Pharmaceutical Compositions Containing GHRP-6

Abstract

A pharmaceutical composition comprising growth hormone releasing peptide 6 (GHRP-6), a tartrate buffer of pH 5.0-6.0, and trehalose as a stabilizing agent, and use of said composition for the manufacture of a medicament. The invention also provides a kit-of-parts comprising said pharmaceutical composition and a method of treating an individual in need thereof, said method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising GHRP-6, a tartrate buffer of pH 5.0-6.0, and trehalose.

Description

[Technical field]

[0001] Technical Field The present invention relates to the pharmaceutical and biomedical industry and to pharmaceutical formulations of peptides and proteins to improve their safety profile. In particular, the present invention relates to a pharmaceutical composition of growth hormone releasing peptide 6 (GHRP-6). It discloses a pharmaceutical formulation of the peptide suitable for its administration by parenteral route.

[0002] prior art GHRP-6 is one of the best studied molecules in the family of growth hormone secretagogues (Casanueva and Dieguez, Trends Endocrinol. Metab., 1999, 10:30-38). Its amino acid sequence is His-Trp-Ala-Trp-Phe-Lys-NH 2 where the second and fifth amino acids are D amino acids. It is a synthetic peptide, an analogue of intestinal met-encephalin and gastric ghrelin, and competes with these substances for somatrotroph anterior pituitary receptors. These peptides act through a protein G-linked receptor known as the growth hormone secretagogue receptor (GHS-R1a), whose natural ligand is ghrelin (Kojima et al., Nature, 1999, 402:656-660). It has been demonstrated that there is an additional binding site for GHRP-6 in non-endocrine tissues via association with the receptor CD36, which belongs to the family of scavenger or type B remover receptors (Bodart et al., Circ. Res., 1999, 85:796-802).

[0003] In studies performed in animal models of ischemia / reperfusion, GHRP-6 showed a strong cytoprotective effect (Murriel and Mochly, Arch. Biochem. Brophys., 2003, 420:246-254). Some preliminary evidence suggests that the mechanism by which tissue damage is reduced may be related to the control of the cellular redox environment. Other forms of cell death, such as apoptosis, also appear to be prevented by GHRP-6 (Colonna et al., EUR. J. Pharmacol., 1997, 334:201-207; Cibrian et al., Clin. Sci., 2006, 110:563-573). Its beneficial effects on cardiovascular function have been demonstrated in diseases such as myocardial infarction and ischemic heart conditions (Berlanga et al., Clin. Sci., 2007, 112:241-250; Berlanga et al., Clin. Med. Insights Cardiol., 2017, 11:1-9), while the combination of GHRP-6 with epidermal growth factor (EGF) has proven to be a promising therapeutic approach in the treatment of ischemic stroke (Subiros et al., Neurol. Res., 2016, 38(3):187-195).

[0004] GHRP-6 has been administered to humans by parenteral route, mainly intravenous route, with the aim of inducing the release of growth hormone. The doses applied by intravenous route in humans varied between 1 and 2 μg / kg body weight (Leal-Cerro et al., EUR. J. Endocrinol., 1995, 132(6):712-715; Loche et al., J. Clin. Endocrinol. Metab., 1995, 80(2):674-678). For the new application in humans by parenteral route, pharmacological studies suggest that the therapeutic dose of GHRP-6 should be 100 times higher. The reported therapeutic dose in a model of acute myocardial infarction in pigs was 400 μg / kg (Berlanga et al., Clin. Sci., 2007, 112:241-250), which represents a dose 200-400 times higher than that used in humans in the 1990s. On the other hand, the reported therapeutic dose of GHRP-6 in a model of ischemic cerebral infarction in gerbils (Meriones unguiculatus) was approximately 600 μg / kg (Subiros et al., Neurol. Res., 2016, 38(3):187-195). In this last example, GHRP-6 was used in combination with EGF.

[0005] Regarding safety in humans, during administration of GHRP-6 by the intravenous route, doses of 2 μg / kg or less are said to be safe. However, to achieve an effective use of the peptide in the treatment of acute myocardial infarction and ischemic cerebral infarction, much higher doses are required, ranging from 50 to 600 μg / kg. Likewise, for said purpose, it is necessary to obtain GHRP-6 preparations of high concentration (1.0 to 10.0 mg / mL) that maintain their quality, stability and therapeutic properties while minimizing adverse and / or toxic effects. A high safety profile of the product is especially necessary in the treatment of patients with acute myocardial infarction and stroke.

[0006] A particular problem with formulations containing high concentrations of peptides or proteins is posed by their tendency to aggregate. As a result of aggregation, precipitation of the product may occur during storage, which may affect the reproducibility of the dose administered to the patient. Also, embolism may occur during parenteral administration of particle-containing solutions.

[0007] Another common problem with pharmaceuticals is the possibility of adverse effects and / or toxicity during the treatment of patients. These undesirable events can be of different types depending on, among other things, the properties of the active ingredient (metabolism, binding to proteins, etc.), the formulation, the route of administration and the characteristics of the patient (Ajayi et al., J. Clin. Pharmacol., 2000, 40(10):1093-1101; Choonara et al., Paediatr. Perinat. Drug Ther., 2001, 5:12-18). The preparation of concentrated formulations of peptides that provide adequate therapeutic effects while maintaining a minimal profile of adverse effects when applied by parenteral routes poses a challenge. Since each peptide exhibits a unique in vivo behavior, a specific formulation for each peptide needs to be developed.

[0008] There is therefore a need to obtain stable pharmaceutical compositions of GHRP-6 that have the necessary concentrations of peptide for therapeutic administration in several conditions, while at the same time exhibiting the required safety profile in humans.

[0009] Description of the invention The present invention solves the above-mentioned problems by providing a pharmaceutical composition comprising GHRP-6, tartaric acid, a pH adjusting buffer at pH 5.0-6.0, and trehalose. GHRP-6 is numbered according to the amino acid sequence identified in the sequence listing as SEQ ID NO: 1. This composition, revealed for the first time in the present invention, exhibits a high safety profile and minimizes the adverse and toxic events detected with previous formulations in both animals and humans.

[0010] When a formulation of GHRP-6 was evaluated in rats, containing a citrate buffer at pH 6.0 and sodium chloride as an isotonicity agent, an element of acute toxicity was observed when a dose of the peptide was administered that was 50 times the therapeutic dose of 400 μg / kg. The toxicity study showed changes in the adrenal glands consisting of focal areas of necrosis at the level of the zona fasciculata with the presence of swollen cells and macrodroplet fatty degeneration. These signs are associated with the administration of high doses of GHRP-6 contained in the citrate / NaCl formulation. Surprisingly, in toxicity studies carried out in rats, the formulations of the invention did not show signs of acute toxicity or the appearance of adverse effects in the animals as a result of the application of doses 100 times higher than the therapeutic dose of 400 μg / kg body weight. None of the formulations of the invention showed signs of toxicity at the systemic level during repeated administration (14 consecutive administrations) of up to 50 times the therapeutic dose. Furthermore, in the rabbit model, which is widely used for the toxicological evaluation of drugs, good local tolerance was observed at doses up to 10 times the therapeutic dose.

[0011] On the other hand, when the dosage of the GHRP-6 formulation containing citrate buffer at pH 6.0 and sodium chloride as an isotonicity agent was scaled up, bradycardia occurred in healthy volunteers receiving doses of 50 μg / kg and 100 μg / kg. Clinically, bradycardia can cause serious problems when the peptide is administered as a drug, especially in patients suffering from conditions such as acute myocardial infarction or ischemic cerebral infarction. Surprisingly, the GHRP-6 formulation of the present invention did not show any signs of toxicity and no bradycardia was reported after administration of doses up to 100 μg / kg.

[0012] In one embodiment of the present invention, the peptide GHRP-6 is in a concentration of 1.0 mg / mL to 10.0 mg / mL in the pharmaceutical composition. In the 1990s, the chemical stability of GHRP-6 was tested only at low concentrations (approximately 100 μg / mL) with respect to pH / buffer system, ionic strength and temperature (Ha et al., Int. J. Pharm., 1996, 144:91-97). In contrast, in the present invention, the stability of GHRP-6 was achieved at high concentrations of 2.5 mg / mL to 10 mg / mL (25-100 times higher than the mentioned report) in the presence of ingredients where the stability of the peptide was not evaluated.

[0013] The pharmaceutical composition disclosed by the present invention exhibits high stability during storage under refrigerated conditions (5±3° C.) despite its high peptide concentration, and also exhibits high stability at room temperature (25±2° C.), allowing the elimination of the cold chain during drug distribution.

[0014] In one embodiment of the present invention, the buffer has a concentration between 10 mmol / L and 100 mmol / L. The tartrate buffers that may be used are essentially all physiologically acceptable ones suitable for reaching the desired pH value, such as sodium and potassium tartrate salts, or mixtures thereof. The buffer preferably consists of one or more tartrate salts and / or free acids (e.g. tartaric acid, sodium bitartrate, potassium bitartrate, disodium tartrate anhydrous, disodium tartrate dihydrate, disodium and potassium tartrate tetrahydrate, calcium tartrate). In a particular embodiment, the tartrate buffer is constituted by sodium salts, potassium salts or a mixture of both.

[0015] In one embodiment of the present invention, the stabilizing compound trehalose is in the range of 2% to 10% (w / v). Preferably, the trehalose used is in the D(+) form and in the dihydrate form.

[0016] The formulations of the invention may be provided as liquids or in a dry form obtained by lyophilization, and therefore the concentrations of peptides, buffers and stabilizers refer to the concentrations that are in liquid form or result from a suspension of a lyophilisate.

[0017] The freeze-drying method is carried out after sterilization (e.g., by filtration). It removes the solvent (e.g., water for injection) used in the system to a level of less than 5%, and further reduces the residual humidity to a value of less than 3%. The method provides a GHRP-6 composition that can be stored for a long period at room temperature. The formulation can remain stable during storage under the action of shear forces acting during preparation, transportation and storage at high temperatures.

[0018] In a preferred embodiment of the present invention, an aqueous solution of trehalose is prepared in a tartrate buffer; dissolution of GHRP-6 in the solution then occurs. The formulation may optionally be lyophilized. The lyophilized compositions described herein are easily reconstituted when contacted with a sufficient amount of a pharma- ceutically acceptable solvent. The lyophilized compositions are reconstituted in less than 2 minutes. Suitable diluents include water for injection, saline, aqueous media in the presence of a buffer, optionally buffers in the presence of sugars, vitamins, and synthetic polymers. In a preferred embodiment, the diluent is water for injection. The lyophilized compositions may be reconstituted to produce compositions that exhibit the desired therapeutic concentration.

[0019] In another aspect, the present invention includes the use of a pharmaceutical composition containing GHRP-6, a tartrate buffer of pH 5.0-6.0, and trehalose for the manufacture of a medicament. A medicament containing GHRP-6 together with other ingredients is useful as a cytoprotectant in the treatment of tissues damaged due to lack of blood perfusion. Thus, in one embodiment of the present invention, a pharmaceutical product containing the aforementioned GHRP-6 formulation is used as a cytoprotectant, cardioprotectant, cardiorepairing or neuroprotective agent, or as an agent for restoring brain injury.

[0020] In one embodiment of the invention, the medicament is a kit of components which also comprises a second pharmaceutical composition.Thus, a kit of components comprising a pharmaceutical composition comprising GHRP-6, a tartrate buffer of pH 5.0-6.0 and trehalose is also an object of the present invention.

[0021] In another aspect, the present invention discloses a method for treating an individual in need thereof, in which a therapeutically effective amount of a pharmaceutical composition comprising GHRP-6, a pH adjusted tartrate buffer of pH 5.0-6.0, and trehalose is administered. In the treatment method of the present invention, the pharmaceutical composition is administered by a route known to those skilled in the art. In one embodiment of the present invention, the pharmaceutical composition containing GHRP-6 is administered by an intravenous, subcutaneous or intramuscular route. [Brief description of the drawings]

[0022] [Figure 1] Reverse-phase high-performance liquid chromatography (RP-HPLC) profiles of GHRP-6 lyophilized in the presence of different excipients at 3% (w / v) in two buffers at 25 mmol / L and pH 5.5: (A) sodium tartrate and (B) histidine-HCl. The concentration of the peptide was 2.5 mg / mL. Samples were stored at 50 ± 2 °C for 6 months before chromatographic analysis. [Diagram 2] Evaluation of the biological activity by inotropic effect of two GHRP-6 preparations in Balb / c mice (n=6). The corresponding placebo of each preparation was used as experimental control. Left ventricular ejection fraction (LVEF) was evaluated by echocardiography. Animals were administered a dose of 100 μg GHRP-6 per kilogram of body weight or an equivalent amount of the corresponding placebo preparation by intraperitoneal route.

[0023] Example For all the studies carried out, GHRP-6 was used as the active pharmaceutical ingredient (API), which has the amino acid sequence identified as SEQ ID NO: 1 in the sequence listing. The peptides were obtained stepwise by the method of linear synthesis of peptides in solid phase (Atherton and Sheppard, 1989. Solid phase peptide synthesis: a practical approach, at: Coligan, J., Dunn, B., Ploegh H. (Eds.), Current Protocols in Protein Science. IRL Press, Oxford). The identity of the peptide batches was confirmed by mass spectrometry in electrospray mode (ESI-MS), while the percentage of the fraction of intact peptides was determined by RP-HPLC and was higher than 98.0%.

[0024] ● Example 1. Obtaining a liquid formulation of GHRP-6 in citrate / sodium chloride Stability at different pH values, ionic species, buffer concentrations and sodium chloride concentrations The pH value of pharmaceutical formulations is relevant for their administration by parenteral route. To test the stability of GHRP-6 at different pH values, citrate-phosphate buffer (citrate / Na 2 HPO 4 ) was used at a concentration of 100 mmol / L and pH values ​​between 4.0 and 8.0. For the preparation of the different formulations, the API was dissolved until the desired concentration of 2.5 mg / mL was reached. The formulations were sterilized by filtration (through a 0.22 μm filter) and dispensed into 2R vials in a ratio of 1.0 mL per vial. Representative samples of the different formulations were incubated at 60 °C and the stability of the peptides was evaluated by RP-HPLC after storage at said temperature for different time intervals.

[0025] The percentage of intact peptide fraction was evaluated by RP-HPLC, C18 column (4.6 × 150 mm, 5 μm), (Vydac, USA). As mobile phase, a mixture of a solution of water for injection containing 0.1% (v / v) trifluoroacetic acid (TFA) (solution A) and acetonitrile containing 0.05% (v / v) TFA (solution B) was used. To separate the degradation products formed during the stress test, a gradient from 0 to 60% of B in A was used for 45 min at a working temperature of 34 °C. The GHRP-6 concentration was determined experimentally by the absorbance at 280 nm and by the concentration of 146.8 mL mg -1 cm -1 Mass absorption coefficient (ε 1% During storage of GHRP-6 formulations (2.5 mg / mL) in citrate-sodium phosphate buffer at 60°C, the highest purity values ​​in RP-HPLC were obtained at pH 6.0.

[0026] Next, the stability of the peptide (2.5 mg / mL) was tested in sodium acetate, sodium citrate, sodium phosphate and citrate-sodium phosphate buffers at the same pH values ​​with buffer concentrations of 25, 50 and 100 mmol / L. In phosphate, citrate and citrate-phosphate buffers, a catalytic effect was observed in the degradation of the peptide, and the purity of the peptide decreased with increasing buffer concentration. No catalytic effect was observed with the use of sodium acetate buffer. The highest purity value of the peptide was observed with the use of sodium citrate buffer at pH 6.0 and a concentration of 25 mmol / L according to RP-HPLC analysis.

[0027] Finally, with the aim of evaluating sodium chloride as a tonicity agent, the effect of its concentrations (2.5, 5.0 and 10.0 mg / mL) on GHRP-6 stability was evaluated. This study was carried out in sodium citrate buffer at pH 6.0 (buffer concentration 25 mmol / L). During storage of 2.5 mg / mL GHRP-6 formulations in 25 mmol / L sodium citrate buffer at 60 °C, the purity values ​​of the peptide were not affected by the concentration of sodium chloride present in the formulation (2.5 to 10 mg / mL). Therefore, this compound may be used as an isotonicity agent in formulations. Table 1 summarizes the results of some of the aforementioned formulations. [Table 1] The pH of each formulation was 6.0.

[0028] Taking the above results into consideration, the composition of the formulation evaluated during scale-up and stability studies was as follows: GHRP-6 at 2.5 mg / mL, sodium citrate buffer at 25 mmol / L and pH of 5.7-6.3, while sodium chloride was used as an isotonicity agent at 6 mg / mL.

[0029] Preparation of a liquid formulation of GHRP-6 at 2.5 mg / mL in citrate / sodium chloride: Scale-up and stability studies Ingredients in a 2.5mg / mL GHRP-6 formulation : GHRP-6 (2.5 g), citric acid (4.8 g), sodium chloride (6.0 g) and water for injection (sufficient for 1.0 L).

[0030] All the components of the formulation were measured and dissolved with water for injection, except for the API constituted by GHRP-6. The pH of the solution was checked and adjusted to a value of 6.0±0.3 with 1.0 mol / L NaOH. The API was added to reach the desired concentration in the solution (2.5 mg / mL). The formulation was then sterilized by filtration (through a 0.22 μm filter) in a class 100 area. The sterile liquid formulation was then dispensed into stoppered and sealed vials in a class 100 area. Samples from the three batches were placed in a cold chamber at controlled temperature (5±3° C.) and in an incubator at 25±2° C. (for accelerated stability testing). At given time intervals, samples were withdrawn to analyze the important quality attributes: product appearance, GHRP-6 purity measured by RP-HPLC, GHRP-6 identity measured by ESI-MS, peptide concentration determined by absorbance at 280 nm, pH, sterility and pyrogenicity. Analytical tests were performed as previously described or as described in the monograph USP - United States Pharmacopeia.

[0031] During storage of GHRP-6 liquid formulation 2.5 mg / mL in citrate / sodium chloride at 5±3°C for 12 months, the percentage of intact peptide fraction remained above 98.04%. During the stability study, the product remained unchanged in its appearance. Also, the tests of sterility, pyrogens and identity by ESI-MS were found to be satisfactory for all batches both at the beginning and end of the study. Similarly, the formulation remained stable during storage at 25±2°C for 6 months. The purity measured by RP-HPLC remained above 95.73% and the tests of sterility, pyrogens and identity by ESI-MS were found to be satisfactory for all batches both at the beginning and end of the study. It is concluded that the obtained formulation was found to be stable during storage at 5±3°C for 1 year and at 25±2°C for 6 months.

[0032] ● Example 2. Preclinical Toxicology Studies Conducted on a Liquid Formulation of GHRP-6 in Citrate / Sodium Chloride In the preclinical trials described below, a liquid formulation of GHRP-6 described in Example 1 was used, consisting of 2.5 mg / mL GHRP-6, 25 mmol / L sodium citrate buffer and a pH of 5.7-6.3, with 6 mg / mL sodium chloride used as an isotonicity agent.

[0033] Assessment of acute toxicity in Sprague-Dawley rats A total of 50 rats (25 females and 25 males) were used, constituting 5 groups of 10 individuals each. The doses corresponding to each group were administered in three applications, one every 6 hours. The formulations were applied by intraperitoneal route on days 1 and 2 of the experimental phase, according to the design shown in Table 2. A therapeutic dosage (TD) of 400 μg / kg was used as the benchmark. The parameters evaluated were body weight, food intake, clinical and macroscopic observations, and histopathological examination of the organs of interest. [Table 2]

[0034] After detailed analysis of the results obtained, it was concluded that the formulation of GHRP-6 in citrate / sodium chloride, at high doses, did not cause acute systemic changes that could compromise the safety of the patient. The evaluated formulations showed a sufficient safety framework up to 30 times the TD of the formulation of GHRP-6 in citrate / sodium chloride for use in clinical trials. Nevertheless, findings considered as elements of toxic events associated with the administration of high doses of GHRP-6 (in this case 50 times the TD) were reported in the adrenal glands (focal areas of necrosis at the level of the zona fasciculata, with the presence of swollen cells and macrodroplet fatty degeneration).

[0035] Evaluation of repeated dose toxicity in Sprague-Dawley rats One hundred young adult rats of both sexes (50 females and 50 males) were used, constituting seven groups. The untreated and satellite groups (administered 9 times the TD) were completed with 10 animals each. The other five groups were completed with 16 animals each. The study design is summarized in Table 3. The satellite group received the highest dose used in the study with the aim of evaluating the reversibility of potential effects. The animals in this group were sacrificed twice as long as the remaining animals. In all the treatment groups constituted, 14 consecutive doses (once a day for 2 weeks) were administered by intraperitoneal route. The parameters evaluated were body weight, food consumption, clinical and gross observations, and histopathological examination of the organs of interest. Similarly, measurements of hematology and biochemistry, as well as absolute and relative weights of the organs were performed. In addition, all organs of each subject were examined by histopathology. [Table 3]

[0036] From this study it was concluded that the formulation of GHRP-6 in citrate / sodium chloride was not toxic at the systemic level at the doses evaluated.

[0037] Evaluation of local tolerability in F1 rabbits A total of 18 female individuals were used, constituting 6 groups of 3 individuals each. Five consecutive doses were administered by the intravenous route, every 72 hours. The distribution of the populations by treatment was as follows: group I, untreated; group II, placebo; group III, TD of GHRP-6 (400 μg / kg); group IV, 5 times the TD of GHRP-6 (2000 μg / kg); group V, 10 times the TD of GHRP-6 (4000 μg / kg); and group VI (satellite), 10 times the TD of GHRP-6 (4000 μg / kg). The parameters evaluated were body weight, food intake, clinical and macroscopic observations, as well as histopathological examination of the organs of interest. The results demonstrated that repeated administration of a formulation of GHRP-6 in citrate / sodium chloride was well tolerated at high doses and that the adverse events observed did not constitute signs of toxicity.

[0038] ● Example 3. Dosage testing of a liquid formulation of GHRP-6 in citrate / sodium chloride in healthy volunteers A phase I non-controlled clinical trial of dose scale-up in healthy volunteers was conducted to evaluate GHRP-6 in a citrate / sodium chloride-containing liquid formulation tested and developed in Example 1. The formulation contained: GHRP-6 at 2.5 mg / mL, sodium citrate buffer at 25 mmol / L and pH of 5.7-6.3. Sodium chloride at 6 mg / mL was used as an isotonicity agent. The peptide was administered in a single dose by intravenous route at six dose scales: 1, 10, 50, 100, 200 and 400 μg / kg body weight. The sample consisted of 18 healthy subjects, male, aged 18-35 years, with body weight within the normal range, who voluntarily agreed to participate in the trial study. At each level of dose scale-up, three subjects were treated. Progression to the next level was dependent on the absence of toxicity (serious adverse events).

[0039] Regarding the safety of the peptide, no serious adverse events were reported. As can be seen in Table 4, six different adverse events were recorded in 12 subjects (66.7%), with sweating (50%), bradycardia (44.4%) and drowsiness (16.7%) being the most frequent. All adverse events showed a causal relationship to the administered medicinal product containing GHRP-6 as API. The number of reported adverse events increased with the dose level of the peptide during the study. [Table 4]

[0040] From the basal values, heart rate values ​​below the normal range (60-100) were observed. Pathological values ​​(less than 60) were reported as an adverse event of bradycardia. This had a duration that varied between approximately 55 minutes and 3 hours, with the largest decrease in heart rate values ​​in groups V (200 μg / kg) and VI (400 μg / kg) during the first 30 minutes following administration of the peptide. For the adverse event that required the most clinical attention (bradycardia), the relationship with the concentration of GHRP-6 was explored in more detail. Although no statistically significant correlation was detected, it was interesting to observe that the tendency for bradycardia became greater as the concentration value of GHRP-6 increased, considering that the heart rate was lower. Clinically, bradycardia can pose serious problems when the peptide is administered to patients. In this study, it occurred at four of the six dosage levels tested, even though the subjects were healthy volunteers.

[0041] Based on the results obtained with dose scale-up in healthy individuals, it became necessary to explore new alternatives for formulations in which the peptide remains concentrated and stable for its evaluation in humans.

[0042] ● Example 4. Obtaining a new formulation of GHRP-6 Effect of tartrate, succinate, and histidine buffers at pH 5.0-6.0 on the stability of GHRP-6 in aqueous solutions Since the peptide is stable in the pH range of 5.0-6.0, tartrate, succinate and histidine buffers were tested, which show good buffering capacity for adjusting the pH in that range of values. The buffers were prepared at a final concentration of 50 mmol / L. Tartaric acid was used for the preparation of the tartrate buffer. The histidine buffer was prepared from the histidine-HCl reagent, and in both cases the pH was adjusted between 5.0 and 6.0 with 1.0 mol / L NaOH. The succinate buffer was prepared from succinic acid and NaOH, in the same way as the tartrate buffer.

[0043] The API (GHRP-6) was dissolved in different buffers. The shaking time was less than 1 hour at 25±2°C. The concentration of GHRP-6 was 2.5 mg / mL as determined by absorbance at 280 nm. The samples were then sterilized by filtration (on a 0.22 μm filter) under aseptic conditions. To evaluate the stability of the peptides, the samples were placed in an incubator for 28 days under stress conditions at 60±2°C. The percentage of intact peptides, the concentration of peptides and pH were evaluated according to the methods described above.

[0044] In succinate buffer, the stability of GHRP-6 increased with increasing pH in the range evaluated (5.0-6.0). After storage of the formulation at 60°C for 28 days, the highest stability was reached in succinate at pH 6.0, where the purity of the peptide was found to be 74.31%. The purity of the peptide was lower than that reached under the same conditions for tartrate and histidine buffers.

[0045] Table 5 shows the results of stability testing of GHRP-6 in tartrate and histidine buffers and shows that purity values ​​in RP-HPLC in sodium tartrate buffer were higher than those observed for histidine-HCl at pH values ​​between 5.5 and 6.0. [Table 5]

[0046] Effect of excipients on the stability of lyophilized GHRP-6 in tartrate and histidine buffers at pH 5.5 It is known that solid-state formulations exhibit higher stability than those in aqueous solutions. To achieve the required stability of GHRP-6, different excipients were evaluated for lyophilization in tartrate and histidine-HCl buffers. Buffers at pH 5.5 and a concentration of 25 mmol / L were prepared as previously described. The excipients used were sucrose, lactose, trehalose, mannitol, glycine, alanine and dextran 40, which were dissolved in the evaluated buffers at a final concentration of 3% (w / v). The peptide was dissolved to a concentration of 2.5 mg / mL as previously described. The formulations were then sterilized by filtration under sterile environmental conditions and subjected to lyophilization. Conventional lyophilization cycles adapted to the group of excipients were applied (Santana et al., Biologicals, 2014, 42:322-333).

[0047] For stability testing, samples of the lyophilized formulations were placed in an incubator under stress conditions at 50±2°C. The highest stability of the lyophilized peptide was observed in the presence of trehalose followed by dextran 40, the results of which are shown in Table 6. The results were similar when sodium tartrate buffer at pH 5.5 and histidine-HCl buffer were used. Surprisingly, cryoprotective excipients widely used for lyophilization of peptides and proteins such as sucrose, lactose and mannitol were found to be ineffective for GHRP-6. Meanwhile, Figure 1 shows the RP-HPLC profile of lyophilized GHRP-6 in the presence of the four excipients. It can be seen that the highest stability of the peptide was achieved when lyophilized in the presence of the cryoprotective excipient trehalose. [Table 6]

[0048] Surprisingly, high chemical stability of GHRP-6 lyophilized in the presence of tartrate and histidine buffers in combination with trehalose was observed in other evaluated formulations. Similarly, these formulations showed low numbers of subvisible particles per volume unit when analyzed using the light obscuration method and were consistent with the United States Pharmacopeia (USP) standard. <788> ,2008) for particles of 10 μm and ≥25 μm. Therefore, the composition of the lyophilized formulation evaluated during scale-up and stability testing was 2.5 mg / mL peptide GHRP-6, 25 mmol / L sodium tartrate buffer, pH 5.0–6.0, and 3% (w / v) trehalose as a stabilizer or tonicity agent.

[0049] ● Example 5. Scale-up and stability testing of GHRP-6 tartrate / trehalose liquid formulations Two liquid formulations of GHRP-6 were developed containing 3.75 g / l tartaric acid, 33.16 g / l alpha-alpha dihydrate trehalose, and enough water for injection to fill 1.0 L. In one formulation, the peptide was at a concentration of 2.5 mg / mL and in the other 10.0 mg / mL. To obtain the 2.5 mg / mL concentration, 2.5 g of GHRP-6 was dissolved, and for the 10.0 mg / mL concentration, 10.0 g of GHRP-6 was dissolved.

[0050] All components of the formulation were measured and dissolved in water for injection, except for the API of GHRP-6. The pH of the solution was checked and adjusted to a value of 5.5±0.3 with 1.0 mol / L NaOH. GHRP-6 was added gradually until the desired concentration (2.5 or 10.0 mg / mL) was obtained. Following this, the formulation was sterilized by filtration and the sterile liquid formulation was dispensed into vials in a class 100 area. Three batches of liquid formulation of the peptide were manufactured in tartrate / trehalose at each concentration of GHRP-6 (2.5 and 10.0 mg / mL). Samples from the batches were placed in a cold chamber at a controlled temperature (5±3°C) or in an incubator at 25±2°C. At given time intervals, the samples were analyzed. Stability testing at 25±2°C was performed for 6 months. Critical quality attributes and analytical methods were evaluated similar to those described above.

[0051] Table 7 contains the results of stability studies of liquid formulations of GHRP-6, which show that during 2 years of storage at 5±3° C., the percentage of intact peptide fraction remained above 97% at a concentration of 2.5 mg / mL and above 98% at 10.0 mg / mL. [Table 7]

[0052] During storage at 25±2°C for 6 months, the percentage of intact GHRP-6 fraction remained above 95% for the 2.5 mg / mL presentation and above 97% for the 10.0 mg / mL presentation. Also, the pH remained within the required range. Appearance, number of sub-visible particles, sterility, pyrogen and identity by ESI-MS were found to be satisfactory for all batches at the start and end of the study, at the two concentrations of peptide and at the two temperature values ​​tested. From the results of the stability study, it was concluded that the evaluated liquid formulations were stable for 24 months at 5±3°C and for 6 months at 25±2°C.

[0053] ● Example 6. Scale-up and stability testing of a lyophilized formulation of GHRP-6 in tartrate / trehalose It is known that proteins and peptides in the solid phase generally show higher stability than in the liquid state as the storage temperature increases. Therefore, two lyophilized formulations of GHRP-6 were tested and developed, containing 3.75 g / l tartaric acid, 33.16 g / l α-α-dihydrate trehalose, and a quantity of water for injection sufficient for 1.0 L. For the concentration of 2.5 mg / mL, 2.5 g of GHRP-6 was dissolved, and for the concentration of 10.0 mg / mL, 10.0 g of GHRP-6 was dissolved. The same manufacturing method as in Example 5 was applied. Three batches of the product in bulk in the liquid state were prepared. The product in bulk was then dispensed into vials to proceed to the lyophilization method. A conventional lyophilization cycle was applied (Santana et al., Biologicals, 2014, 42:322-333). Based on the described procedure, three batches of lyophilized formulation of GHRP-6 in tartrate / trehalose were manufactured at each strength of GHRP-6 (2.5 or 10.0 mg / vial). Samples collected from the batches were placed in a cold chamber at controlled temperature (5±3°C) or in an incubator at 25±2°C. At given time intervals, samples of the lyophilized formulation were analyzed. Stability studies at 25±2°C were performed for 6 months. The critical quality attributes and analytical methods evaluated were similar to those described above.

[0054] Table 8 contains the stability results of the lyophilized formulations of GHRP-6. During 2 years of storage at 5±3° C., the percentage of intact peptide fraction remained above 99% for both presentations (2.5 mg / vial and 10.0 mg / vial). The peptide concentration, residual moisture and pH of all samples corresponding to both formulations remained at appropriate values ​​during the tested intervals. [Table 8]

[0055] During storage at 25±2°C for 6 months, the percentage of intact GHRP-6 fraction remained above 99.4% for both presentations. The peptide concentration, residual moisture and pH of all samples remained at appropriate values ​​during the time intervals tested. Also, the appearance, number of particles not visible to the naked eye, sterility, pyrogen and identity tests by ESI-MS were found to be satisfactory for all batches at the two concentrations of peptide and the two temperature values ​​tested, at the beginning and end of the study.

[0056] Based on this stability study, it was concluded that the evaluated lyophilized formulations were very stable for 24 months at 5±3° C. and 6 months at 25±2° C. The percentage of intact peptide remained above 99% for both presentations for 6 months at 25±2° C., justifying that the peptides may be stored even without a cold chain during product distribution.

[0057] ● Example 7. Evaluation of the biological activity of a formulation of GHRP-6 in tartrate / trehalose in an animal model The in vivo biological activity of two formulations of GHRP-6 was evaluated for a formulation containing the peptide at a concentration of 2.5 mg / mL. As controls, samples of the formulation of GHRP-6 in citrate / NaCl (developed in Example 1) and the formulation of GHRP-6 in tartrate / trehalose, prepared as described in Example 5, with 2.5 mg / mL of peptide, were used. As controls, placebos corresponding to each formulation, prepared in the same manner but without the addition of GHRP-6 peptide, were also evaluated.

[0058] The inotropic activity of the peptides was evaluated in Balb / c male mice (6 per group). A dose of 100 μg / kg of the GHRP-6 preparation or an equivalent amount of placebo was administered by intraperitoneal route. For each group, echocardiograms were obtained at time 0 and every 5 minutes up to 15 minutes after administration under the same conditions of monitoring, temperature and sedation.

[0059] M-mode two-dimensional ultrasound images were obtained using a high-resolution imaging system (Vevo770™, Visual Sonics Inc.). The system was equipped with a 40 MHz scaled matrix linear magnetic amplifier and acquired data at frequencies up to 250 photograms / second with an axial resolution of up to 30 microns between focal regions. Data processing and analysis were performed applying the software Vevo770 (version 3.0.0, Visual Sonics Inc.) taking into account the parasternal long axis at an angle of 45° and caudal angulation of the platform. Left ventricular ejection fraction (LVEF) was calculated by Teichold's formula.

number

[0060] Measurements were performed according to a specific echocardiogram, with diastolic parameters measured at the peak of the R wave and systolic parameters measured at the peak of the T wave. Measurements were completed over 3 to 5 consecutive cardiac cycles, in accordance with the recommendations of the American Society of Echocardiography (Langy et al., EUR. J. Echocardiogr., 2006, 7(2):79-108).

[0061] According to Figure 2, for individuals treated with the peptide in a formulation in tartrate / trehalose, LVEF increased significantly over time and remained elevated for at least the 15 minutes tested. This effect was not observed in individuals treated with a placebo formulation. LVEF was higher for individuals receiving the formulation in tartrate / trehalose compared to individuals treated with other formulations of the peptide. That is, the formulation of GHRP-6 in tartrate / trehalose showed higher inotropic activity in the animal model used.

[0062] ● Example 8. Preclinical Toxicity Study of Formulations of GHRP-6 in Tartrate / Trehalose After the desired biological activity was demonstrated, the lyophilized formulation containing sodium tartrate, trehalose and 5 mg of GHRP-6 per vial was reconstituted and subjected to toxicity testing in the following animal models:

[0063] Assessment of acute toxicity in Sprague-Dawley rats A total of 50 rats (25 females and 25 males) were used, with 5 groups of 10 individuals each. The doses corresponding to each group were administered in three applications, one every 6 hours. The products were applied by intraperitoneal route on days 1 and 2 of the experimental phase. A TD of 400 μg / kg was used as a guide. Table 9 shows the design of the study carried out. [Table 9]

[0064] After detailed analysis of the results obtained, it was concluded that the lyophilized formulation of the peptide GHRP-6 in tartrate / trehalose did not cause any acute systemic changes compromising the safety of patients at the doses evaluated, thus providing a sufficient safety framework up to 100 times the TD to allow the formulation in tartrate / trehalose to be submitted to clinical trials.

[0065] Evaluation of repeated dose toxicity in Sprague-Dawley rats 100 young adult rats of both sexes (50 females and 50 males) were used, constituting 7 groups. Table 10 shows the design of the study carried out. The non-treated and satellite groups (administered 50 times the TD) consisted of 10 animals each. The other 5 groups were completed with 16 animals each. The satellite group received the highest dose used in the study, with the aim of evaluating the reversibility of potential effects, and the animals of said group were sacrificed after twice the time of the remaining animals of the study. In all the treatment groups constituted, 14 consecutive doses (once a day for 2 weeks) were administered by intraperitoneal route. [Table 10]

[0066] The obtained results were analyzed in detail and it was concluded that the lyophilized preparation of GHRP-6 in tartrate / trehalose was not toxic at systemic levels up to 50 times the TD.

[0067] Evaluation of local tolerability in F1 rabbits A total of 18 females were used, constituting 6 groups of 3 animals each. Five consecutive doses were administered by intravenous route every 72 hours. The distribution of the populations by treatment was as follows: Group 1, no treatment; Group II, placebo; Group III, TD of GHRP-6 (400 μg / kg); Group IV, 5 times the TD of GHRP-6 (2000 μg / kg); Group V, 10 times the TD of GHRP-6 (4000 μg / kg); and Group VI, 10 times the TD of GHRP-6 (4000 μg / kg).

[0068] Analysis of the results obtained in this study concluded that repeated administration of the preparation of GHRP-6 in tartrate / trehalose was well tolerated and that the adverse events observed did not constitute signs of toxicity.

[0069] ● Example 9. Evaluation of the safety of a GHRP-6 formulation in tartrate / trehalose in patients with acute myocardial infarction The study used a lyophilized formulation of GHRP-6 containing sodium tartrate, trehalose, and 5 mg of GHRP-6 per vial. A prospective, multicenter, open, randomized, phase IB clinical trial (exploratory) was conducted in patients with acute myocardial infarction undergoing primary percutaneous transluminal coronary angioplasty. After reconstitution of the lyophilized formulation, the peptide was randomly administered as a bolus via the intravenous route at a dose of 50 or 100 μg per kg of patient body weight. The first dose was administered at the time the patient entered the study. The product was then administered every 12 hours for 7 days, up to a total of 14 doses of the peptide. The sample consisted of 19 patients, both male and female, over the age of 30, who volunteered to participate in the study. The primary objective of the study was to evaluate the safety of a lyophilized formulation containing GHRP-6.

[0070] Table 11 shows a summary of the registered adverse events, intended to be compared with those observed in a study of dose scale-up in healthy volunteers carried out with a formulation of GHRP-6 in citrate / NaCl. In this table it can be seen that in this study no adverse events were observed caused by the citrate / NaCl formulation as reflected in Example 3. The other events were of mild or moderate intensity, most of which had only a low causal relationship to the peptide under study and were attributable to the patient's baseline illness. [Table 11]

[0071] Thus, the conclusions from the clinical evaluation of a formulation of GHRP-6 in tartrate / trehalose are that it does not cause bradycardia, can be used in the treatment of patients suffering from cardiovascular or cerebrovascular diseases, and provides the necessary stability at high concentrations of the peptide for at least two years. [Sequence List Free Text]

[0072] Sequence Listing 1 <223> Description of the artificial sequence: GHRP-6, Trp2 and Phe5 are D amino acids <223> Amidation

Claims

1. A pharmaceutical composition comprising growth hormone releasing peptide 6 (GHRP-6) at a concentration of 1.0 mg / mL to 10.0 mg / mL, a tartrate buffer of pH 5.0 to 6.0, and trehalose, wherein the pharmaceutical composition is lyophilized and stable at room temperature (not under refrigerated conditions).

2. The composition of claim 1, wherein the buffer has a concentration of 10 mmol / L to 100 mmol / L.

3. The composition according to claim 1 , wherein the tartrate buffer is formed by a sodium salt, a potassium salt or a mixture of both.

4. 2. The composition of claim 1, wherein the concentration of trehalose is in the range of 2% to 10% (w / v).

5. The composition described in claim 1, which is a result of lyophilization.

6. A pharmaceutical composition according to any one of claims 1 to 5 for use as a medicine.

7. The pharmaceutical composition according to any one of claims 1 to 5 for use as a cell protective agent, a cardioprotective agent, a cardiorestorative agent or a neuroprotective agent, or as an agent for restoring brain damage.

8. A pharmaceutical composition described in any one of claims 1 to 5, which is administered by intravenous, subcutaneous or intramuscular route.

9. A kit of parts comprising a pharmaceutical composition according to any one of claims 1 to 5.

10. The kit of parts described in claim 9, comprising a second pharmaceutical composition.

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