Patent Publication Number: US-6211152-B1

Title: Formulations for peptide release

Description:
This is a Continuation of Application Ser. No. 08/981,285, filed Dec. 19, 1997, now U.S. Pat. No. 5,925,619, which is a 371 of PCT/AU96/00370, filed Jun. 20, 1996. 
    
    
     The present invention relates to pharmaceutical and veterinary formulations for the sustained release of deslorelin which is an agonist of the peptide gonadotropin releasing hormone (GnRH). Uses of the formulations include prevention of reproductive function, particularly in dogs and cats, and treatment, particularly in humans, of prostate and breast cancer and other diseases or conditions where suppression of testosterone or estradiol levels is beneficial. 
     Uncontrolled reproduction in domestic pets is a world wide problem. In less developed countries, reproduction of domestic cats and dogs is relatively uncontrolled. Sporadic programs of work exist aimed at controlling reproduction in these animals by surgical castration. In the more developed countries, reproduction is controlled more by ovarectomy in females and in some cases, by orchidectomy in males, or by physically locking away animals to prevent mating. 
     Surgical techniques, no matter how minor, carry some risk. Many pet owners are also loathe to have their animal surgically modified and will tolerate the problems of uncontrolled reproduction and associated behaviour. To remove the ability to reproduce from domestic pets without the use of surgery and without resorting to lengthy kennelling procedures has been an objective of the small animal research industry for some years. Drugs which are currently available for this process, are steroid-based drugs. They produce unpleasant side effects, particularly after lengthy use, and they are not widely used. 
     The peptide gonadotrophin releasing hormone (GnRH) has been the subject of intensive research for many years. It is a hypothalamic decapeptide which is synthesised and stored in neurosecretory cells of the medial basal hypothalamus. The releasing hormone is released in a pulsatile manner into the hypophysial portal circulation and is transported to the anterior pituitary. Here, it regulates the secretion of the gonadotrophins, leuteinising hormone (LH) and follicle stimulating hormone (FSH), into the systemic circulation. Thus, GnRH is a humoral link between the neural and endocrine components of reproductive function (for review see Conn PM (ed) 1996 Gonadotropin-releasing hormone Endocrine Review 7:1). GnRH binds to a single class of receptors on gonadotrope cells. Prolonged exposure of these cells to the GnRH results in loss of responsiveness to the hormone, through receptor alteration (reviewed in Hazum E and Conn PM (1988) Endocrine Review 9: 379-866). The outcome of down-regulation of responsiveness to GnRH is suppression of circulating levels of gonadotropins and sex hormones. This has the consequence of suppressing reproductive function and other processes affected by sex hormone levels. 
     For many years, researchers have tried to develop a commercial vaccine, based on forming antibodies to GnRH, to cut this hormone axis and hence act as a contraceptive. The present applicants have commercialised such a vaccine; however, the developed technology is not suitable for contraception in pets. This lack of suitability is due to the biological variation of response in individual pets to a vaccine and the lack of predictability of the length of effect of the vaccine. 
     It is generally accepted in the marketplace that for a pet contraceptive to be successful, it would be preferably efficacious in all treated animals and its length of response time would be predictable. This response should preferably either be for six or twelve months. Reversibility of the effect would be an additional desirable benefit. 
     In 1987, Brian Vickery from Louisiana (Vickery, B. H. and Nestor, J. J. (1987) In LHRH and its Analogues, Part 2, p517-543), demonstrated that overdosing dogs/bitches with the superagonist of GnRH, nafarelin, shut down reproductive function for a variable period of three to eighteen months. The difficulties facing product development in this area have been: 
     (i) to have available a source of GnRH, or an agonist, at a cost effective price; and 
     (ii) to have a cost-effective delivery system for a peptide which releases at a controlled rate over six to twelve months, at a rate and dose that will shut down animals predictably and reliably for six or twelve months. 
     The present applicants have developed a formulation comprising deslorelin as the active agent which, when administered to animals, prevents reproductive function over an extended and predictable period of time. The formulation also allows the restoration of reproductive function following termination of administration. Whilst the formulation is particularly described in relation to dogs, it is believed that the formulation will be useful in other animals such as humans. 
     In addition, the use of GnRH analogues, including deslorelin, for the suppression of hormone levels in humans is well documented. Van Leusden H.A.I.M. (Gynecol Endocrinol 8 (1994) 215-222) has reviewed the use of a variety of GnRH agonist peptides for suppression of estradiol levels in female patients and use for the treatment of endometriosis and leiomyoma. From a survey of a large body of published work, these authors concluded that many GnRH analogues, including deslorelin, were effective in suppressing estradiol levels and hence in treating these sex hormone-accelerated conditions provided that the peptide was delivered so as to maintain a constant minimum blood level. The prerequisite for a peptide to be active was the ability to disturb the pulsatile release of endogenous GnRH. This required a constant minimum plasma level (this level was not defined). They suggested that a mode of delivery was more important than minor differences in potency between different GnRH analogues. These authors also concluded that in a suppressed pituitary, the dose of GnRH analogue needed to maintain suppression gradually decreased with the duration of treatment (also explored in Sandow J and Donnez T (1990) in Brosens I, Jacobs HS and Rennebaum B (eds) LHRH analogues in Gynaecology pp 17-31 Camforth: Parthenon Publishing). 
     Similarly, the use of GnRH agonists including deslorelin, in the treatment of sex hormone dependent tumours, including breast cancer and prostate cancer, has been described. Redding et al, (1984) Proc Natl Acad Sci USA 81 5845-5848 described the use of a GnRH analogue [D Trp 6 ] LH-RH for suppression of prostate cancer in rats and demonstrated that a microencapsulated form of the peptide, delivering a controlled dose over a 30 day period was more effective in suppressing serum testosterone levels and prostate tumour weight than daily subcutaneous administration of equivalent or double doses of the free peptide. The value of this analogue in human prostate cancer patients to suppress testosterone levels and show tumour progression has been demonstrated by Parmar H et al (1985) The Lancet Nov 30, 1201-1205. This one month depot injection of a GnRH agonist has now been registered for use and tested and used widely in the treatment of breast, ovarian and prostate cancer, endometriosis, myoma and in precocious puberty in children, as have other GnRH agonists. (Nelson J R and Corson S L (1993) Fertil Steril 59: 441-3; Paul D, Conte F A, Grumbach, M M and Kaplan S L (1995) J Clin Endocrin Metab 80: 546-551). 
     A three month depot preparation of a GnRH agonist has also been described (Okada H, Doken Y, Ogawa Y and Toguchi H (1994) Pharm Res (US) 11: 1199-1203.). Linear drug release from the injected microspheres was obtained with persistent suppression of serum LH, FSH (rats) and testosterone (rats and dogs) for over 16 weeks. Doses of GnRH analogues used to suppress sex hormone levels in males and females are the same (e.g. Plosker, G. L. and Brogden, R. V. (1994) Drugs Vol. 48, pages 930-967). Thus, the demonstration of suppression of sex hormone levels in one sex is predictive of similar suppression in the other sex. 
     Accordingly, the abovementioned deslorelin formulation developed by the present applicants, is also useful for treating a range of hormone dependent diseases and conditions in animals (including humans) such as those mentioned above. The formulation offers an improved treatment for these hormone dependent diseases and conditions, by continuing to deliver the GnRH analogue over a period of 12 months or more, thus reducing the need for frequent subcutaneous injections or implant insertions. 
     Thus, in a first aspect, the present invention provides a pharmaceutical and/or veterinary formulation comprising deslorelin and an excipient, the formulation being characterised in that, in vitro, it releases deslorelin into phosphate buffered saline, as hereinafter described, at 37° C. at a rate of about 2-80 μg/day for at least 200 days but preferably for at least 300 days. 
     In a second aspect the present invention consists in a method of preventing reproductive function in animals for at least 3 months, the method comprising administering to the animal the formulation of the first aspect of the invention. 
     In a third aspect, the present invention consists in a method of treating a disease or condition for which suppression of sex hormone levels is beneficial in an animal, the method comprising administering to the animal the formulation of the first aspect of the invention. 
     In a preferred embodiment of the present invention the formulation comprises about 2-10%(w/w) deslorelin (on an active basis), about 0.5-2.5%(w/w) lecithin and about 85-97.5%(w/w) stearin. 
     More preferably, the formulation comprises about 5-10% (w/w) deslorelin (on an active basis), about 0.5-1.5% (w/w) lecithin and about 89-94% (w/w) stearin. 
     Particularly preferred formulations are; 
     (I) 94% (w/w) stearin, 5% (w/w) deslorelin (on an active basis) and 1% (w/w) lecithin, and 
     (II) 93% (w/w) stearin, 5% (w/w) deslorelin (on an active basis) and 2% (w/w) lecithin. 
     In a still further preferred embodiment of the present invention the formulation is for administration to humans, or dogs and/or cats. 
     The formulation will typically exist in the form of rods which have been extruded. The rods will then be cut into predetermined lengths for implantation in the animal. As will be readily appreciated the length of rod will determine the rate and dose of deslorelin. As opposed to implanting longer rods more than one rod can be implanted in each animal. 
     The disease or condition referred to in the method of the third aspect of the invention may be any disease or condition wherein reduction of sex hormone (testosterone or estradiol) levels over a prolonged period is beneficial. Examples include prostate cancer, ovarian and breast cancer, benign hormone-dependent disorders such as endometriosis, myoma and premenstrual tension, and precocious puberty in children. 
     Deslorelin is described in U.S. Pat. No. 4,218,439. Deslorelin has the formula [6-D-tryptophan-9-(N-ethyl-L prolinamide)-10-deglycinamide] or P Glutamine—Histidine—Tryptophan—Serine—Tyrosine—D Tryptophan—Leucine—Arginine—Proline—ethylamide. 
     Stearin is partially hydrogenated palm oil. Its principle fatty acids are C16:0(45%) and C18:0(53%). Melting point is about 55° C. 
     Lecithin is phosphatidylcholine. It is a mixture of diglycerides of stearic, palmitic and oleic acids linked to the choline ester of phosphoric acid. Both stearin and lecithin are found in plants and animals. 
     In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples and accompanying figures. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG.  1 : Provides a graph showing the average daily in vitro release profile from three 60 mg rods of formulation I, demonstrating an initial rapid release of the agent and then continued release extending over a prolonged period. 
     FIG.  2 : Provides a graph showing the cumulative in vitro release profile from three 60 mg rods of formulation I, demonstrating the reproducible release observed, extending for at least 250 days. 
     FIG.  3 : Provides graphical results of normal plasma testosterone levels in a control dog. 
     FIGS.  4 - 7 : Show: 
     (i) the effect of deslorelin release from formulation II on down-regulating pituitary function, thereby lowering testosterone levels to zero; 
     (ii) the length of the effect; and 
     (iii) the reversibility of the effect. 
     FIG.  8 : Provides graphical results of plasma testosterone levels from two dogs implanted with rods of the deslorelin formulation. Reduction in the plasma testosterone levels after 13 days indicates contraception. 
     FIGS.  9  and  10 : Provides graphical results of testicular size data from dogs implanted with the deslorelin formulation up to eight months post implantation. Testicular atrophy is seen in response to pituitary down-regulation. 
     FIG.  11 : Provide graphical results of testicular size data from a controlled dog, up to nine weeks post implantation with a placebo (i.e. identical formulation II less deslorelin). 
     FIG.  12  and  13 : Provides graphical results of plasma testosterone levels from dogs implanted with the deslorelin formulation. These dogs were euthanased after it was observed that the formulation was active, in order to collect tissues. 
    
    
     EXAMPLE 1: DOG CONTRACEPTIVE FORMULATION I 
     A formulation comprising 94% stearin, 5% deslorelin (on an active basis) and 1% lecithin was evaluated in dogs. This formulation was produced as follows: 
     Stearin (supplied as free flowing beads of 1 mm or less in diameter made by Vandenberg Foods) and lecithin (supplied as a deep brown viscous syrup from R P Schearer) were hand mixed using a spatula in a small beaker. The deslorelin was then added and thoroughly mixed into the excipients. The mixed material was transferred to the barrel of a ram extruder that has a 1 mm nozzle attached and is equilibrated to 55° C. The ram extrusion pressure is 40 psi. The ram was attached and pressure applied until the product began to extrude. At this point the pressure was backed off and the product allowed to reach 55° C. The product was then extruded—3g over a 30 second period. The resulting exudate was allowed to cool and then broken up and re-extruded through a 1 mm nozzle. This step was included to ensure uniformity of content throughout the matrix. The 1 mm nozzle was then replaced with a 2.3 mm diameter nozzle. The same product temperature equilibration procedure was conducted prior to extrusion. The product was then extruded and after cooling the long rods produced could be sectioned into lengths of the required weight. 
     The rods produced were implanted into male dogs using standard techniques. Results obtained demonstrated that the release of deslorelin from the rods in vitro followed a reproducible path and continued for up to 250 days. In the dogs a continued decline in testicular size was seen for at least 5 months and suppression of plasma testosterone levels for at least 4 months were observed. 
     FIGS. 1 and 2 provide results of in vitro deslorelin release with 60 mg rods. The assay involved immersing the rod into a container with 1 ml of phosphate buffered saline (prepared as described below) which is placed in a reciprocating water bath at 37° C. The saline is replaced daily and the withdrawn saline assayed for deslorelin with HPLC. 
     Phosphate buffered saline (PBS—pH 7.3) referred to herein, is prepared by dissolving 8.00 g of sodium chloride, 1.00 g di-sodium hydrogen phosphate anhydrous, 0.40 g sodium dihydrogen phosphate dihydrate (0.31 g if anhydrous), and 0.05 g sodium azide in 1 litre of deionised water. 
     EXAMPLE 2: Dog Contraceptive Formulation II 
     A formulation comprising 93% stearin, 5% deslorelin (on an active level) and 2% lecithin was evaluated in dogs. This formulation was produced as follows: 
     Stearin beads (ADMUL PO 58 from Quest International Australasia Limited) and lecithin (Topcithin 300, Bronson &amp; Jacobs, Australia) were hand mixed using a spatula in a small beaker. The deslorelin was then added and thoroughly mixed into the excipients. The material was transferred to the barrel of a ram extruder that has a 1 mm nozzle attached and is equilibrated to 55.8° C. The ram extrusion pressure is 40 psi. The ram was then attached and pressure applied until the product began to extrude. At this point the pressure was backed off and the product allowed to reach 55.8° C. The product was then extruded—3g over a 30 second period. The resulting extrudate was allowed to cool and then broken up before re-extruding the mixed granulation through the 1 mm nozzle at 58.3° C. and into an injectable mould that generates a finished rod product that is 2.3 mm in diameter and approximately 25 mm long. The rods are then sterilised by gamma irradiation. 
     The rods produced were implanted into male and female dogs (0.5, 1 or 2×120 mg rod containing 6 mg of deslorelin). The results obtained with the dogs are set out in FIGS. 9 to  13  and Tables 1 to 4. The results show that the formulation is able to suppress testosterone levels in dogs for 12 months or more and in bitches for at least 5 months. Accordingly, the formulation of the present invention is able to prevent reproductive function in dogs over an extended period of time. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Progesterone positive bitches - non pregnant 
               
            
           
           
               
               
               
               
            
               
                   
                 Dogs 
                 Implanted 
                 Observation (after 1 month) 
               
               
                   
                   
               
               
                   
                 BA1/6 
                 May 1996 
                 No post treatment oestrus 
               
               
                   
                 BA2/6 
                 May 1996 
                 No post treatment oestrus 
               
               
                   
                 BA316 
                 May 1996 
                 No post treatment oestrus 
               
               
                   
                 PW1/6 
                 May 1996 
                 Mild oestrus display 
               
               
                   
                   
                   
                 Progesterone low 
               
               
                   
                 PW4/12 
                 May 1996 
                 Progesterone dropped by treatment 
               
               
                   
                   
               
            
           
         
       
     
     Results for this group of 5 bitches demonstrate rapid suppression of reproductive function and of plasma sex hormone levels over the first few weeks post implantation. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Progesterone positive bitches - pregnant 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Observation (after 
               
               
                   
                 Dogs 
                 Implanted 
                 0.5 to 1 months) 
               
               
                   
                   
               
               
                   
                 BA1/61 
                 June 1996 
                 No oestrus 
               
               
                   
                 PW1/12 
                 May 1996 
                 No oestrus 
               
               
                   
                   
               
            
           
         
       
     
     These are controls in pregnant bitches. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Progesterone positive bitches 
               
            
           
           
               
               
               
               
            
               
                   
                 Dogs 
                 Implanted 
                 Observation (after 2 to 5 months) 
               
               
                   
                   
               
               
                   
                 BB1/6 
                 January 1996 
                 Display of oestrus then no activity 
               
               
                   
                 BB2/6 
                 February 1996 
                 Display of oestrus then no activity 
               
               
                   
                 BB3/6 
                 February 1996 
                 Display of oestrus then no activity 
               
               
                   
                 BB4/6 
                 March 1996 
                 Display of oestrus then no activity 
               
               
                   
                 BB1/31 
                 April 1996 
                 Display of oestrus then no activity 
               
               
                   
                 BB1/61 
                 April 1996 
                 Prolonged oestrus 
               
               
                   
                   
               
            
           
         
       
     
     Results for this group of 5 bitches show that reproductive behaviour and plasma hormone levels (not shown) can be suppressed for at least 5 months following implantation. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Implant Safety in dogs 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Return to Fertility 
               
               
                   
                 Dog No. 
                 Histology, at Implant Site 
                 (after 12 months) 
               
               
                   
                   
               
               
                   
                 Dog 46 
                 No cellular changes detected 
                 Yes 
               
               
                   
                 Dog 79 
                 No cellular changes detected 
                 Yes 
               
               
                   
                 Dog 40 
                 No tissue taken 
                 Yes 
               
               
                   
                 Dog 47 
                 No tissue taken 
                 Yes 
               
               
                   
                   
               
            
           
         
       
     
     These results show no negative pathological changes at the implantation site and return to fertility of 4 dogs implanted with deslorelin implants for 12 months.