CA2353798A1 - Controlled release formulation comprising gnrh-ii - Google Patents
Controlled release formulation comprising gnrh-ii Download PDFInfo
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- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
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Abstract
A pharmaceutical formulation for the controlled release of a therapeutic peptide or a salt thereof, which peptide has the sequence pyroGlu-His-Trp-Ser-Xaa1-Gly-Xaa2-Xaa3-Pro-Gly-NH2 wherein Xaa1 is His or Tyr, Xaa2 is Trp or Leu, and Xaa3 is Tyr or Arg, provided that when Xaa1 is Tyr and Xaa2 is Leu, then Xaa3 is not Arg, and which formulation further comprises a pharmaceutically acceptable biodegradable polymer. The formulation can be used for treating bone and prostate disorders.
Description
CONTROLLED RELEASE FORMULATION COMPRISING GNRH-II
FIELD OF INVENTION -The present invention relates to a pharmaceutical preparation that releases a therapeutic agent over an extended period.
BACKGROUND TO THE INVENTION
Studies on the physiology of the hypothalamic-pituitary-gonadal axis have resulted in the recognition of gonadotropin releasing hormone (GnRH. otherwise known as luteinizing hormone releasing hormone, LHRH) as a key regulatory hormone. GnRH
is released by the hypothalamus and acts on the pituitary to stimulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). More recently, a peptide with homology to GnRH has been identified (White et al., Proc. Natl.
Acad.
Sci. USA 95 305-309, 1998). This peptide has been called GnRH-II. The sequences of the two peptides are compared below.
GnRH pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ I.D. No.S) GnRH-II pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NHZ (SEQ I.D. No.6) The name "GnRH-II" is, to some extent, misleading. The new peptide is a separate gene product, and is clearly distinguishable from GnRH in its tissue distribution. It seems unlikely that GnRH-II acts as an endogenous releaser of LH and FSH.
Since no clear evidence for a physiological role for GnRH-II has been presented, no attentionhas been paid to the practical aspects of using this peptide as a therapeutic agent.
SUMMARY OF THE INVENTION
We have now found that GnRH-II has an important role in the function of a number of organs. For example, it influences osteogenesis and it modulates the proliferation of prostatic epithelial cells. Accordingly, we have considered the means by which this agent and its analogues might usefully be delivered in a clinical situation.
and it is an object of the present invention to provide suitable formulations for achieving this purpose. The formulations according to the present invention rely on the use of a SUBSTITUTE SHEET (RULE 26) biodegradable polymer to hold the peptide in a depot, from which it is released into the systemic circulation at a controlled rate. These formulations comprise two key elements, the biologically active peptide and the biodegradable polymer. The biologically active peptide is a decapeptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Gly-Xaa~-Xaa'-Pro-Gly-NHZ (SEQ I.D. No.7) wherein Xaa' is His or Tyr, Xaa~ is Trp or Leu, and Xaa' is Tyr or Arg, provided that when Xaa' is Tyr and Xaa= is Leu, then Xaa' is not Arg. The polymer is any pharmaceutically acceptable biodegradable polymer, and preferably a co-polymer of glycolic and lactic acids. The invention further comprises the use of the formulations for the treament of human pathologies.
Figure 1 shows the effect of increasing doses of GnRH-II on serum calcium concentrations in ovariectomised rats.
DESCRIPTION OF THE INVENTION
As used herein, abbreviations referring to amino acids have their conventional meanings and indicate the natural L-isomer (except for the achiral amino acid glycine).
In a first aspect, the invention as disclosed herein comprises a pharmaceutical formulation that releases a therapeutic peptide at a controlled rate and for an extended period of time (i.e. for a period of at feast one day, preferably several days, and more preferably at least one week), particularly for the treatment of diseases of the bone and prostate. The therapeutic peptide is a decapeptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Gly-Xaa~-Xaa'-Pro-Gly-NHZ (7) SUBSTITUTE SHEET (RULE 26) wherein Xaa' is either His or Tyr. XaaZ is either Trp or Leu. and Xaa' is either Tyr or Arg, provided that when Xaa' is Tyr and Xaa~ is Leu, then Xaa' is not Arg.
Preferably, Xaa' is His, Xaa= is Trp, and Xaa' is Tyr. It will be recognised that such a peptide can form salts with acids (for example, acetic acid, trifluoroacetic acid.
ben2oic acid, hydrochloric acid, phosphoric acid and the like). To the extent that such salts are formed with pharmaceutically acceptable acids. they are included within the scope of the invention.
A second essential component of the formulation is a biodegradable, pharmaeutically acceptable polymer. Such polymers are known in the art. They can either be homopolymers {i.e. polymers of a single monomer) or copolymers (i.e. formed from two or more different monomers). Suitable monomers include amino and hydroxy dervatives of carboxylic acids. In a preferred embodiment of the present invention, the polymer is composed of hydroxyacyl monomeric units, and more preferably of a-hydroxyacyl units. Most preferabiy, the polymer is a poly(glycolic acid), a poly(lactic acid) or a copolymer of glycolic and lactic acids. Such a polymer has the following chemical structure.
R O R O
w0 ~ O O
O R O R
where R is hydrogen in poly(glycolic acid), methyl in poly(lactic acid), and randomly hydrogen or methyl in the copolymer.
Two complementary methods for making the formulation of the present invention can be distinguished. The peptide can either be incorporated into a matrix of the polymer, or, more preferably, it can be encapsulated by the polymer. In this second case, the peptide that is encapsulated may be either a solid or in solution.
It is preferred for the peptide to be a solid.
This formulation is useful in the treatment of human pathologies, including disorders of bone growth (including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status, primary and secondary hyperparathyroidism, disuse osteoporosis. diabetes-related osteoporosis, and glucocorticoid-related SUBSTTTUTE SHEET (RULE Z6) osteoporosis) and prostate growth (including benign prcs;atic hyperplasia and prostate cancer).
In a second aspect, the invention as disclosed herein com,:,rises a method for the treatment of an individual suffering from a disorder of bone or prostate growth, or considered to be at risk of so suffering. This method of treatment comprises the administration to said individual of a therapeutically effective amount of a formulation containing, as an active principal, a peptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Giy-Xaa~-Xaa'-Pro-Gly-NH~ (7) or a pharmaceutically acceptable salt thereof, wherein Xaa'. Xaaz and Xaa' are as defined above, and as a second component, a pharmaceutically acceptable biodegradable polymer, which formulation releases the peptide into the systemic circulation as the polymer is eroded. The method of treatment may comprise a single administration of the formulation, but is more likely to comprise a course of repeated administrations. The frequency of the administrations may be from once per day to once per month. The amount of active peptide in each dose will depend on the dosing schedule and the route of administration. Generally, it will be between one milligram (1 mg) and one gram (1 g). The supervising physician will determine the precise dose depending on the parameters generally considered in the art to be relevant. The formulation is administered by intramuscular or subcutaneous injection.
The peptides that comprise the active agents of the compositions of the present invention can be prepared by the methods generally known in the art. For example, the peptides may be prepared by solid-phase synthesis. This involves the sequential addition of amino acid residues to a resin-bound intermediate according to the following strategy.
1. Formation of resin-bound first intermediate PG-Aaa-OH + FG-Res - PG-Aaa-L-Res Aaa = amino acid PG = protecting group FG = functional group Res = polymeric resin SUBSTITUTE SHEET (RULE 26) L = linker group ( -O- or -NH- ) 2. Deprotection PG-Aaa-L-Res - H-Aaa-L-Res 3. Chain extension PG-Bbb-OH + H-Aaa-L-Res - PG-Bbb-Aaa-L-Res 4. Repeat steps 2 and 3 as necessary PG-Bbb-Aaa-L-Res - - - PG-Nnn-...-Bbb-Aaa-L-Res 5. Cleaveldeprotect PG-Nnn-...-f3bb-Aaa-L-Res - H-Nnn-...-Bbb-Aaa-OH (or -NH2) In step one, a protected amino acid is reacted with a functionalised resin.
The protecting group (PG) is most commonly tert-butyloxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc). The functional group on the resin (FG) is commonly a chloroalkyl group, a hydroxyl group or an amine group. When FG is a chforoalkyl or hydroxyl group, the Pinker group (L) is an oxygen atom ( -O- ).
When FG is an amine group, L is -NH-.
In step two, the protecting group (PG) is removed from the a-amino group. When PG is Boc, this can be accomplished by treating the resin with acids such as trifluoroacetic acid or hydrogen chloride in dichloromethane. When PG is Fmoc, the deprotection can be accomplished by treating the resin with bases such as piperidine.
In step three, the peptide chain is extended by one amino acid residue. A
protected amino acid is coupled to the amine group liberated in step two. Many reagents are known in the art for achieving this conversion. One combination is dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt). Generally, a base will also be necessary. Suitable bases include triethylamine and N.N-diisopropylethylamine. The solvent will generally be dichloromethane, dimethylformamide, or a mixture of these.
SUBSTTTUTE SHEET (RULE 26) If the side chains of the amino acids (Aaa - Nnn) contain reactive groups (for example amino groups. carboxylic acid groups, hydroxyl groups) then these will need protecting. The protecting groups chosen for the side chains are generally those that are stable under the conditions required to remove the protecting group (PG) from the a-amino group. If PG is Fmoc, then the side chain protecting groups can conveniently be based on tert-butyl chemistry. On the other hand, if PG is Boc, then the side chain protecting groups can be based on fluorenylmethyl chemistry.
Other protecting groups known in the art can also be used.
In step four, the deprotection/chain extension cycle is repeated until the desired peptide sequence has been constructed.
In step five, the completed peptide is liberated from the resin. Protecting groups are removed from the side chains either before or after the cleavage. When L is -NH-, the peptide liberated is in the form of the C-terminal amide. When L is -O-, the peptide liberated is often the C-terminal free acid and a second step is required to form the C-terminal amide.
The peptides may also be prepared by solution-phase synthesis, and this may be more convenient when large quantities of material are needed.
The polymers required for the formulation are generally well known in the art.
As stated previously, the formulation may take the form of a simple dispersion of the peptide in a matrix of the polymer, or the peptide may be microencapsulated with the polymer. Dispersions can be prepared by mixing the peptide (as a solid) and the polymer to homogeneity, then compressing the mixture to form a solid mass. It may be necessary to add a binding agent to the mixture in order to achieve a suitably cohesive composition. The mass can then be ground up to give particles suitable for suspension in a biologically compatible liquid (such as water or isotonic saline) and injection.
Microencapsulated formulations can be prepared either from the solid peptide (as a powder) or from a solution, and particularly an aqueous solution, of the peptide. The polymer is first dissolved in a suitable organic solvent. The peptide is then added to this solution and the mixture is vigorously stirred to disperse the peptide in the organic phase. A second organic solvent is then added. This second solvent is SUBSTTTUTE SHEET (RULE 26) chosen to reduce the solubility of the polymer in the organic phase. The polymer comes out of solution to form a coating around the particles of solid peptide (or around the droplets of dispersed aqueous solution). The resultant microcapsules are then hardened by washing to remove traces of the organic solvents. They are then ready to be suspended in an appropriate liquid for administration.
The above general description is further elaborated below in a number of examples.
These are intended to illustrate certain aspects of the invention. They are not intended to be limiting in any way.
EXAMPLES
Example 1 - Synthesis of GnRH-II
1 A. Preparation of resin-bound protected peptide.
pyroGlu-His(Bom)-Trp(CHO)-Ser(Bzl)-His(Bom)-Gly-Trp(CHO)-Tyr(Bzl)-Pro-Gly-Ores This peptide was prepared using standard solid-phase methods starting from Boc-Gly-esterified Merrifield resin (60 g, 1 mmollg). The synthesis was performed in a manual synthesizer, with a total solvent and reagent volume of 300 mL for each operation. The standard deprotection/washlcoupling protocol is summarised in Table 1.
SUBSTITUTE SHEET (RULE 26) Table 1 Step Reagent Time (min)Number of Operations Deprotection of HCIIDCM* 60 ( 1 Boc Washing DCM 2 - 4 ~ 3 Neutralisation 10% DIPEA/DCM 4 ~ 2 Washing DCM 2 - 4 1 Coupling Activated ester60 - 120**1 - 2 Washing DCM 2 - 4 3 * Gaseous hydrogen chloride was bubbled through a suspension of the resin in DCM
** Completeness of reaction was determined by a negative ninhydrin test Benzotriazolyl esters were used as the activated esters throughout the synthesis.
These were prepared from the corresponding protected amino acids by reaction with 1-hydroxybenzotriazole (1 eq.) and dicyclohexyfcarbodiimide (1 eq.). The quantities used (in relation to the resin substitution capacity) are listed in Table 2.
Table 2 Cycle Amino acid derivativeMolar no. excess 1 Boc-Pro-OH 1. 8 2 Boc-Tyr(Bzl}-OH 1. 8 3 Boc-Trp(CHO)-OH 1. 8 4 Boc-Gly-OH 1. 8 Boc-His(Bom)-OH 1. 8 6 Boc-ser(Bzl)-OH 2.0 7 Boc-Trp(CHO)-OH 2.0 8 Boc-His(Bom)-OH 2.0 g pyroGlu-OH 2.0 SUBSTTTUTE SHEET (RULE 26) Following the final coupling. the resin was washed with dichloromethane t3 x 3 L) and dried under reduced pressure at +40°C to constant weight.
Amino acid analysis: Consistent with proposed sequence 1 B. Cleavage and deprotection pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2 (6) The peptidoresin prepared in Example 1A was placed in a linen bag in a pressure vessel. The vessel was then charged with gaseous ammonia to a final pressure of 4 atm. After 72h the excess ammonia was vented and the resin was extracted with acetic acid (3x 1 OOmL) and ethanol (3x 1 OOmL). The combined extracts were degassed with nitrogen, 10% palladium-on-carbon was added, and the mixture was stirred under an atmosphere of hydrogen. When the reaction was complete (as judged by HPLC}, the mixture was filtered and the filtrate was evaporated. The residue was purified by reverse-phase HPLC to give the title compound.
Example 2- Microencapsulation of peptide Copoly(D,L-lactic acid, glycolic acid) with a lactic acidlglycolic acid ratio of 50/50 is used. To a solution of this polymer (3.7g) in dichloromethane (100mL) in a reaction vessel equipped with a stirrer is added GnRH-ll acetate (0.158, prepared by dissolving the peptide of example 1 in acetic acid and lyophilising the resultant solution). The mixture is stirred at 500revolutions/minute, then silicone oil (Dow Corning 360 Medical Fluid~, 45g) is added over 10 minutes. The mixture is then introduced as a thin jet into capryiic-capric acid-triglyceride (Miglyol~ 812, 3.3L) with continuous stirring at 1000revolutionslminute. When addition is complete, stirring is continued for 1 hour, then the microcapsules are collected by filtration, washed twice with isopropanol, and finally dried.
Example 3 -Analysis of the effects of GnRH-II and analogues on Osteogenic cell populations in vitro.
(a) Human osteoblasts were isolated from cancerous bone from orthopaedic surgery (Niisson et al., 1995) according to standard procedures known in the art. The bone SUBSTTTLJTE SHEET (RULE 26) explants were minced into small bone chips and then washed extensively in Dulbecco's modified Eagle's medium (DMEM)IF12 (1:1 Gibco, Paisley, U.K).
These osteoblast like cells, Murine osteoblastic MC3T3-E1 cells and human clonal osteosarcoma cell lines MG-63 (non-mineralising) and SaOS-2 (mineralising osteosarcoma) were cultured in DMEM:F12, 1:1 with the addition of 10% fetal calf serum (FCS, Gibco), fungizone (500mg11), gentamycin sulphate (50mg/l). L-glutamine (2mM) and I-ascorbic acid (100mg/i) in a humidified COZ chamber at 37' C.
(b) Human bone marrow stromal cells were isolated from bone fragments rinsed in phosphate-buffered saline. Bone~marrow cells were collected and spun through a column of Ficoll Hypaque (Kimble et al J. Clin. Invest. 93 1959-1967, 1994) Cells at the interface were pelleted, counted and seeded into 75cmz flasks. The cells were incubated in a humidified COZ chamber at 37°C and the medium changed weekly. At confluence, the cells were harvested using trypsin EDTA and re-seeded in a-minimum essential medium (a-MEM) supplemented with 10% fetal calf serum (FCS, Gibco), penicillin (100UIml), streptomycin (100mglml), fungizone and L-glutamine (2mM).
(c) All cells were serum-starved for 48h before addition of GnRH-I and GnRH-II. Cells were placed in DMEM without phenol red (in order to avoid oestrogen-like effects of phenol red) containing 10% charcoal-stripped serum for 48 hours in 12 well plates.
Dose dependent effects of GnRH-I and GnRH-II and analogues of the peptides were studied following the addition of peptides at final concentrations ranging from 10'9 to 10'~M. 1 mM dibutyryl CAMP was used as a control. The cells were incubated for 24, 48 and 96h with the peptide being replaced every 24 hours.
(d) TO assess the effects of the peptides on cell proliferation, ['HJthymidine was added at 1 mCi/ml for an additional 24hours and ['H]thymidine incorporation was determined. Radioisotope incorporation was determined using a scintillation counter and the results were calculated as cpm/mg of total protein.
(e) Expression of osteoblastic differentiation markers was also determined (Tintut Y et al,. J Biol Chem 273 7547-53, 1998). Total RNA was isolated at several stages io SUBSTITUTE SHEET (RULE 26) before treatment, at 24. 48. 72 and 96 hours after acaition of peptides. Type I
procollagen, osteopontin and 28S RNA (used as an internal control) expression was determined by Northern blot analyses. Alkaline phospha;ase, matrix GLA
protein.
osteoclastin and GAPDH (as an internal control) were determined by RT-PCR with specific primers designed for each gene.
The peptides of the invention caused significant effects at concentrations below 100NM.
Example 4 - Analysis of the effects of GnRH-II and analogues on Osteoclast populations in vitro.
(a) Human clonal cell lines of osteoclast precursors (FLG 29.1 ) were used as an in vitro model of osteoclast differentiation (Gattei V et al., Cell Growth Differ 7 753-63, 1996). In addition, co-cultures of FLG 29.1 and osteoblastic cells (Saos-2) were evaluated for migratory, adhesive, cytochemical, morphological, and biochemical changes. Dose dependent effects of GnRH-I and GnRH-II and analogues of the peptides were studied following addition at final concentrations ranging from 10-9 to 10~M to FLG 29.1 cultures and to co-cultures. Parathyroid hormone was added as a control. Potentiation (or inhibition) of the differentiation of the preosteoclasts (fusion into large multinucleated elements) and a number of other factors were measured (Orlandini ef al,. Cell Tissue Res. 281 33-42, 1995). These included:
1. Positive staining for tartrate-resistant acid phosphatase in FLG 29.1 cells 2. A decrease of the alkaline phosphatase activity expressed by Saos-2 cells 3. The appearance of typical ultrastructural features of mature osteoclasts in FLG
29.1 cells 4. The release into the culture medium of granulocyte-macrophage colony stimulating factor.
5. To assess the effects the peptides on cell proliferation, ['H]thymidine was added at 1 mCilml for an additional 24hours and ['H]thymidine incorporation was determined as described above.
(b) Bone marrow cells removed from human bone fragments were cultured in the presence of 10nM 1,25-(OH)Z vitamin D~ for seven days to generate multinucleated osteoclasts using standard techniques known in the art (Takahashi et at., Endocrinol 122 1473-1482, 1988). The culture medium (a-MEM) was removed and replaced by a fresh phenol red free medium supplemented with antibiotics and 10% charcoal-stripped heat-inactivated FCS containing GnRH-I, GnRH-I! or SUBSTITUTE SHEET (RULE 26) analogues. and the cultures were maintained for a further 24 hours. Floating cells were harvested and osteoclasts stained for tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation i Hughes et al..
Nat. Med.
_2 1132-1135. 1996) 1. Cells were incubated in 0.2M acetate buffer, pH 4.7-5.~~. containing tartaric acid and 2% naphthol AS-BI phosphate (dissolved at 20mg/ml in ethylene glycol monomethyl ether) for l5min at 37°C. The cells were then transferred to a second solution consisting of the same buffer and concentration of tartaric acid with 0.1 % pararosanoiline chloride (hexazotised by mixing with an equal volume of 4% sodium nitrite for 5min at room temperature) for 10min at 37°C.
This treatment causes a red cytoplasmic stain in cells expressing TRAP. Harris' hematoxylin was used as a nuclear counterstain.
2. Apoptotic multinulceated osteoclasts were identified by strong expression of TRAP, larger size than accompanying viable TRAP-positive cells. Confirmation of apoptosis was carried out using acridine orange stain. Viable osteoclasts were counted after fixation in 95% ethanol and TRAP hematoxylin staining, and apoptotic osteoclasts were expressed as a percentage of the total number of multinucleated osteoclasts (viable and apoptotic) in each culture well.
The peptides of the invention caused significant effects at concentrations below 100NM.
Example 5- Expression analysis of GnRH mRNA in osteogenic and osteoclast cell populations Total RNA was extracted from cells cultured as described above:
1. osteoblast like cells, isolated from cancerous bone 2. murine osteoblastic MC3T3-E1 cells 3. MG-63 (non-mineralising) 4. SaOS-2 (mineralising osteosarcoma) 5. human bone marrow stromal cells 6. human FLG 29.1 osteoclast precursor cells 7. multinucleated osteoclasts generated from bone marrow Expression of GnRH-I and GnRH-II was determined by RT-PCR using PCR primers outlined in SEQ I.D. No 1-4. The integrity of the cDNA generated was determined by assessing the relative level of actin amplification.
SUBSTITUTE SHEET (RULE 26) Example 6 - Effect of GnRH-II on bone mineral density in the ovariectomised rat (a) Female adult (8 weeks old, 200-215g) Sprague Dawley rats were bilaterally ovariectomised (OVX). Animals were kept for 4 weeks post-delivery before commencing treatment. Purina rat chow (1.00% calcium. 0.61% phosphorous) and water were provided ad libitum. Each study consisted of 6 weight-matched groups (n = Blgroup).
(b) Treatment started 4 weeks post-OVX. After 4 weeks, a baseline control OVX
group was sacrificed (Group A). The remaining groups were injected once a day with vehicle (Group B), l~cgJkg body weight (Group C), l0~cglkg body weight (Group D), 100~g/kg body weight (Group E) of GnRH-II, and 80~glkg body weight (Group F) of hPTH(1-34).
(c) All rats were weighed every fourth day and dosages adjusted for 50g increase in mean group weight. Rats were given alternate subcutaneous injections of caicein (30mg/kg) or tetracyclin (30mglkg) in 2% sodium bicarbonate-saline, respectively to label mineralization surfaces on days 10, 19 and 26, following treatment with drug.
Bone mineral density was assessed by dual energy x-ray absorptometry-DEXA).
On day 28 serum calcium levels were determined by colorimetric assay using a commercial kit.
(d) Success of OVX was confirmed at necropsy by failure to detect ovarian tissue and by observation of marked atrophy of the uterine horns. Both legs were disarticulated at the hip. The left tibia and femur were cleaned of excess muscle and soft tissue and placed in 70% ethanol. The anterior eminence of the right tibia metaphysis was shaved with a razor blade, barely exposing bone marrow.
Both right femur and tibia were then placed in 10% phosphate-buffered formalin for 24h and transferred to 70% ethanol.
Ovariectomised animals treated daily with 10 and 100itg/kg of GnRH-II and 801rglkg PTH for 28days have pronounced hypercalcemia. Results are shown in Figure 1.
~3 SUBSTITUTE SHEET (RULE 26) Example 7 - Cellular localisation of GnRH-II in paraffin sections of normal rat bone and human bone.
(a) Frozen and/or paraffin-embedded human and rat bone sections were fixed for 36h depending on size (3-5h at room temperature, then approx 24h at 4°C) and then soaked in 0.1M Tris + 5 % EDTA (12.118 + 508 EDTA) pH 7.3 until decalicified.
(b) Sections were then processed for antibody staining (rabbit polycfonal anti-GnRH-II antibody) using standard techniques.
Staining for GnRH-II was observed in platelets, megakaryocytes at the growth plate (especially proliferating chondrocytes). Some staining was also seen in the bone-forming cells particularly in active osetoblasts as well as new osteoid.
Example 1 demonstrates the preparation of the peptides of the invention, which can then be formulated as illustrated in Example 2. Examples 3 to 7 demonstrate the biological activity of the peptides of interest. The scope of the invention is not intended to be limited in any way by these Examples. In particular, it will be realised that variety of controlled release formulations of these peptides can be prepared by varying the polymer and/or the physical nature of the combination of the peptide and polymer.
However, these variations give formulations with equivalent biological properties, and are intended to be within the scope of the invention as defined in the following Claims.
SEQ I.D. Nos. 1 to 4 referred to in Example 5 are as follows CTG CAG CTG CCT GAA GGA C (1 ) GGG CGG GGC GGG GCT CTC G (2) ATT CTA CTG ACT TGG TGC GTG (3) GGA ATA TGT GCA ACT TGG TGT (4) Id SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
<110> Ferring BV
<120> Controlled Release Formulation <130> 40325 <140> CA 2,353,798 <141> 1999-12-02 <150> PCT/GB99/04045 <151> 1999-12-02 <150> GB 9826662.0 <151> 1998-12-03 <160> 7 <170> PatentIn Ver. 2.1 <210> 1 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Synthetic PCR
primer <400> 1 ctgcagctgc ctgaaggag 19 <210> 2 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic PCR
primer <400> 2 gggcggggcg gggctctcg 19 <210> 3 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic PCR
primer <400> 3 attctactga cttggtgcgt g 21 <210> 4 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Synthetic PCR
primer <400> 4 ggaatatgtg caacttggtg t 21 <210> 5 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Analogue of GnRH
<220>
<221> SITE
<222> (1) <223> Glu in first position is pyroGlu <400> 5 Glu His Trp Ser Tyr Gly Leu Arg Pro Gly <210> 6 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Analogue of GnRH-II
<220>
<221> SITE
<222> (1) <223> Glu in first position is pyroGlu <400> 6 Glu His Trp Ser His Gly Trp Tyr Pro Gly <210> 7 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Analogue of GnRH-II
<220>
<221>SITE
<222>(1) <223>Glu first is pyroGlu in position <220>
<221>SITE
<222>(5) <223>Xaa His Tyr is or <220>
<221>SITE
<222>(7) <223>Xaa Trp Leu is or <220>
<221>SITE
<222>(8) <223>Xaa Tyr Arg is or <400>7 Glu is Ser Gly Xaa Pro H Trp Xaa Xaa Gly
FIELD OF INVENTION -The present invention relates to a pharmaceutical preparation that releases a therapeutic agent over an extended period.
BACKGROUND TO THE INVENTION
Studies on the physiology of the hypothalamic-pituitary-gonadal axis have resulted in the recognition of gonadotropin releasing hormone (GnRH. otherwise known as luteinizing hormone releasing hormone, LHRH) as a key regulatory hormone. GnRH
is released by the hypothalamus and acts on the pituitary to stimulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). More recently, a peptide with homology to GnRH has been identified (White et al., Proc. Natl.
Acad.
Sci. USA 95 305-309, 1998). This peptide has been called GnRH-II. The sequences of the two peptides are compared below.
GnRH pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ I.D. No.S) GnRH-II pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NHZ (SEQ I.D. No.6) The name "GnRH-II" is, to some extent, misleading. The new peptide is a separate gene product, and is clearly distinguishable from GnRH in its tissue distribution. It seems unlikely that GnRH-II acts as an endogenous releaser of LH and FSH.
Since no clear evidence for a physiological role for GnRH-II has been presented, no attentionhas been paid to the practical aspects of using this peptide as a therapeutic agent.
SUMMARY OF THE INVENTION
We have now found that GnRH-II has an important role in the function of a number of organs. For example, it influences osteogenesis and it modulates the proliferation of prostatic epithelial cells. Accordingly, we have considered the means by which this agent and its analogues might usefully be delivered in a clinical situation.
and it is an object of the present invention to provide suitable formulations for achieving this purpose. The formulations according to the present invention rely on the use of a SUBSTITUTE SHEET (RULE 26) biodegradable polymer to hold the peptide in a depot, from which it is released into the systemic circulation at a controlled rate. These formulations comprise two key elements, the biologically active peptide and the biodegradable polymer. The biologically active peptide is a decapeptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Gly-Xaa~-Xaa'-Pro-Gly-NHZ (SEQ I.D. No.7) wherein Xaa' is His or Tyr, Xaa~ is Trp or Leu, and Xaa' is Tyr or Arg, provided that when Xaa' is Tyr and Xaa= is Leu, then Xaa' is not Arg. The polymer is any pharmaceutically acceptable biodegradable polymer, and preferably a co-polymer of glycolic and lactic acids. The invention further comprises the use of the formulations for the treament of human pathologies.
Figure 1 shows the effect of increasing doses of GnRH-II on serum calcium concentrations in ovariectomised rats.
DESCRIPTION OF THE INVENTION
As used herein, abbreviations referring to amino acids have their conventional meanings and indicate the natural L-isomer (except for the achiral amino acid glycine).
In a first aspect, the invention as disclosed herein comprises a pharmaceutical formulation that releases a therapeutic peptide at a controlled rate and for an extended period of time (i.e. for a period of at feast one day, preferably several days, and more preferably at least one week), particularly for the treatment of diseases of the bone and prostate. The therapeutic peptide is a decapeptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Gly-Xaa~-Xaa'-Pro-Gly-NHZ (7) SUBSTITUTE SHEET (RULE 26) wherein Xaa' is either His or Tyr. XaaZ is either Trp or Leu. and Xaa' is either Tyr or Arg, provided that when Xaa' is Tyr and Xaa~ is Leu, then Xaa' is not Arg.
Preferably, Xaa' is His, Xaa= is Trp, and Xaa' is Tyr. It will be recognised that such a peptide can form salts with acids (for example, acetic acid, trifluoroacetic acid.
ben2oic acid, hydrochloric acid, phosphoric acid and the like). To the extent that such salts are formed with pharmaceutically acceptable acids. they are included within the scope of the invention.
A second essential component of the formulation is a biodegradable, pharmaeutically acceptable polymer. Such polymers are known in the art. They can either be homopolymers {i.e. polymers of a single monomer) or copolymers (i.e. formed from two or more different monomers). Suitable monomers include amino and hydroxy dervatives of carboxylic acids. In a preferred embodiment of the present invention, the polymer is composed of hydroxyacyl monomeric units, and more preferably of a-hydroxyacyl units. Most preferabiy, the polymer is a poly(glycolic acid), a poly(lactic acid) or a copolymer of glycolic and lactic acids. Such a polymer has the following chemical structure.
R O R O
w0 ~ O O
O R O R
where R is hydrogen in poly(glycolic acid), methyl in poly(lactic acid), and randomly hydrogen or methyl in the copolymer.
Two complementary methods for making the formulation of the present invention can be distinguished. The peptide can either be incorporated into a matrix of the polymer, or, more preferably, it can be encapsulated by the polymer. In this second case, the peptide that is encapsulated may be either a solid or in solution.
It is preferred for the peptide to be a solid.
This formulation is useful in the treatment of human pathologies, including disorders of bone growth (including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status, primary and secondary hyperparathyroidism, disuse osteoporosis. diabetes-related osteoporosis, and glucocorticoid-related SUBSTTTUTE SHEET (RULE Z6) osteoporosis) and prostate growth (including benign prcs;atic hyperplasia and prostate cancer).
In a second aspect, the invention as disclosed herein com,:,rises a method for the treatment of an individual suffering from a disorder of bone or prostate growth, or considered to be at risk of so suffering. This method of treatment comprises the administration to said individual of a therapeutically effective amount of a formulation containing, as an active principal, a peptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Giy-Xaa~-Xaa'-Pro-Gly-NH~ (7) or a pharmaceutically acceptable salt thereof, wherein Xaa'. Xaaz and Xaa' are as defined above, and as a second component, a pharmaceutically acceptable biodegradable polymer, which formulation releases the peptide into the systemic circulation as the polymer is eroded. The method of treatment may comprise a single administration of the formulation, but is more likely to comprise a course of repeated administrations. The frequency of the administrations may be from once per day to once per month. The amount of active peptide in each dose will depend on the dosing schedule and the route of administration. Generally, it will be between one milligram (1 mg) and one gram (1 g). The supervising physician will determine the precise dose depending on the parameters generally considered in the art to be relevant. The formulation is administered by intramuscular or subcutaneous injection.
The peptides that comprise the active agents of the compositions of the present invention can be prepared by the methods generally known in the art. For example, the peptides may be prepared by solid-phase synthesis. This involves the sequential addition of amino acid residues to a resin-bound intermediate according to the following strategy.
1. Formation of resin-bound first intermediate PG-Aaa-OH + FG-Res - PG-Aaa-L-Res Aaa = amino acid PG = protecting group FG = functional group Res = polymeric resin SUBSTITUTE SHEET (RULE 26) L = linker group ( -O- or -NH- ) 2. Deprotection PG-Aaa-L-Res - H-Aaa-L-Res 3. Chain extension PG-Bbb-OH + H-Aaa-L-Res - PG-Bbb-Aaa-L-Res 4. Repeat steps 2 and 3 as necessary PG-Bbb-Aaa-L-Res - - - PG-Nnn-...-Bbb-Aaa-L-Res 5. Cleaveldeprotect PG-Nnn-...-f3bb-Aaa-L-Res - H-Nnn-...-Bbb-Aaa-OH (or -NH2) In step one, a protected amino acid is reacted with a functionalised resin.
The protecting group (PG) is most commonly tert-butyloxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc). The functional group on the resin (FG) is commonly a chloroalkyl group, a hydroxyl group or an amine group. When FG is a chforoalkyl or hydroxyl group, the Pinker group (L) is an oxygen atom ( -O- ).
When FG is an amine group, L is -NH-.
In step two, the protecting group (PG) is removed from the a-amino group. When PG is Boc, this can be accomplished by treating the resin with acids such as trifluoroacetic acid or hydrogen chloride in dichloromethane. When PG is Fmoc, the deprotection can be accomplished by treating the resin with bases such as piperidine.
In step three, the peptide chain is extended by one amino acid residue. A
protected amino acid is coupled to the amine group liberated in step two. Many reagents are known in the art for achieving this conversion. One combination is dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt). Generally, a base will also be necessary. Suitable bases include triethylamine and N.N-diisopropylethylamine. The solvent will generally be dichloromethane, dimethylformamide, or a mixture of these.
SUBSTTTUTE SHEET (RULE 26) If the side chains of the amino acids (Aaa - Nnn) contain reactive groups (for example amino groups. carboxylic acid groups, hydroxyl groups) then these will need protecting. The protecting groups chosen for the side chains are generally those that are stable under the conditions required to remove the protecting group (PG) from the a-amino group. If PG is Fmoc, then the side chain protecting groups can conveniently be based on tert-butyl chemistry. On the other hand, if PG is Boc, then the side chain protecting groups can be based on fluorenylmethyl chemistry.
Other protecting groups known in the art can also be used.
In step four, the deprotection/chain extension cycle is repeated until the desired peptide sequence has been constructed.
In step five, the completed peptide is liberated from the resin. Protecting groups are removed from the side chains either before or after the cleavage. When L is -NH-, the peptide liberated is in the form of the C-terminal amide. When L is -O-, the peptide liberated is often the C-terminal free acid and a second step is required to form the C-terminal amide.
The peptides may also be prepared by solution-phase synthesis, and this may be more convenient when large quantities of material are needed.
The polymers required for the formulation are generally well known in the art.
As stated previously, the formulation may take the form of a simple dispersion of the peptide in a matrix of the polymer, or the peptide may be microencapsulated with the polymer. Dispersions can be prepared by mixing the peptide (as a solid) and the polymer to homogeneity, then compressing the mixture to form a solid mass. It may be necessary to add a binding agent to the mixture in order to achieve a suitably cohesive composition. The mass can then be ground up to give particles suitable for suspension in a biologically compatible liquid (such as water or isotonic saline) and injection.
Microencapsulated formulations can be prepared either from the solid peptide (as a powder) or from a solution, and particularly an aqueous solution, of the peptide. The polymer is first dissolved in a suitable organic solvent. The peptide is then added to this solution and the mixture is vigorously stirred to disperse the peptide in the organic phase. A second organic solvent is then added. This second solvent is SUBSTTTUTE SHEET (RULE 26) chosen to reduce the solubility of the polymer in the organic phase. The polymer comes out of solution to form a coating around the particles of solid peptide (or around the droplets of dispersed aqueous solution). The resultant microcapsules are then hardened by washing to remove traces of the organic solvents. They are then ready to be suspended in an appropriate liquid for administration.
The above general description is further elaborated below in a number of examples.
These are intended to illustrate certain aspects of the invention. They are not intended to be limiting in any way.
EXAMPLES
Example 1 - Synthesis of GnRH-II
1 A. Preparation of resin-bound protected peptide.
pyroGlu-His(Bom)-Trp(CHO)-Ser(Bzl)-His(Bom)-Gly-Trp(CHO)-Tyr(Bzl)-Pro-Gly-Ores This peptide was prepared using standard solid-phase methods starting from Boc-Gly-esterified Merrifield resin (60 g, 1 mmollg). The synthesis was performed in a manual synthesizer, with a total solvent and reagent volume of 300 mL for each operation. The standard deprotection/washlcoupling protocol is summarised in Table 1.
SUBSTITUTE SHEET (RULE 26) Table 1 Step Reagent Time (min)Number of Operations Deprotection of HCIIDCM* 60 ( 1 Boc Washing DCM 2 - 4 ~ 3 Neutralisation 10% DIPEA/DCM 4 ~ 2 Washing DCM 2 - 4 1 Coupling Activated ester60 - 120**1 - 2 Washing DCM 2 - 4 3 * Gaseous hydrogen chloride was bubbled through a suspension of the resin in DCM
** Completeness of reaction was determined by a negative ninhydrin test Benzotriazolyl esters were used as the activated esters throughout the synthesis.
These were prepared from the corresponding protected amino acids by reaction with 1-hydroxybenzotriazole (1 eq.) and dicyclohexyfcarbodiimide (1 eq.). The quantities used (in relation to the resin substitution capacity) are listed in Table 2.
Table 2 Cycle Amino acid derivativeMolar no. excess 1 Boc-Pro-OH 1. 8 2 Boc-Tyr(Bzl}-OH 1. 8 3 Boc-Trp(CHO)-OH 1. 8 4 Boc-Gly-OH 1. 8 Boc-His(Bom)-OH 1. 8 6 Boc-ser(Bzl)-OH 2.0 7 Boc-Trp(CHO)-OH 2.0 8 Boc-His(Bom)-OH 2.0 g pyroGlu-OH 2.0 SUBSTTTUTE SHEET (RULE 26) Following the final coupling. the resin was washed with dichloromethane t3 x 3 L) and dried under reduced pressure at +40°C to constant weight.
Amino acid analysis: Consistent with proposed sequence 1 B. Cleavage and deprotection pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2 (6) The peptidoresin prepared in Example 1A was placed in a linen bag in a pressure vessel. The vessel was then charged with gaseous ammonia to a final pressure of 4 atm. After 72h the excess ammonia was vented and the resin was extracted with acetic acid (3x 1 OOmL) and ethanol (3x 1 OOmL). The combined extracts were degassed with nitrogen, 10% palladium-on-carbon was added, and the mixture was stirred under an atmosphere of hydrogen. When the reaction was complete (as judged by HPLC}, the mixture was filtered and the filtrate was evaporated. The residue was purified by reverse-phase HPLC to give the title compound.
Example 2- Microencapsulation of peptide Copoly(D,L-lactic acid, glycolic acid) with a lactic acidlglycolic acid ratio of 50/50 is used. To a solution of this polymer (3.7g) in dichloromethane (100mL) in a reaction vessel equipped with a stirrer is added GnRH-ll acetate (0.158, prepared by dissolving the peptide of example 1 in acetic acid and lyophilising the resultant solution). The mixture is stirred at 500revolutions/minute, then silicone oil (Dow Corning 360 Medical Fluid~, 45g) is added over 10 minutes. The mixture is then introduced as a thin jet into capryiic-capric acid-triglyceride (Miglyol~ 812, 3.3L) with continuous stirring at 1000revolutionslminute. When addition is complete, stirring is continued for 1 hour, then the microcapsules are collected by filtration, washed twice with isopropanol, and finally dried.
Example 3 -Analysis of the effects of GnRH-II and analogues on Osteogenic cell populations in vitro.
(a) Human osteoblasts were isolated from cancerous bone from orthopaedic surgery (Niisson et al., 1995) according to standard procedures known in the art. The bone SUBSTTTLJTE SHEET (RULE 26) explants were minced into small bone chips and then washed extensively in Dulbecco's modified Eagle's medium (DMEM)IF12 (1:1 Gibco, Paisley, U.K).
These osteoblast like cells, Murine osteoblastic MC3T3-E1 cells and human clonal osteosarcoma cell lines MG-63 (non-mineralising) and SaOS-2 (mineralising osteosarcoma) were cultured in DMEM:F12, 1:1 with the addition of 10% fetal calf serum (FCS, Gibco), fungizone (500mg11), gentamycin sulphate (50mg/l). L-glutamine (2mM) and I-ascorbic acid (100mg/i) in a humidified COZ chamber at 37' C.
(b) Human bone marrow stromal cells were isolated from bone fragments rinsed in phosphate-buffered saline. Bone~marrow cells were collected and spun through a column of Ficoll Hypaque (Kimble et al J. Clin. Invest. 93 1959-1967, 1994) Cells at the interface were pelleted, counted and seeded into 75cmz flasks. The cells were incubated in a humidified COZ chamber at 37°C and the medium changed weekly. At confluence, the cells were harvested using trypsin EDTA and re-seeded in a-minimum essential medium (a-MEM) supplemented with 10% fetal calf serum (FCS, Gibco), penicillin (100UIml), streptomycin (100mglml), fungizone and L-glutamine (2mM).
(c) All cells were serum-starved for 48h before addition of GnRH-I and GnRH-II. Cells were placed in DMEM without phenol red (in order to avoid oestrogen-like effects of phenol red) containing 10% charcoal-stripped serum for 48 hours in 12 well plates.
Dose dependent effects of GnRH-I and GnRH-II and analogues of the peptides were studied following the addition of peptides at final concentrations ranging from 10'9 to 10'~M. 1 mM dibutyryl CAMP was used as a control. The cells were incubated for 24, 48 and 96h with the peptide being replaced every 24 hours.
(d) TO assess the effects of the peptides on cell proliferation, ['HJthymidine was added at 1 mCi/ml for an additional 24hours and ['H]thymidine incorporation was determined. Radioisotope incorporation was determined using a scintillation counter and the results were calculated as cpm/mg of total protein.
(e) Expression of osteoblastic differentiation markers was also determined (Tintut Y et al,. J Biol Chem 273 7547-53, 1998). Total RNA was isolated at several stages io SUBSTITUTE SHEET (RULE 26) before treatment, at 24. 48. 72 and 96 hours after acaition of peptides. Type I
procollagen, osteopontin and 28S RNA (used as an internal control) expression was determined by Northern blot analyses. Alkaline phospha;ase, matrix GLA
protein.
osteoclastin and GAPDH (as an internal control) were determined by RT-PCR with specific primers designed for each gene.
The peptides of the invention caused significant effects at concentrations below 100NM.
Example 4 - Analysis of the effects of GnRH-II and analogues on Osteoclast populations in vitro.
(a) Human clonal cell lines of osteoclast precursors (FLG 29.1 ) were used as an in vitro model of osteoclast differentiation (Gattei V et al., Cell Growth Differ 7 753-63, 1996). In addition, co-cultures of FLG 29.1 and osteoblastic cells (Saos-2) were evaluated for migratory, adhesive, cytochemical, morphological, and biochemical changes. Dose dependent effects of GnRH-I and GnRH-II and analogues of the peptides were studied following addition at final concentrations ranging from 10-9 to 10~M to FLG 29.1 cultures and to co-cultures. Parathyroid hormone was added as a control. Potentiation (or inhibition) of the differentiation of the preosteoclasts (fusion into large multinucleated elements) and a number of other factors were measured (Orlandini ef al,. Cell Tissue Res. 281 33-42, 1995). These included:
1. Positive staining for tartrate-resistant acid phosphatase in FLG 29.1 cells 2. A decrease of the alkaline phosphatase activity expressed by Saos-2 cells 3. The appearance of typical ultrastructural features of mature osteoclasts in FLG
29.1 cells 4. The release into the culture medium of granulocyte-macrophage colony stimulating factor.
5. To assess the effects the peptides on cell proliferation, ['H]thymidine was added at 1 mCilml for an additional 24hours and ['H]thymidine incorporation was determined as described above.
(b) Bone marrow cells removed from human bone fragments were cultured in the presence of 10nM 1,25-(OH)Z vitamin D~ for seven days to generate multinucleated osteoclasts using standard techniques known in the art (Takahashi et at., Endocrinol 122 1473-1482, 1988). The culture medium (a-MEM) was removed and replaced by a fresh phenol red free medium supplemented with antibiotics and 10% charcoal-stripped heat-inactivated FCS containing GnRH-I, GnRH-I! or SUBSTITUTE SHEET (RULE 26) analogues. and the cultures were maintained for a further 24 hours. Floating cells were harvested and osteoclasts stained for tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation i Hughes et al..
Nat. Med.
_2 1132-1135. 1996) 1. Cells were incubated in 0.2M acetate buffer, pH 4.7-5.~~. containing tartaric acid and 2% naphthol AS-BI phosphate (dissolved at 20mg/ml in ethylene glycol monomethyl ether) for l5min at 37°C. The cells were then transferred to a second solution consisting of the same buffer and concentration of tartaric acid with 0.1 % pararosanoiline chloride (hexazotised by mixing with an equal volume of 4% sodium nitrite for 5min at room temperature) for 10min at 37°C.
This treatment causes a red cytoplasmic stain in cells expressing TRAP. Harris' hematoxylin was used as a nuclear counterstain.
2. Apoptotic multinulceated osteoclasts were identified by strong expression of TRAP, larger size than accompanying viable TRAP-positive cells. Confirmation of apoptosis was carried out using acridine orange stain. Viable osteoclasts were counted after fixation in 95% ethanol and TRAP hematoxylin staining, and apoptotic osteoclasts were expressed as a percentage of the total number of multinucleated osteoclasts (viable and apoptotic) in each culture well.
The peptides of the invention caused significant effects at concentrations below 100NM.
Example 5- Expression analysis of GnRH mRNA in osteogenic and osteoclast cell populations Total RNA was extracted from cells cultured as described above:
1. osteoblast like cells, isolated from cancerous bone 2. murine osteoblastic MC3T3-E1 cells 3. MG-63 (non-mineralising) 4. SaOS-2 (mineralising osteosarcoma) 5. human bone marrow stromal cells 6. human FLG 29.1 osteoclast precursor cells 7. multinucleated osteoclasts generated from bone marrow Expression of GnRH-I and GnRH-II was determined by RT-PCR using PCR primers outlined in SEQ I.D. No 1-4. The integrity of the cDNA generated was determined by assessing the relative level of actin amplification.
SUBSTITUTE SHEET (RULE 26) Example 6 - Effect of GnRH-II on bone mineral density in the ovariectomised rat (a) Female adult (8 weeks old, 200-215g) Sprague Dawley rats were bilaterally ovariectomised (OVX). Animals were kept for 4 weeks post-delivery before commencing treatment. Purina rat chow (1.00% calcium. 0.61% phosphorous) and water were provided ad libitum. Each study consisted of 6 weight-matched groups (n = Blgroup).
(b) Treatment started 4 weeks post-OVX. After 4 weeks, a baseline control OVX
group was sacrificed (Group A). The remaining groups were injected once a day with vehicle (Group B), l~cgJkg body weight (Group C), l0~cglkg body weight (Group D), 100~g/kg body weight (Group E) of GnRH-II, and 80~glkg body weight (Group F) of hPTH(1-34).
(c) All rats were weighed every fourth day and dosages adjusted for 50g increase in mean group weight. Rats were given alternate subcutaneous injections of caicein (30mg/kg) or tetracyclin (30mglkg) in 2% sodium bicarbonate-saline, respectively to label mineralization surfaces on days 10, 19 and 26, following treatment with drug.
Bone mineral density was assessed by dual energy x-ray absorptometry-DEXA).
On day 28 serum calcium levels were determined by colorimetric assay using a commercial kit.
(d) Success of OVX was confirmed at necropsy by failure to detect ovarian tissue and by observation of marked atrophy of the uterine horns. Both legs were disarticulated at the hip. The left tibia and femur were cleaned of excess muscle and soft tissue and placed in 70% ethanol. The anterior eminence of the right tibia metaphysis was shaved with a razor blade, barely exposing bone marrow.
Both right femur and tibia were then placed in 10% phosphate-buffered formalin for 24h and transferred to 70% ethanol.
Ovariectomised animals treated daily with 10 and 100itg/kg of GnRH-II and 801rglkg PTH for 28days have pronounced hypercalcemia. Results are shown in Figure 1.
~3 SUBSTITUTE SHEET (RULE 26) Example 7 - Cellular localisation of GnRH-II in paraffin sections of normal rat bone and human bone.
(a) Frozen and/or paraffin-embedded human and rat bone sections were fixed for 36h depending on size (3-5h at room temperature, then approx 24h at 4°C) and then soaked in 0.1M Tris + 5 % EDTA (12.118 + 508 EDTA) pH 7.3 until decalicified.
(b) Sections were then processed for antibody staining (rabbit polycfonal anti-GnRH-II antibody) using standard techniques.
Staining for GnRH-II was observed in platelets, megakaryocytes at the growth plate (especially proliferating chondrocytes). Some staining was also seen in the bone-forming cells particularly in active osetoblasts as well as new osteoid.
Example 1 demonstrates the preparation of the peptides of the invention, which can then be formulated as illustrated in Example 2. Examples 3 to 7 demonstrate the biological activity of the peptides of interest. The scope of the invention is not intended to be limited in any way by these Examples. In particular, it will be realised that variety of controlled release formulations of these peptides can be prepared by varying the polymer and/or the physical nature of the combination of the peptide and polymer.
However, these variations give formulations with equivalent biological properties, and are intended to be within the scope of the invention as defined in the following Claims.
SEQ I.D. Nos. 1 to 4 referred to in Example 5 are as follows CTG CAG CTG CCT GAA GGA C (1 ) GGG CGG GGC GGG GCT CTC G (2) ATT CTA CTG ACT TGG TGC GTG (3) GGA ATA TGT GCA ACT TGG TGT (4) Id SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
<110> Ferring BV
<120> Controlled Release Formulation <130> 40325 <140> CA 2,353,798 <141> 1999-12-02 <150> PCT/GB99/04045 <151> 1999-12-02 <150> GB 9826662.0 <151> 1998-12-03 <160> 7 <170> PatentIn Ver. 2.1 <210> 1 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Synthetic PCR
primer <400> 1 ctgcagctgc ctgaaggag 19 <210> 2 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic PCR
primer <400> 2 gggcggggcg gggctctcg 19 <210> 3 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic PCR
primer <400> 3 attctactga cttggtgcgt g 21 <210> 4 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Synthetic PCR
primer <400> 4 ggaatatgtg caacttggtg t 21 <210> 5 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Analogue of GnRH
<220>
<221> SITE
<222> (1) <223> Glu in first position is pyroGlu <400> 5 Glu His Trp Ser Tyr Gly Leu Arg Pro Gly <210> 6 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Analogue of GnRH-II
<220>
<221> SITE
<222> (1) <223> Glu in first position is pyroGlu <400> 6 Glu His Trp Ser His Gly Trp Tyr Pro Gly <210> 7 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Analogue of GnRH-II
<220>
<221>SITE
<222>(1) <223>Glu first is pyroGlu in position <220>
<221>SITE
<222>(5) <223>Xaa His Tyr is or <220>
<221>SITE
<222>(7) <223>Xaa Trp Leu is or <220>
<221>SITE
<222>(8) <223>Xaa Tyr Arg is or <400>7 Glu is Ser Gly Xaa Pro H Trp Xaa Xaa Gly
Claims (13)
1. A pharmaceutical formulation for the controlled release of a therapeutic peptide or a salt thereof, which peptide has the sequence pyroGlu-His-Trp-Ser-Xaa1-Gly-Xaa2-Xaa3-Pro-Gly-NH2 wherein Xaa1 is His or Tyr, Xaa2 is Trp or Leu, and Xaa3 is Tyr or Arg, provided that when Xaa1 is Tyr and Xaa2 is Leu, then Xaa3 is not Arg, and which formulation further comprises a pharmaceutically acceptable biodegradable polymer.
2. The pharmaceutical composition according to Claim 1, wherein the peptide is pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2
3. The formulation according to Claim 1, wherein the polymer is a polymer of a hydroxy derivative of a carboxylic acid, or a copolymer of such derivatives.
4. The formulation according to Claim 3, wherein the polymer is a polymer of glycolic acid, a polymer of lactic acid, or a copolymer of lactic and glycolic acids.
5. The formulation according to Claim 1 wherein the peptide is microencapsulated by the polymer.
6. A method for the treatment of a human medical condition, which method comprises the administration to an individual in need of such treatment of a therapeutically effective amount of a controlled release formulation of a peptide according to any of the preceding Claims.
7. A formulation according to any of claims 1 to 5 for treatment of or for protection against disorder of bone growth or disorder of prostate growth.
8. The use of a peptide or salt as defined in claim 1 or 2, together with a pharmaceutically acceptable biodegradable polymer, for the preparation of a controlled release medicament for the treatment of or protection against disorder of bone growth or disorder of prostate growth,
9. A use according to claim 8 wherein said polymer is [a] a polymer of a hydroxy derivative of a carboxylic acid, or a co-polymer of such derivatives, or [b] a polymer of glycolic acid, a polymer of lactic acid, or a co-polymer of lactic and glycolic acids.
10. A use according to claim 8 or 9 wherein said disorder is selected from age-related osteoporosis, osteoporosis associated with post-menopausal hormone status, primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, glucocorticoid-related osteoporosis, benign prostatic hyperplasia and prostate cancer.
11. A formulation according to claim 7 for the treatment of or protection against disorder selected from age-related osteoporosis, osteoporosis associated with post-menopausal hormone status, primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, glucocorticoid-related osteoporosis, benign prostatic hyperplasia and prostate cancer.
12. A method for treating or protecting against a human disorder of bone growth or of prostate growth, which method comprises the administration to an individual in need of such treatment or protection of a therapeutically effective amount on a formulation according to any of claims 1 to 5.
13. A method according to claim 12 wherein said disorder is selected from age-related osteoporosis, osteoporosis associated with post-menopausal hormone status, primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, glucocorticoid-related osteoporosis, benign prostatic hyperplasia and prostate cancer.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9826662A GB2344287A (en) | 1998-12-03 | 1998-12-03 | Controlled release pharmaceutical formulation |
| GB9826662.0 | 1998-12-03 | ||
| PCT/GB1999/004045 WO2000032218A1 (en) | 1998-12-03 | 1999-12-02 | Controlled release formulation comprising gnrh-ii |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2353798A1 true CA2353798A1 (en) | 2000-06-08 |
Family
ID=10843631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002353798A Abandoned CA2353798A1 (en) | 1998-12-03 | 1999-12-02 | Controlled release formulation comprising gnrh-ii |
Country Status (22)
| Country | Link |
|---|---|
| EP (1) | EP1140133A1 (en) |
| JP (1) | JP2002531411A (en) |
| KR (1) | KR20010089538A (en) |
| CN (1) | CN1332635A (en) |
| AU (1) | AU770676B2 (en) |
| BR (1) | BR9915943A (en) |
| CA (1) | CA2353798A1 (en) |
| CZ (1) | CZ20011893A3 (en) |
| EE (1) | EE200100293A (en) |
| GB (1) | GB2344287A (en) |
| HR (1) | HRP20010421A2 (en) |
| HU (1) | HUP0104943A3 (en) |
| IL (1) | IL143496A0 (en) |
| MX (1) | MXPA01005543A (en) |
| NO (1) | NO20012636L (en) |
| NZ (1) | NZ511984A (en) |
| PL (1) | PL348575A1 (en) |
| RU (1) | RU2233170C2 (en) |
| SK (1) | SK7552001A3 (en) |
| TR (1) | TR200102273T2 (en) |
| WO (1) | WO2000032218A1 (en) |
| ZA (1) | ZA200104530B (en) |
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| WO2011032099A1 (en) | 2009-09-11 | 2011-03-17 | The Board Of Trustees Of The University Of Illinois | Methods of treating diastolic dysfunction and related conditions |
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| CN103068842B (en) | 2010-06-16 | 2016-10-19 | 印第安纳大学研究及科技有限公司 | Insulin receptor INSR is had highly active single-chain insulin agonist |
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| EP2655401B1 (en) | 2010-12-20 | 2016-03-09 | The Regents of the University of Michigan | Inhibitors of the epidermal growth factor receptor-heat shock protein 90 binding interaction |
| MA34885B1 (en) | 2010-12-22 | 2014-02-01 | Indiana Unversity Res And Technology Corp | GLUCAGON ANALOGS HAVING A GIP RECEPTOR ACTIVITY |
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| CN104114183A (en) | 2011-12-20 | 2014-10-22 | 印第安纳大学研究及科技有限公司 | CTP-based insulin analogs for treatment of diabetes |
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| CA2877358A1 (en) | 2012-06-21 | 2013-12-27 | Indiana University Research And Technology Corporation | Glucagon analogs exhibiting gip receptor activity |
| KR20150039748A (en) | 2012-06-21 | 2015-04-13 | 인디애나 유니버시티 리서치 앤드 테크놀로지 코퍼레이션 | Analogs of glucagon exhibiting gip receptor activity |
| JP6387008B2 (en) | 2012-09-26 | 2018-09-05 | インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーションＩｎｄｉａｎａ Ｕｎｉｖｅｒｓｉｔｙ Ｒｅｓｅａｒｃｈ Ａｎｄ Ｔｅｃｈｎｏｌｏｇｙ Ｃｏｒｐｏｒａｔｉｏｎ | Insulin analog dimer |
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| WO2015120187A1 (en) | 2014-02-05 | 2015-08-13 | The University Of Chicago | Chimeric antigen receptors recognizing cancer-spevific tn glycopeptide variants |
| CN108271356A (en) | 2014-09-24 | 2018-07-10 | 印第安纳大学研究及科技有限公司 | Duodenin-insulin conjugate |
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| WO2020191342A1 (en) | 2019-03-20 | 2020-09-24 | The Regents Of The University Of California | Claudin-6 antibodies and drug conjugates |
| MX2020013206A (en) * | 2019-03-26 | 2021-02-26 | Novel Pharma Inc | Long-acting fatty acid-binding gnrh derivative and pharmaceutical composition comprising same. |
| US20220160872A1 (en) | 2019-04-09 | 2022-05-26 | The Board Of Trustees Of The University Of Illinois | Drug Adsorbed Highly Porous Activated Carbon for Enhanced Drug Delivery |
| US20230190725A1 (en) | 2019-04-29 | 2023-06-22 | The Board Of Trustees Of The University Of Illinois | Mek inhibitors for corneal scarring and neovascularization |
| CA3132656A1 (en) | 2019-04-30 | 2020-11-05 | Instituto De Medicina Molecular Joao Lobo Antunes | Rank pathway inhibitors in combination with cdk inhibitors |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH661206A5 (en) * | 1983-09-23 | 1987-07-15 | Debiopharm Sa | PROCESS FOR THE PREPARATION OF A MEDICINAL PRODUCT FOR THE TREATMENT OF HORMONDEPENDENT DISEASES. |
| DE3414595A1 (en) * | 1984-04-18 | 1985-10-31 | Hoechst Ag, 6230 Frankfurt | USE OF GONADOLIBERIN AND GONADOLIBERINAGONISTS FOR TREATING CLIMATE COMPLAINTS |
| US4540513A (en) * | 1984-09-25 | 1985-09-10 | Kanegafuchi Kagaku Kogyo Kabushiki Kaisha | Decapeptide having gonadotropin releasing activity |
| US4721775A (en) * | 1985-08-26 | 1988-01-26 | Board Of Regents, The University Of Texas System | Effective peptides related to the luteinizing hormone releasing hormone from L-amino acids |
| NO302481B1 (en) * | 1990-10-16 | 1998-03-09 | Takeda Chemical Industries Ltd | Polymer for an extended release preparation, as well as an extended release preparation |
| IT1243390B (en) * | 1990-11-22 | 1994-06-10 | Vectorpharma Int | PHARMACEUTICAL COMPOSITIONS IN THE FORM OF PARTICLES SUITABLE FOR THE CONTROLLED RELEASE OF PHARMACOLOGICALLY ACTIVE SUBSTANCES AND PROCEDURE FOR THEIR PREPARATION. |
| CA2192773C (en) * | 1995-12-15 | 2008-09-23 | Hiroaki Okada | Production of sustained-release preparation for injection |
| AU3388597A (en) * | 1996-06-13 | 1998-01-07 | University Of Cape Town | Human type ii gonadotropin-releasing hormone receptor |
-
1998
- 1998-12-03 GB GB9826662A patent/GB2344287A/en not_active Withdrawn
-
1999
- 1999-12-02 NZ NZ511984A patent/NZ511984A/en unknown
- 1999-12-02 BR BR9915943-0A patent/BR9915943A/en not_active IP Right Cessation
- 1999-12-02 IL IL14349699A patent/IL143496A0/en unknown
- 1999-12-02 TR TR2001/02273T patent/TR200102273T2/en unknown
- 1999-12-02 CA CA002353798A patent/CA2353798A1/en not_active Abandoned
- 1999-12-02 MX MXPA01005543A patent/MXPA01005543A/en unknown
- 1999-12-02 SK SK755-2001A patent/SK7552001A3/en unknown
- 1999-12-02 CN CN99815183A patent/CN1332635A/en active Pending
- 1999-12-02 RU RU2001118040/15A patent/RU2233170C2/en not_active IP Right Cessation
- 1999-12-02 AU AU15732/00A patent/AU770676B2/en not_active Ceased
- 1999-12-02 KR KR1020017006883A patent/KR20010089538A/en not_active Application Discontinuation
- 1999-12-02 CZ CZ20011893A patent/CZ20011893A3/en unknown
- 1999-12-02 JP JP2000584909A patent/JP2002531411A/en active Pending
- 1999-12-02 PL PL99348575A patent/PL348575A1/en not_active Application Discontinuation
- 1999-12-02 HU HU0104943A patent/HUP0104943A3/en unknown
- 1999-12-02 EE EEP200100293A patent/EE200100293A/en unknown
- 1999-12-02 WO PCT/GB1999/004045 patent/WO2000032218A1/en not_active Application Discontinuation
- 1999-12-02 EP EP99958357A patent/EP1140133A1/en not_active Withdrawn
-
2001
- 2001-05-29 NO NO20012636A patent/NO20012636L/en not_active Application Discontinuation
- 2001-06-01 ZA ZA200104530A patent/ZA200104530B/en unknown
- 2001-06-01 HR HR20010421A patent/HRP20010421A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| KR20010089538A (en) | 2001-10-06 |
| NO20012636D0 (en) | 2001-05-29 |
| WO2000032218A1 (en) | 2000-06-08 |
| IL143496A0 (en) | 2002-04-21 |
| BR9915943A (en) | 2001-08-21 |
| AU770676B2 (en) | 2004-02-26 |
| HRP20010421A2 (en) | 2002-06-30 |
| TR200102273T2 (en) | 2001-12-21 |
| HUP0104943A3 (en) | 2002-08-28 |
| JP2002531411A (en) | 2002-09-24 |
| ZA200104530B (en) | 2002-06-04 |
| EP1140133A1 (en) | 2001-10-10 |
| CN1332635A (en) | 2002-01-23 |
| NZ511984A (en) | 2002-11-26 |
| PL348575A1 (en) | 2002-06-03 |
| MXPA01005543A (en) | 2003-07-14 |
| NO20012636L (en) | 2001-07-12 |
| EE200100293A (en) | 2002-08-15 |
| GB2344287A (en) | 2000-06-07 |
| HUP0104943A2 (en) | 2002-06-29 |
| AU1573200A (en) | 2000-06-19 |
| GB9826662D0 (en) | 1999-01-27 |
| CZ20011893A3 (en) | 2002-05-15 |
| RU2233170C2 (en) | 2004-07-27 |
| SK7552001A3 (en) | 2002-02-05 |
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