.9      1.8     2.2   2.31      hr      315.5    168.0   196.3 226.62      hrs     551.2    280.6   296.9 376.24      hrs     756.8    462.2   466.7 561.96      hrs     1007.1   593.1   624.6 741.61      day     70.4     91.5    142.5 101.52      days    29.0     36.0    41.1  35.43      days    21.3     23.8    26.2  23.84      days    15.3     11.4    18.5  15.15      days    19.2     8.3     14.3  13.96      days    22.0     5.4     11.9  13.18      days    21.7     8.5     8.7   12.910     days    21.6     12.3    7.2   13.713     days    16.3     19.9    4.1   13.415     days    17.0     19.2    3.1   13.117     days    14.5     17.5    2.2   11.420     days    16.0     14.4    2.3   10.9______________________________________