MXPA00010640A - Protein formulations - Google Patents

Abstract

The present invention relates to pharmaceutical formulations comprising human parathyroid hormone at a concentration from 0.3 to 10 mg/ml, a pharmaceutically acceptable buffer having a pH from 4 to 6, and at least one tonicity modifier. The said pharmaceutical formulations are useful for the treatment of bone related disorders such as osteoporosis.

Description

PROTEIN FORMULATIONS TECHNICAL FIELD The present invention relates to pharmaceutical formulations 5 comprising human parathyroid hormone (PTH), useful for the treatment of disorders related to bones such as osteoporosis.

TECHNICAL BACKGROUND 10 Parathyroid Hormone Human parathyroid hormone is a protein of 84 amino acids involved in calcium and phosphorus homeostasis and the control of bone growth and density. Equivalent terms for parathyroid hormone are PTH and less frequently used parathyrin and parathormone. Human PTH can be obtained through tissue extraction, from peptide synthesis or from yeast, battery host cells or genetically engineered mammals. Essentially pure human PTH is described in EU 5,208,041. The recombinant production of PTH in E. coli is described in v. g. , EU 5,223,407, EU 5,646, 015 and EU 5,629,205. The production of PTH. the recombinant in yeast is described in EP-B-0383751. Synthetic human PTH is commercially available from Bachem Inc., Bubendorf, Switzerland. 25 In mammals, the balance between bone formation, associated • < J * aj ^ -M ^ "- * - •> - '" • * • - •• "-' - '-f * * - l lr; i] ^ .__. Ag _ ^ _ _ gíg« ^ «With the activity of osteoblasts, on the one hand, and bone loss, associated with the activity of osteoclasts, on the other hand, it is disturbed in several diseases affecting the bones, such as osteoporosis.Parathyroid hormone has been shown to have a Potential therapeutic role in osteoporosis The anabolic actions of parathyroid hormone in bones are reviewed by Dempster, D. and collaborators in Endocrine reviews vol 14 (6), 690-709, 1993. For many proteins it is common that increasing protein concentration increases the propensity for protein aggregation and precipitation, either specific or non-specific, such reactions may be rapid, or may proceed slowly, so aggregation may not be clearly evident. of protein can reveal small autocatalytic activities p significant, that increase the degradation of proteins. Independently, aggregation and precipitation affect not only the available protein drug concentration but also the physical and biopharmaceutical properties of the formulation. From a pharmaceutical point of view, this is highly undesirable. A common prejudice, therefore, is to maintain low concentrations of protein. It is known that PTH forms aggregates when concentration is increased. One strategy to overcome this problem is to modify the pH to values either very low or very high. However, this often stimulates the chemical degradation of the protein, and also HEMÉ MÉ adds discomfort during administration, v. g. , subcutaneous injection. The diluted formulations need larger volumes to obtain the same dose in, for example, parenteral formulations. This not only leads to larger syringes and packages, but also to discomfort during administration and higher production costs. From this reasoning, it is concluded that a protein formulation should be as concentrated as possible. In addition, the development of a non-parenteral dosage form benefits that the protein concentration is as high as possible. Hence, there is a need for a formulation that allows a high concentration of protein without aggregation.

PTH Formulations Unlike other proteins that have been formulated successfully, PTH is particularly sensitive to various forms of degradation. For example, oxidation may occur in methionine residues at positions 8 and 18, giving rise to the oxidized PTH species ox- (8) -PTH and ox-M (18) -PTH, while deamidation may occur at the position 16, giving rise to d16-PTH. The polypeptide chain is truncated by breaking peptide bonds, both at the N- and C- terminals. In addition, PTH can also be adsorbed on surfaces, form aggregates and / or non-specific precipitates, thus reducing the available concentration of the drug. All these degradation reactions, and their combinations, lead < * - '- - - - - - - «« faith »** .-.» ^ A partial or complete loss of PTH bioactivity A PTH formulation must therefore avoid these degradation reactions. 17207 (Holthuis et al.) Discloses a PTH preparation comprising an excipient, eg, mannitol, which co-lyophilizes with PTH to give an amorphous cake, and a non-volatile buffering agent, eg, a citrate source. Description, PTH is desirably incorporated into an aqueous solution in a concentration range from 25 to 250 μg / ml, preferably 50 to 150 μm / ml. EU 5,563, 122 (Endo et al.) describes a lyophilized composition comprising PTH (1-34), sodium chloride and sugar When the freeze-dried samples of PTH (1-34) were stored at + 40 ° C for three months, it was found that the preparations containing combinations of sodium chloride and sugar were more stable than control preparations containing chloride of sodium only or sugar alone. In these experiments, each sample contained 36 μg of PTH (1-34) together with 5 to 10 mg of sugar and 0.1 to 1 mg of NaCl.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: stability of PTH at different protein concentrations. Figure 2: PTH stability at different pH values. Figure 3: PTH stability in the presence of NaCl.

*. . . . TO . '^,. . yr. . .. ... . .-. . . .. ^ ... ^ li tl i,. ^^^ - DESCRI PTION OF THE INVENTION Contrary to established knowledge, it has been found that relatively high concentrations of PTH can be used in a pharmaceutically acceptable formulation. The formulation may be in liquid form, but it may also be lyophilized, and reconstituted before administrations either single or multiple. The formulation reduces discomfort during administration due to a small volume of injection, since the formulation also has a pH and a buffering capacity that reduces pain upon administration. The use of high concentrations of PTH also offers the possibility to develop non-parenteral dosage forms, such as nasal, inhalable or oral formulations, or transdermal formulations. The possibility of a high concentration of PTH also allows a safe and economical multiple dose formulation, which is suitable for v. g. , self-administration. Accordingly, the present invention provides in a first aspect a pharmaceutical formulation comprising human parathyroid hormone (PTH) at a concentration of or above 0.3 mg / ml, such as from 0.3 to 10 mg / ml; a pharmaceutically acceptable buffer having a pH of from 4 to 6; and at least one tonicity modifier. The term "parathyroid hormone" (PTH) encompasses naturally occurring human PTH, as well as synthetic or recombinant PTH (rPTH). In addition, the term "parathyroid hormone" encompasses the PTH of »^ -», .t.i. , j. ^ - ^ ,. .. ^^^ " ^^. ^ _. ., .._, _ * ..., ".... -. . ^ - .. ^ .- s s.iafc¿aaia & *;. total length (1-84) as well as PTH fragments. Thus it will be understood that fragments of PTH variants can be incorporated, in amounts that give biological activity equivalent to PTH (1-84), in the formulations according to the invention, if desired. The 5 PTH fragments incorporate at least the PTH amino acid residues necessary for a biological activity similar to that of intact PTH. Examples of such fragments are PTH (1-31), PTH (1-34), PTH (1-36), PTH (1-37), PTH (1-38), PTH (1-41), PTH (28). -48) and PTH (25 and 139). The term "parathyroid hormone" also encompasses functional variants and analogs of PTH. The present invention then includes pharmaceutical formulations comprising such variants and functional analogues of PTH, which carry modifications such as substitutions, deletions, insertions, inversions or delations, but nevertheless that have substantially the biological activities of the parathyroid hormone. PTH variants improved in stability are known in the technique of v. g. , WO 92/1 1286 and WO 93/20203. PTH variants can incorporate v. g. , amino acid substitutions improve the stability and half-life of PTH, such as waste replacement of methionine in positions 8 and / or 18, and the replacement of asparagine in position 16. Cyclized PTH analogs are described in v. g., WO 98/05683. In this context, the term "biologically active" should be understood as causing a sufficient response in a bioassay for PTH activity, such as the cell-based assay of ^ j ^^^ ^ glgi ^ j &jgj ^ jíg ^ osteosarcoma rat for production of adenylate cyclase stimulated with PTH (see Rodan et al (1 983) J. Clin. Invest. 72, 1 51 1; and Rabbani and collaborators (1988) Endrocrinol, 123, 2709). The PTH to be used in the pharmaceutical formulations according to the invention is preferably recombinant human PTH, such as full-length recombinant human PTH. In the preferred forms of the invention, the concentration of said human parathyroid hormone may be 0.3-5 mg / ml or 0.3-3 mg / ml; or above 1 mg / ml, such as 1 -10 mg / ml, 1-5 g / ml; 1 -3 mg / ml; 10 or 1 -2 mg / ml. The said pharmaceutically acceptable buffer can be v. g. , a buffer of an acetate, a citrate, a phosphate or a carbonate. Preferably, the buffer is a citrate buffer at a concentration of 5 to 20 mM. The said pharmaceutically acceptable buffer 15 preferably has a pH between 5 and 6, most preferably around 5.5. The said tonicity modifier may be v. g. , sorbitol, glycerol, sucrose, or preferably, sodium chloride and / or mannitol. In a preferred form of the invention, the PTH formulation may comprise 1-3 mg / ml of parathyroid hormone, 2 to 5 mg / ml of NaCl, 20 to 50 mg / ml of mannitol, 5 to 10 mM of citrate buffer at a pH between 4 and 6, and optionally a preservative, such as benzyl alcohol or m-cresol. The pharmaceutical formulations according to the invention are useful in the treatment or prevention of bone disorders, in -M ^ Mji? 8 particular osteoporosis. In a further aspect, the present invention provides a process for the preparation of a pharmaceutical formulation as described above, said process comprising dissolving human parathyroid hormone, at a concentration of 0.3 to 10 mg / ml, and at least one modifier of tonicity, in a pharmaceutically acceptable buffer having a pH between 4 and 6. In another aspect, the invention provides the use of parathyroid hormone, at a concentration of 0.3 to 10 mg / ml, in the manufacture of a pharmaceutical formulation for the treatment or prevention of bone disorders, in particular osteoporosis, said pharmaceutical formulation further comprises a pharmaceutically acceptable buffer having a pH between 4 and 6, and at least one tonicity modifier. In still another aspect, the invention provides a method for treating or preventing disorders related to bones, in particular osteoporosis, which comprises administering to a mammal, including man, in need of such treatment or prevention an effective amount of a pharmaceutical formulation. of PTH as described above.

EXPERIMENTAL METHODS The full-length human PTH (1-84) used in the studies of the formulation was produced and secreted from an Escherichia Coli strain by known methods (see v. G, EU 5,223,407, EU 5, 646,015 and EU). 5,629,205). Briefly, the gene for human parathyroid hormone in plasmid pJT42 was fused with protein A secretion signal (ompA) DNA from the outer membrane of E. coli. Also present in the plasmid is a lactose repressor gene (laclq). Translation of the ompA-rhPTH mRNA and further processing by endogenous peptidase resulted in the production of mature 84-amino acid human parathyroid hormone which was harvested from the bacterial culture broth. The recombinant expression was followed by purification of PTH for a preparation essentially free of contaminants. The purification process, which involved methods known in the art (see v. G., EU 5,208,041), involved cell separation, filtration, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography, preparative reverse phase HPLC, and a second ion exchange chromatography followed by desalination to give a liquid mass. The resulting preparation typically had a purity of 95%, or better, as assayed by reverse phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). 20 PTH solutions were analyzed by RP-HPLC to evaluate the concentration of PTH, purity and formation of oxidized PTH, using trichloroacetic acid and acetonitrile in the mobile phase. Deamidated PTH was determined by cation exchange HPLC, using a KJ gradient. Retention times for and quantities of PTH, ox- 25 M (8) -PTH, ox-M (18) -PTH and d16-PTH were determined using the Appropriate reference standards. The effect of temperature on a regime for a process can be described by the term Q 0: Qv > - '-. { ?) where Ri and R2 are the regimes observed at temperatures Ti and T2, respectively (cf Chang, R: Physics-chemistry with applications to biological systems, New York, Macmillan Publishing Co., Inc., 1977, Schmidt-Nielsen K. Animal Physiology: Adaptation and environment.

Cambridge edition, Cambridge University Press, 1983). If Q10 = 2, then the regime doubles as long as the temperature increases by 10 degrees; if Q? 0 = 3, then the regime triples every 10 degrees. Since the activation energy is very constant for a temperature range, most reactions and processes have a Q10 between 2 and 3. Aggregation and precipitation were determined by visual inspection of the bottles against a white or black background, and by comparison with a suitable reference solution. The bioactivity of PTH was measured using the cell assay of rat osteosarcoma (UMR 106) for production of adenylate cyclase stimulated with PTH (Rodan et al., J. Clin Invest., 1983, 72: 151 1; Rabbiani et al., Endocrinol., 1988, 123: 2709).

EXAMPLE 1 - Effect of protein concentration on the stability of PTH Different concentrations of PTH were formulated in 10 mM citrate buffer. In a series of experiments, aliquots of the different formulations were lyophilized and stored at + 37 ° C - + 40 ° C. In another series, aliquots were stored as a liquid at + 4 ° C. At various time points (at least three), the purity of PTH was determined by rp-HPLC. The results were expressed as% area. The information for each time series, representing different concentrations of PTH, was then normalized, placing the purity of PTH at time 0 for 100%, to allow comparisons between the series. The PTH loss regimen, expressed as% per month, was calculated from the slope of the linear regression line of% PTH vs. time, using the formula: where x corresponds to time (months) and and corresponds to% of PTH purity that remains from the beginning. As shown in Figure 1 and in Tables I and ll, the stability of PTH increases markedly, when the concentration of PTH is increased, for both formulations lyophilized (Table I) and liquid (Table II). The stability is higher than PTH concentrations above 0.3 mg / ml, for both liquid and iiofilized formulations.

EXAMPLE 2 - pH Stability Six different formulations consisting of PTH were prepared (0.2 mg / ml), sodium citrate / citric acid (10 mM) at a pH of 4, 4.5, 5, 5.5, 6 and 6.5, respectively, and mannitol (50 mg / ml). Aliquots were sealed (1 ml) in 2 ml glass bottles under nitrogen and analyzed for PTH purity by RP-HPLC. The results are shown in Table III and in Figure 2. The degradation was accelerated at higher temperatures, as expected for most reactions. An approximate calculation of this temperature effect showed a Q10 = 2 in the temperature range from + 4o C to + 25 ° C, and Q10 = 3 for the temperature range from + 25 ° C to + 37 ° C, at all pH values tested. This range of values of Q10 is consistent with other biochemical reactions, and justifies the use of higher temperatures for degradation studies. accelerated. The results indicate that formulations at a pH of 4.5 to 5.5 have advantageous properties in terms of PTH stability. All the formulations appeared as a clear colorless solution after storage. In addition, the formulations were stable at + 4 ° C at a pH between 4 and 6. EXAMPLE 3 - Effect of ionic strength on the stability of PTH Formulations consisting of PTH (0.2 mg / ml), sodium citrate / citric acid (10 mM), pH 6, and mannitol (50 mg / ml). NaCl was added to the final concentrations indicated in Figure 3. formulations with added NaCl were hypertonic with respect to ^ ^ Blood plasma, in addition to having an increased ionic strength. Aliquots (1 ml) were sealed in 2 ml glass bottles under nitrogen and analyzed for PTH purity by RP-HPLC. The results, shown in Figure 3, indicate that at higher temperatures, a small increase in stability with increased ionic strength was obtained. At + 4 ° C, however, the formulations were not affected by increased ionic strength in terms of PTH stability. All the formulations appeared as a clear colorless solution after storage.

EXAMPLE 4 - Effect of Tonicity Modifiers on PTH Stability Formulations were prepared comprising PTH (0.2 mg / ml), sodium citrate / citric acid (10 mM), in the presence of a tonicity modifier (5% mannitol) or 0.9% NaCl). Aliquots (0.7 ml) were sealed under nitrogen in 3 ml glass bottles. After storage at the indicated temperatures, the flasks were broken and the PTH formulation was analyzed for purity, oxidized PTH (ox-M (8) -PTH and ox-M (18-PTH), deamidated PTH (d 16-PTH). ) and bioactivity using the adenylate cyclase assay The results, shown in Table IV, show that the degradation was not affected by the tonicity modifier.The bioactivity of PTH was preserved at all temperatures studied.All the formulations appeared as a Clear colorless solution after storage The formation of oxidized and deamidated PTH • Mi ^ iMte ^ K-. ^^^^ g ^ ^ ¿^ ^ ^ *! »« £ * as a decrease in PTH concentration was negligible after 18 risks at + or C.

EXAMPLE 5 - Effect of oxidative stress on PTH in prefilled syringes Formulations were prepared comprising PTH (0.2 mg / ml), sodium citrate / citric acid, pH 5.5, (10 mM), and NaCl (9 mg / ml). Aliquots of 0.4 ml were filled into syringes equipped with a crowned needle, and a rubber plunger was mounted contacting the solution. The syringes were stored in either ambient air or in an atmosphere of protective nitrogen, as indicated in Table V. The content of the syringes was analyzed for PTH purity, oxidation and deamidation. The results, shown in Table V, indicate that the presence of air does not appreciably affect oxidation, deamidation or purity of PTH. In addition, the PTH formulations are stable at + 4 ° C in a product container.

Example 6 - Effect of preservatives on PTH stability (a) Formulations were prepared comprising PTH (1.3 mg / ml), sodium citrate / citric acid, pH 5.51 (10 mM) and mannitol (50 mg / ml). Some formulations further comprised a preservative (3 mg / ml m-cresol or 1 mg / ml EDTA). Aliquots (0.5 ml) of the formulations were sealed under nitrogen 25 in 3 ml glass jars, and stored in a refrigerator (+ 2o) C to + 8o C). After two weeks, the formulations were tested for purity of PTH and oxidized forms of PTH by RP-HPLC. The results, shown in Table IV, indicate that the formulations were not affected by m-cresol or EDTA. (b) Formulations consisting of PTH (0.8 mg / ml), sodium citrate / citric acid, pH 5.5 (10 mM) and NaCl (9 mg / ml) were prepared. The preservatives m-cresol or benzyl alcohol were added to some of the formulations at a concentration of 3 mg / ml, respectively. Aliquots of 0 5 ml were sealed under nitrogen in 3 ml glass bottles, and stored in an inverted position, allowing the liquid to be in contact with the rubber plugs. After storage at + 4 ° C, the formulations were tested for PTH, oxidized PTH and deamidated PTH. The results are shown in Table VII. The stability of PTH in the presence of m-cresol was comparable to that of the control. In the presence of benzyl alcohol, the stability of PTH decreased, attributable mainly to the formation of oxidized M8 and M 18 forms of PTH. The concentration of PTH after storage was not affected by the presence of preservative (information not shown). Previous experiments have shown that the bioactivity of PTH is not affected by the presence of these preservatives. In addition, these preservatives gave satisfactory protection against microbial growth after the challenge, according to methods described in U. S. Pharmacopeia and European Pharmacopeia (information not shown). In the tested formulations, PTH is stable in the presence of efficient preservatives, thus allowing the use of the formulation in a multiple dose product. fifteen twenty TABLE Stability of PTH in freeze-dried formulations n. d. = not determined TABLE II Stability of PTH in liquid formulations n tí = not determined. *** »» .-. , A »a.Mtn» wd «-,.

TABLE ll l Stability of PTH at different pH values n .d. = not determined TABLE IV 5 pH stability in the presence of tonicity modifiers n.d .: not detected; M8 = ox-M (8) -PTH; M18 = ox-M (18) -PTH? GI * ^^^^^ i # ^ '' < * »* ^ - 'TABLE V Stability of PTH in the presence of air < d. = level of detection from below; n.d. = not determined; 8 = ox- (8) -PTH; M18 = ox- (18) -PTH TABLE VI Stability of PTH in the presence of preservatives Refr. = refrigerator; RT = Temp. ambient; 8 = ox- (8) -PTH; M18 = ox-M (18) -PTH ^^^ g ^ j ^ gj ^ A TABLE V Stability of PTH in the presence of conservatives < d.l. = detection level below; M8 = ox-M (18) -PTH; M18 = ox-M (18) -PTH , ».« - M ^ .. * g

Claims (18)

1. CLAIMS 1. A pharmaceutical formulation comprising human parathyroid hormone in a concentration of or above 0.3 mg / ml to 10 mg / ml, a pharmaceutically acceptable buffer having a pH of 4 to 6, and at least one tonicity modifier .
2. 2. The formulation according to claim 1 wherein said human parathyroid hormone is human recombinant parathyroid hormone.
3. 3. The formulation according to claim 1 or 2 wherein said human parathyroid hormone is full-length parathyroid hormone.
4. 4. The formulation according to any one of claims 1 to 3 wherein the concentration of said human parathyroid hormone is from 0.3 mg / ml to 5 mg / ml.
5. 5. The formulation according to claim 4 wherein the concentration of said human parathyroid hormone is from 1 mg / ml to 3 mg / ml.
6. 6. The formulation according to any one of claims 1 to 5 wherein said pharmaceutically acceptable buffer is a citrate buffer at a concentration of 5 to 20 mM. The formulation according to any one of claims 1 to 6 wherein said pharmaceutically acceptable buffer has a pH between 5 and 6. The formulation according to any one of the '' • - - ** • - «* ''» * '- "-" •; .- ... .. .. . .-. *** * ... ... * «? ÍÍiMlt? Í *. ~~.?. ~. . . . . . •. " ,and V . Claims 1 to 7 wherein the said tonicity modifier is sodium chloride and / or mannitol. 9. The formulation according to any one of claims 1 to 8 comprising from 1 to 3 mg / ml of 5 parathyroid, 2 to 5 mg / ml of NaCl, 20 to 50 mg / m of mannitol, 5 to 10 mM of citrate buffer at a pH between 4 and 6, and optionally a preservative. 10. The formulation according to any one of claims 1 to 9 in liquid form. 10 1 1. The formulation according to any one of claims 1 to 9 in a thiofilized form. 12. A process for the preparation of a pharmaceutical formulation according to any one of claims 1 to 11, which comprises dissolving human parathyroid hormone, to a 15 concentration of 0.3 to 10 mg / ml, and at least one tonicity modifier, in a pharmaceutically acceptable buffer having a pH between 4 and 6. 13. A pharmaceutical formulation according to any one of claims 1 to 11 for use in the treatment or prevention of 20 disorders of the bones. 14. A pharmaceutical formulation according to any one of claims 1 to 1 for use in the treatment or prevention of osteoporosis. 15. The use of parathyroid hormone in a concentration of 0.3 25 to 10 mg / ml, in the manufacture of a pharmaceutical formulation for the treatment or prevention of bone disorders, said pharmaceutical formulation further comprising a pharmaceutically acceptable buffer having a pH between 4 and 6, and at least one tonicity modifier. 16. The use according to claim 15 for the treatment or prevention of osteoporosis. 17. A method for treating or preventing disorders related to bones which comprises administering to a mammal, including man, in need of such treatment or prevention an effective amount of a formulation according to either one of claims 1 or 1. . 18. The method according to claim 17 for treatment or prevention of osteoporosis.