Stable, high-concentration formulation of the antibody nimotuzumab

Abstract

The present invention relates to the fields of biotechnology and medicine and describes high-concentration pharmaceutical formulations containing the humanized monoclonal antibody nimotuzumab in the concentration range of 50-200 mg / mL. The low viscosity of these solutions allows for their use subcutaneously or intramuscularly in cancer treatment. These formulations are stable in both liquid and lyophilized form.

Description

[Technical Field]

[0001] The present invention relates to the fields of biotechnology and medicine, and in particular to obtaining highly concentrated and stable formulations of the humanized monoclonal antibody nimotuzumab intended for subcutaneous (SC) or intramuscular (IM) administration as a cancer treatment. [Background technology]

[0002] Nimotuzumab is a humanized IgG1 isotype monoclonal antibody (mAb) that recognizes the human epidermal growth factor receptor Her1. It was obtained by cloning in frame the DNA of the hypervariable region of the murine mAb ior egf / r3 and the variable and constant regions of the heavy and light chains of human origin (REI and NEWM, respectively) (Mateo, C. et al. (1997), Immunotechnology 3:71-81). The efficacy of nimotuzumab has been demonstrated in clinical trials in patients with head and neck tumors (Crombet, T. et al. (2004) J Clin Oncol. 22:1646-1654), gliomas (Ramos, T. et al. (2006) Cancer Biol Ther. 5:375-379, MacDonald, T. et al. (2011) Neuro Oncol. 13:1049-1058), and esophageal tumors (Ramos-Suzarte, M. et al. (2012) Cancer Biology & Therapy 13:600-605). This antibody (Ab) is currently being tested in phase III clinical trials in nasopharyngeal carcinoma, locally advanced esophageal cancer, and esophageal squamous cell carcinoma (Galluzzi, L. et al. (2012) OncoImmunology. 1:28-37).

[0003] Nimotuzumab mAb is administered intravenously (IV) as an infusion using a liquid formulation containing the mAb at a concentration of 5 mg / mL (https: / / www.cecmed.cu / registro / rcp / cimaher-nimotuzumab). This administration is performed by dissolving four vials of nimotuzumab at a concentration of 5 mg / mL in a bag of 0.9% NaCl solution. The composition of the commercial formulation of nimotuzumab also includes a 15 mM sodium phosphate buffer solution at a pH of approximately 7, 150 mM sodium chloride, and 0.02% polysorbate 80 (Revista Cubana de Farmacia. 2012;46(3):379-380). In clinical settings, nimotuzumab is administered weekly for six weeks at a dose of 200 mg (400 mg for pancreatic cancer) in combination with radiation therapy and chemotherapy. A maintenance dose of 200 mg is then administered every 15 days until the patient's clinical condition permits. It is therefore important to have a route of administration that facilitates this continuous use of nimotuzumab in patients outside of a hospital setting.

[0004] As is known, IV administration has several drawbacks, such as the long time required to administer the drug, the need for qualified personnel trained in the administration procedure, the risk of infection, and severe adverse reactions (Michael F. Haller (2007) Pharmaceutical Technology, 31(10):118-132).

[0005] For this reason, there is an increasing global trend toward SC administration of mAbs (Viola M, Sequeira J et al. (2018) J Control Release;286:301-314). This route offers advantages such as easier manipulation and administration of a single vial in the required amount (Haller MF (2007) Pharmaceutical Technology,31(10):118-132), reduced time required for administration and the volume administered, reduced risk of infection, and even the possibility of self-administration by patients. IM administration also has advantages compared to IV administration, such as lower treatment costs and less intensive care required. On the other hand, the IM route is associated with a lower risk of infection and a shorter administration time than the IV route. It is also known that drugs administered via the IM route are less likely to cause adverse effects or overdose. It should also be noted that the absorption and bioavailability of drugs after IM administration are very high, almost as high as those reported using the IV route. (Jing-fen Jin et al.,Patient Preference and Adherence,2015,9:923-942).

[0006] Among the mAbs currently in clinical use in SC formulations are trastuzumab (U.S. Pat. No. 9,345,661) and rituximab (U.S. Pat. No. 10,280,227); in both formulations, the enzyme hyaluronidase is used to increase the injection volume to greater than 1.5 mL and achieve good biodistribution of the mAb, thus ensuring that the dose required for therapeutic effect is reached (Shpilberg, O. and C. Jackisch. (2013) British Journal of Cancer. 109:1556-1561).

[0007] Another high-concentration formulation using hyaluronidase enzyme is described in patent application CN 107898756A, which claims a highly concentrated formulation of nimotuzumab mAb for SC or IM use. As reported in the patent, the presence of hyaluronidase in this formulation is necessary to ensure that the necessary permeability is achieved.

[0008] Previous reports have described highly concentrated formulations of mAbs against epidermal growth factor receptor (EGFR), such as the formulation described in International Publication No. WO 2011080209, which claims a highly concentrated liquid formulation obtained by ultrafiltration containing an anti-EGFR mAb. While this patent states that the claimed formulation can be used for nimotuzumab mAb, no actual evidence is provided to support this claim. While this patent application claims mAb concentrations ranging from 1 mg / ml to 200 mg / ml, it only reaches a maximum concentration of 50 mg / ml for mAb hu-ICR 62. Furthermore, the range of excipients and concentrations claimed in this patent application is too broad, as it includes almost all of the most commonly used excipients and concentrations in the state of the art. In the present invention, it has been demonstrated that some combinations of excipients and concentrations within the range claimed in International Publication No. WO 2011080209 are not the most stable or best formulation options for nimotuzumab mAb.

[0009] A prime example of a mAb administered by the IM route is palivuzumab mAb, which is presented as a lyophilized formulation that, after reconstitution in water, has a concentration of 100 mg / mL of palivuzumab mAb in addition to excipients such as histidine, glycine, and mannitol and is used at a dose of 15 mg / kg for the treatment of respiratory syncytial virus.

[0010] Achieving a mAb formulation that maintains stability, low turbidity, and viscosity is a challenge that requires appropriate screening methodologies. Finding the right combination of excipients and conditions required for mAbs to meet these requirements at high concentrations is not a trivial task. To date, it is not possible to predict in advance which combination will be most effective for a particular mAb, as each combination is different and behaves differently with respect to its primary degradation mechanism (Manning MC et al. (2018) Advances in Protein Chemistry and Structural Biology, 112:1-59; Viola M, Sequeira J, et al. (2018) J Control Release; 286:301-314).

[0011] The present inventors have discovered formulations for nimotuzumab mAb that differ from previously reported ones, with a specific combination of excipients that ensure mAb stability, low turbidity, and low viscosity. These formulations are also less complex because they do not require the use of hyaluronidase enzymes to ensure the stability and permeability of SC formulations. These specific formulations also achieve nimotuzumab concentrations of 100-180 mg / mL, thus enabling increased mAb doses without increasing administration volume. Administration of these formulations via SC or IM routes has the added advantage of ease of use and the ability for patients to self-administer them when chronic administration is required, at doses of 200 mg-360 mg for SC and 200-720 mg for IM. Summary of the Invention

[0012] In one embodiment, an object of the present invention is a highly concentrated and stable pharmaceutical formulation of nimotuzumab monoclonal Ab, comprising nimotuzumab mAb in a concentration range of 100-210 mg / mL, a buffer substance in the range of 5-30 mM, a pH value of 6.5±0.5, a surfactant in the range of 0.02-0.06%, an amino acid or a mixture thereof in the range of 30-150 mM, and optionally a carbohydrate as a stabilizer in the range of 2-6%.

[0013] In particular, the buffer substance of the formulation is selected from histidine buffer and sodium phosphate. The surfactant is selected from polysorbate 20 and polysorbate 80. The amino acid is selected from L-methionine and glycine. The carbohydrate used in the formulation is sucrose.

[0014] More specifically, the formulation may be in liquid or lyophilized form. A liquid formulation at a concentration of 150 mg / mL has a viscosity of 5 cP or less.

[0015] A further object of the present invention is the use of the above-described pharmaceutical preparation in the treatment of cancer. In particular, a method of treating a patient in need thereof is described, comprising administering the pharmaceutical preparation described in the present invention at a dose of 200 mg / 70 kg to 750 mg / 70 kg via the SC or IM route. SC administration of this mAb is performed using an injection volume of 1.3 to 2 mL and a concentration of 150 to 200 mg / mL. Furthermore, the mAb can be administered at more than one injection site if a higher dose of mAb is required. For the IM route, a dose of 200 to 750 mg / 70 kg is achieved with an injection volume of 1.3 to 5 mL at a concentration of 150 mg / mL. A dose of 260 to 1000 mg / 70 kg is achieved using a concentration of 200 mg / mL and an intramuscular volume of 1.3 to 5 mL. DETAILED DESCRIPTION OF THE INVENTION

[0016] Obtaining a stable, highly concentrated formulation of nimotuzumab mAb The present invention relates to a high-concentration formulation of the mAb nimotuzumab, also referred to herein as hR3. The concentration of nimotuzumab in the formulation described herein is in the range of 100-210 mg / ml, preferably 100-180 mg / ml. The nimotuzumab formulation disclosed in this patent application comprises a buffering substance, a surfactant, an amino acid, and optionally a carbohydrate. The buffering substance may be histidine or sodium phosphate in the range of 5-30 mM, which allows the pH of the formulation to be maintained in the pH range of 6.5±0.5. The formulation of the present invention may contain polysorbate 20 or polysorbate 80 as a surfactant in the concentration range of 0.02-0.06%. The carbohydrate may be sucrose or trehalose in the concentration range of 2-6%. Furthermore, the amino acid is selected from L-methionine, glycine, or both in the concentration range of 30-150 mM.

[0017] To obtain these formulations, an Amicon Ultra ultrafiltration-diafiltration (UF / DF) system with cellulose membranes having pore sizes of 30-50 kD can be used. To diafilter or concentrate the sample, a centrifugation speed ranging from 900 x g to 1100 x g at a temperature of 2-8°C can be used. The sample is subjected to sterile filtration, with the type of filter selected depending on the sample volume. Another method for preparing the formulations described in this invention is by using a laboratory- or pilot-scale tangential flow filtration system. For this purpose, membranes with pore sizes of 30 and 50 kD can be used. Diafiltration of the sample to change buffer is performed using 10-15 diavolumes of final buffer. To concentrate the sample, the initial volume must be reduced to a volume smaller than the calculated volume corresponding to the desired concentration.

[0018] These formulations can be obtained by a stepwise screening strategy, consisting of a first step in which the best buffer and pH value are selected. In a second step, the most appropriate salts, surfactants, and carbohydrates are selected, followed by a third step in which the best amino acids for the formulation are selected. Another method that can be used is simultaneous screening, which involves the use of different types of excipients or their combinations according to a factorial design. When using this method, interactions may occur between two or more excipients in the same formulation.

[0019] In another aspect, the present invention provides a method for lyophilizing and reconstituting the above formulation, as well as a method for preparing a stable, isotonic, reconstituted formulation, comprising reconstituting the lyophilized mixture of mAb and stabilizer in the above buffer so that the mAb concentration in the reconstituted formulation is at least 100 mg / mL to 210 mg / mL, i.e., 3-5 times higher than the mAb concentration in the mixture before lyophilization.

[0020] Determining the stability of high-concentration formulations of nimotuzumab To determine the most stable formulation variant and the effect of each excipient on stability, a study of parameters predicting stability was performed using dynamic light scattering (DLS). These parameters, as mentioned above, are the diffusion interaction parameter (kD), zeta potential, and aggregation temperature (Tagg). kD was measured by measuring the diffusion coefficient of nimotuzumab at concentrations from 12 mg / ml to 2 mg / ml. Tagg was determined from an initial sample at a temperature ramp from 25 to 76 °C, and zeta potential was determined by measuring samples at the initial concentration.

[0021] Another method used to determine the best formulation is the stress test, in which samples are subjected to a temperature of 50°C in a thermostatic chamber for 10 to 20 days and then analyzed at different time intervals by physicochemical and biological determinations.

[0022] Among these determinations is turbidity, which is measured by the absorbance of a sample in the UV spectrum from 340 to 450 nm, allowing the detection of large aggregates. Another example is the determination of particle size by DLS, which is performed by measuring the initial concentration of an intact, undiluted sample to detect the formation of aggregates or particles in solution. Viscosity measurements are performed for highly concentrated samples using DLS size standards with a nominal diameter range of 100 to 500 nm, preferably a nominal diameter of 200 nm.

[0023] Samples were also analyzed by SDS-PAGE to determine whether fragment formation occurred during the stress test. The analysis was performed under non-reducing conditions using 7.5% polyacrylamide gels and Coomassie blue or silver nitrate staining. Monomer purity was also determined by size-exclusion high-performance liquid chromatography (SEC-HPLC).

[0024] The biological activity of the mAb is determined by the relative potency to the nimotuzumab reference substance and can be measured by flow cytometry or by inhibition of cell proliferation in cell lines expressing human Her1.

[0025] Treatment method The formulation of the present invention can be used to treat patients with head and neck, glioma, esophageal, lung, and pancreatic tumors. For therapeutic use, the formulation should be administered to the diseased subject as a monotherapy or in combination with conventional therapies used to treat tumors, such as radiation therapy or chemotherapy, to enhance the therapeutic effect.

[0026] These formulations are administered via the SC or IM route. For the SC route, a minimum sample volume of 1.33 mL should be used for a 200 mg / 70 kg dose. For larger doses, the injection volume can be increased by up to 2 mL until a maximum dose of 300 mg / 70 kg is reached. If a dose greater than 300 mg / 70 kg is required, a mAb concentration greater than 150 mg / mL can be used, or the dose can be split and administered into two separate injection sites. Injection sites that can be used for this administration include, but are not limited to, the deltoid region, abdominal region, and anterior thigh region. If the dose used in the clinic is 200 mg per week, a nimotuzumab concentration of 134 mg / mL will be sufficient to administer the treatment. If a higher dose is required, a higher concentration of nimotuzumab, up to 210 mg / mL, can be used. For example, for the treatment of pancreatic cancer requiring a dose of 400 mg, two separate injection sites should be used.

[0027] The literature reports that the maximum acceptable IM administration volume is 5 mL; therefore, using this route of administration, injection volumes can be 1.3 to 5 mL, resulting in doses of 200 to 750 mg / 70 kg when the mAb concentration is 150 mg / mL. If a 200 mg / mL concentration is used, doses can be up to 1000 mg / 70 kg.

[0028] The present invention will be further described in detail using the following examples and figures, which should not, however, be construed as limiting the scope of the present invention. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 shows kD values ​​of hR3 mAb formulation variations determined by DLS. [Figure 2] FIG. 1 shows the kD values ​​of different formulations of hR3 mAb containing amino acids as determined by DLS. [Figure 3] FIG. 1 shows particle size measured by DLS of hR3 mAb formulations containing amino acids. [Figure 4] FIG. 1 shows particle size measured by DLS for hR3 mAb formulations at a concentration of 150 mg / mL stored at 4° C. for 3 months. [Figure 5] FIG. 1 shows the viscosity measured by DLS of hR3 mAb formulations at a concentration of 150 mg / mL stored at 4° C. for 3 months. [Figure 6] FIG. 1 shows the monomer purity measured by SEC-HPLC of hR3 mAb formulations at a concentration of 150 mg / mL stored at 4° C. for 3 months. [Figure 7] FIG. 1 shows the antitumor effect of nimotuzumab mAb administered by IV and SC routes at a dose of 50 mg / kg to athymic Balb / c mice. [Example]

[0030] Example 1. Screening of formulation excipients for nimotuzumab mAb. Nine formulation variations were prepared from a four-factor and half-fraction factorial design, as shown in Table 1. The hR3sol9 variation corresponds to the original formulation of hR3 mAb. After the solutions were prepared, they were filtered through a 0.2 μm pore size membrane filter and stored at 4°C. [Table 1]

[0031] Once the solution was prepared, buffer exchange was performed by UF / DF in an Amicon Ultra Centrifugal Filter Unit with a 50 kD pore size, regenerated cellulose membrane, and a 15 mL sample volume. Samples were centrifuged at 936 × g at 4 °C. mAb hR3 was used as the starting product in a 15 mM sodium phosphate buffer, 150 mM sodium chloride, and 0.2 mg / mL Tween 80, pH 7 solution. After preparing a formulation at a 10 mg / mL Ab concentration, the diffusion interaction parameter (kD) was measured by DLS.

[0032] As can be seen in Figure 1, the variants with higher kD values, and therefore more stable protein-protein interactions and less tendency to aggregate, are the hR3sol8 and hR3sol3 variants, followed by the hR3sol4 variant, demonstrating the stabilizing effect of carbohydrates in the formulation. During this testing, it was also observed that the kD value of the hR3sol8 variant was much higher than that of the hR3sol3 variant, suggesting that the combination of histidine and sucrose has greater stability than the combination of phosphate and trehalose. NaCl was absent in any of the best variants, indicating that NaCl compromises the stability of the hR3mAb.

[0033] Another experiment aimed at verifying which variant was most stable was a stress test conducted at 50°C for 14 days. After stress, samples were analyzed by SEC-HPLC to determine the purity of the monomers. Table 2 shows that the variants with the highest monomer purity were hR3sol8 and hR3sol3, which is consistent with the kDa value measurements. The variants with the lowest monomer purity were hR3sol9 and hR3sol2, i.e., the variants with sodium chloride in their composition, further supporting previous results showing the negative effect of this excipient on stability, regardless of the type of buffer used. [Table 2]

[0034] Based on previous results, it was concluded that the most stable hR3 mAb formulation variants were those with several carbohydrates, such as sucrose and trehalose, in the absence of sodium chloride. Furthermore, the combination of sucrose and histidine is significantly more stable than the combination of phosphate and trehalose.

[0035] Example 2. Screening of amino acids for formulation of hR3 mAb. Different solutions were prepared from the two best variants obtained after excipient screening, and amino acids (L-arginine, L-methionine, glycine) were added to each. The preparation methods were similar to those described in Example 1 for excipient screening. Table 3 shows the formulation variants of hR3 mAb containing different amino acids. [Table 3]

[0036] The results of the kD measurements performed by DLS are shown in Figure 2. As can be seen, the variants with the highest kD values ​​are hR3sol80, hR3sol81, hR3sol82, and hR3sol32, and the most stable variant among these is the one with the histidine buffer. It should also be noted that in both buffers, the presence of methionine leads to high kD values ​​that do not occur with other amino acids.

[0037] The different formulations were subjected to stress in a thermostatic bath at 50°C for 15 days, and particle size was measured by DLS on days 0, 5, 12, and 15. Figure 3 shows the results of these measurements. As can be seen, the variants with smaller particle size, and therefore less aggregated, were the hR3sol80, hR3sol82, and hR3sol83 variants. This confirms that formulations with histidine buffer were more stable than those with phosphate buffer, and that variants containing L-methionine or glycine were found to be more stable than those containing L-arginine.

[0038] Additionally, Table 4 shows the monomer purity values ​​of the formulations stressed at 50°C. [Table 4]

[0039] Table 4 shows that the variants with the highest monomer purity after stress were hR3sol82 and hR3sol80. This result indicates that the variants with histidine buffer are more stable than those with phosphate buffer. It also suggests that the formulations containing methionine are more stable than those without amino acids. The formulations with the lowest purity were hR3sol81 and hR3sol90. This suggests that both L-arginine and sodium chloride have a destabilizing effect on the hR3 mAb formulation. In accordance with previous results, the hR3 mAb formulation with histidine buffer at pH 6, sucrose, polysorbate 80, and L-methionine is the most stable.

[0040] These results demonstrate that the hR3sol2, hR3sol6, hR3sol31, hR3sol81, and hR3sol90 variants within the claimed combinations of WO 2011080209 are not recommended for nimotuzumab mAb because their poor stability adversely affects their efficacy and the safety of their administration to patients. This supports the notion reported in the literature that a formulation stable for one protein is not necessarily stable for another due to structural differences between proteins.

[0041] Example 3. Shelf-life stability study of high concentration hR3 mAb stored at 4°C Stability characterization was performed on hR3sol80, hR3sol82, and hR3sol90 variants at a mAb concentration of 150 mg / mL stored at 4° C. Samples underwent physicochemical analysis of particle size and viscosity by DLS, and monomer purity analysis by HPLC at 0 and 3 months.

[0042] Figure 4 shows the results of particle size measured by DLS for the different variants. As can be seen in the figure, the diameter remained constant for 3 months in each of the formulations, indicating the absence of aggregation in the highly concentrated samples. However, it should be noted that the particle size of the hR3sol90 variant was initially approximately 25 nm, which is larger than the particle size of the other formulations. This suggests that this variant has a greater tendency to self-associate intermolecularly compared to the hR3sol80 and hR3sol82 variants.

[0043] Figure 5 shows viscosity values ​​measured by DLS using 200 nm particle size standards. In all cases, the viscosity had very little variation, indicating the absence of intermolecular aggregation effects or self-association. Furthermore, it is very important to emphasize that all values ​​were less than 5 cP, which is well below the established limit for achieving adequate injectability and manufacturability (Li Li, Sandeep Kumar et al. Pharm Res (2014) 31:3161-3178). The viscosity values ​​are also lower than those reported for other Abs at a concentration of 150 mg / mL.

[0044] Figure 6 shows a graph of the monomer purity measured by SEC-HPLC. As can be seen, the purity of the monomer did not change appreciably, suggesting that the formulation of hR3 mAb at a concentration of 150 mg / mL stored at 4°C is stable.

[0045] Additionally, the biological activity of the hR3 mAb formulation was determined at a concentration of 150 mg / mL without exposure to stress at time 0 and 3 months. Biological activity was measured by hR3 mAb recognition of human epidermal growth factor receptor (Her1) in a flow cytometry assay and inhibition in a cell proliferation assay. To perform the flow cytometry assay, 2 × 10 H292 cells per well were used, and a concentration curve was generated starting at 81 μg / mL at a 1 / 3 dilution. The mean fluorescence intensity (MFI) was then determined. The half maximal effective concentration (EC50) was determined from the MFI value, and relative potency was obtained by comparing it to a commercially available hR3 reference material. Cell proliferation assays were performed using H292 cells and cell proliferation reagent WST-1 solution as the dye. The resulting absorbance was compared to a commercially available hR3 reference material to determine relative potency. Results from both measurements at time 0 are shown in Table 5, and results performed 3 months after the start of the study are shown in Table 6. [Table 5]

[0046] Table 5 shows that the relative potencies of the hR3sol80, hR3sol82, and hR3sol90 variants, as measured by flow cytometry assay, were 79-83%, indicating that they maintain their biological activity even at high concentrations. Meanwhile, the relative potency values ​​obtained in the inhibition of cell proliferation assay were 93-104%, confirming the results from cytometry and demonstrating that high-concentration samples retain their biological activity. It should be noted that each value falls within a different confidence interval due to inherent variability in each assay. Nevertheless, the values ​​are within the acceptable range for each assay. [Table 6]

[0047] Table 6 shows the biological activity results of hR3 formulations at a concentration of 150 mg / mL stored for 3 months at 4° C. As can be noted, the samples with the highest relative potency values ​​were hR3sol80 and hR3sol90, with values ​​ranging from 78 to 81% in recognition by cytometry assay and 77 to 86% in inhibition of proliferation assay.

[0048] Example 4. Stress testing at 50° C. of the best formulation variant of hR3 mAb at a concentration of 150 mg / mL. For formulation variants hR3sol80, hR3sol82, and hR3sol90, hR3 was concentrated to 150 mg / mL by UF / DF in Amicon Ultra Centrifugal Filter Units with a 50 kD pore size, regenerated cellulose membrane, and 15 mL sample volume. Samples were centrifuged at 936 g at a temperature of 4°C. Upon reaching a concentration of 150 mg / mL, the samples were filtered through a 0.2 μm pore size filter and placed in a 50°C incubator. Sampling of the different formulations was performed on days 0, 4, 10, and 15 to measure physicochemical parameters. Table 7 shows the results of the monomer purity measurements performed on the different sampling dates. [Table 7]

[0049] As can be seen in Table 7, after 4 days of stress, the hR3sol90 variant completely precipitated to form a white solid, while the hR3sol80 variant had a monomer purity of 45.3% and the hR3sol82 variant had a monomer purity of 58.9%. From these results, it can be concluded that the most stable formulation at high concentrations is hR3sol82, while the least stable formulation is hR3sol90. This result is consistent with the results obtained at lower concentrations. After 10 days of stress, the hR3sol80 variant formed a yellow gel, while the hR3sol82 variant remained in solution with a monomer purity of 43.9%. Sampling performed on day 15 also showed that the hR3sol82 variant also formed a yellow gel. Previous results demonstrate that hR3sol82 formulations with L-methionine in their composition are more stable at higher concentrations than those lacking this amino acid. These results suggest that the most stable formulations over time are hR3sol80 and hR3sol82, while the hR3sol90 formulation is the least stable.

[0050] Table 8 shows the percentage of monomers and dimers in the sample at a concentration of 150 mg / mL without stress exposure. [Table 8]

[0051] As can be seen in the table, the hR3sol82 formulation is the formulation with the highest monomer percentage (94.1%) and the lowest dimer percentage (5.2%), demonstrating that this formulation has a trend toward a reduced number of associations between molecules contained in the formulation, which promotes stability. The hR3sol80 and hR3sol90 variants have monomer percentages of 93.0% and 92.0%, respectively.

[0052] The biological activity of the hR3 mAb formulation at a concentration of 150 mg / mL was also determined by flow cytometry and by inhibition of cell proliferation assays before being subjected to stress at 50° C. The same methods described in Example 3 were used to make both determinations.

[0053] Example 5 Bioavailability of nimotuzumab mAb administered by SC route at different dose levels to Balb / c mice. Bioavailability studies were performed using the hR3sol90 variant, given that the same Ab and administration route were used and that changes in formulation components did not significantly affect the pharmacokinetic behavior of the Ab. To perform this assay, female Balb / c mice weighing approximately 20 g were used. The animals were divided into four groups that received four different dose levels of the hR3sol90 formulation (25, 50, 100, and 200 mg / kg body weight) via SC. Prior to its administration, the mAb was isotopically iodine-125 ( 125 The mice were labeled with 1000 mg / kg IV (I). Blood was then extracted from each individual mouse and the radioactivity, which is proportional to the Ab concentration in the blood, was measured. Table 9 shows the bioavailability values ​​obtained at different dose levels compared to the 25 mg / kg IV dose. The following formula was used to calculate bioavailability (F): F=(SC-AUC / IV-AUC)*(IVd / SCd), where, SC-AUC is the area under the curve (AUC) of the SC pathway; IV-AUC is the AUC (area under the curve) for the IV route; IVd is the IV dose; SCd represents the SC dose. In the case of the IV route, 100% of the parent drug enters the systemic circulation, so the bioavailability is 1. [Table 9]

[0054] The results in Table 9 show that in all cases, bioavailability exceeded the 75% acceptable for this type of administration route. It should be noted that even though bioavailability does not actually increase as dose increases, the total mass of mAb and AUC (area under the curve) increase. Extrapolating these results to humans, for example, for a 100 mg / kg SC dose, this means that even with a bioavailability of 78.7%, the total mass of mAb that would reach the bloodstream would be 630 mg, more than three times the dose used in the clinic (200 mg).

[0055] Example 6. Antitumor efficacy of nimotuzumab mAb administered by IV and SC routes at a dose level of 50 mg / kg in athymic Balb / c mice. Female athymic Balb / c mice weighing approximately 20 g were divided into three groups of five animals each. All groups received 2 x 10 A431 cells on days 0 and 10, 12, 14, and 16, Group 1 received 1 mg total hR3sol90 formulation mass administered SC, Group 2 received 1 mg total nimotuzumab mAb mass IV, and the control group, Group 3, received phosphate-buffered saline.

[0056] Figure 7 shows that tumor volumes were similar in mice administered nimotuzumab via the SC and IV routes. This demonstrates that despite the fact that the absorption rate and pharmacokinetic properties of each route are different, there was no difference in the efficacy of treatment when using these two routes. Meanwhile, the control group had a much higher tumor growth rate, which was significantly different from the growth rates of the other two groups. This demonstrates the effectiveness of administering nimotuzumab mAb via the SC route.

[0057] Example 7. Purity of lyophilized nimotuzumab mAb at a concentration of 150 mg / mL. The hR3sol80, hR3sol82, and hR3sol90 formulations were lyophilized at a concentration of 150 mg / mL and a volume of 500 μL. For this purpose, a laboratory lyophilizer was used at a temperature of -30°C and vacuum was applied for 30 hours until a dry powder was obtained. The formulations were then reconstituted in 500 μL of ultrapure water until the initial concentration was reached, resulting in a clear, particle-free solution.

[0058] Table 10 shows the purity data obtained from different nimotuzumab mAb formulations after lyophilization and subsequent reconstitution. [Table 10]

[0059] As can be seen, after lyophilization and reconstitution, the hR3sol82 formulation reached greater than 98% monomer purity, while the hR3sol80 variant reached approximately 94% monomer purity and the hR3sol90 variant reached greater than 93% monomer purity. It should also be noted that the dimer percentage varied depending on the type of formulation. Furthermore, the table shows that the monomer + dimer purity of these lyophilized and reconstituted formulations was greater than 99% in all cases, indicating that the nimotuzumab mAb did not lose its stability even when stressed during the lyophilization process. It should be noted that the nimotuzumab mAb has a balance between monomer and dimer due to the characteristics of this mAb, and this balance can vary depending on the type of interactions occurring in solution. The present invention includes the following preferred embodiments. (1) A highly concentrated and stable pharmaceutical formulation of the monoclonal antibody (mAb) nimotuzumab, comprising: a) nimotuzumab mAb at a concentration in the range of 100 to 210 mg / mL; b) a buffer substance in the pH range of 6.5±0.5 and at a concentration in the range of 5 to 30 mM; c) surfactants in the range of 0.02 to 0.06%; d) an amino acid or a mixture thereof in the range of 30 to 150 mM, and e) optionally, carbohydrates as stabilizers in the range of 2-6% The pharmaceutical preparation characterized by comprising: (2) The buffer substance is histidine buffer, and Sodium phosphate The pharmaceutical formulation according to (1), selected from the group consisting of: (3) The surfactant is Polysorbate 20, and Polysorbate 80 The pharmaceutical formulation according to (1), selected from the group consisting of: (4) The amino acid L-methionine, and glycine The pharmaceutical formulation according to (1), selected from the group consisting of: (5) 10. The pharmaceutical formulation according to claim 1, wherein the carbohydrate is sucrose. (6) The pharmaceutical preparation according to any one of (1) to (5), which is in a liquid form. (7) The pharmaceutical preparation according to any one of (1) to (5) above, in a freeze-dried form. (8) The pharmaceutical formulation according to (6), characterized in that it has a viscosity of 5 cP or less at a concentration of 150 mg / ml. (9) Use of the pharmaceutical preparation according to any one of (1) to (8) for treating cancer. (10) A method for treating a patient in need thereof, comprising subcutaneously administering the pharmaceutical formulation according to any one of (1) to (8) at a dosage level of 200 mg / 70 kg to 400 mg / 70 kg using an injection volume of 1.3 to 2 mL, wherein the dose of the pharmaceutical formulation can be divided and administered at two or more separate injection sites. (11) A method for treating a patient in need thereof, comprising intramuscularly administering the pharmaceutical formulation according to any one of (1) to (8) at a dosage level of 150 mg / 70 kg to 1000 mg / 70 kg using an injection volume of 1.3 to 5 mL.

Claims

1. A highly concentrated and stable pharmaceutical formulation of the monoclonal antibody (mAb) nimotuzumab, comprising: The pharmaceutical preparation comprises: a) nimotuzumab mAb at a concentration in the range of 100-210 mg / mL; b) a buffer substance at a concentration in the range of 5 to 30 mM, in a pH range of 6.5±0.5, the buffer substance being selected from the group consisting of histidine buffer and sodium phosphate buffer; c) a surfactant in the range of 0.02-0.06 w / v % selected from the group consisting of polysorbate 20 and polysorbate 80; d) an amino acid or mixture thereof in the range of 30-150 mM, wherein the amino acid or mixture thereof is selected from the group consisting of L-methionine and glycine; and e) 5% w / v of a carbohydrate as a stabilizer, the carbohydrate being sucrose and The pharmaceutical preparation does not contain a hyaluronidase enzyme. The above pharmaceutical preparation.

2. 10. The pharmaceutical formulation of claim 1 in liquid form.

3. 3. The pharmaceutical formulation of claim 2, characterized in that it has a viscosity of 5 cP or less at a concentration of 150 mg / ml.

4. The pharmaceutical preparation according to any one of claims 1 to 3 for treating cancer.

5. 4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the pharmaceutical formulation is administered subcutaneously at a dose level of 200 mg / 70 kg to 400 mg / 70 kg using an injection volume of 1.3 to 2 mL, and wherein the dose of the pharmaceutical formulation is divided and administered at two or more separate injection sites.

6. 4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the pharmaceutical formulation is administered intramuscularly at a dose level of 150 mg / 70 kg to 1000 mg / 70 kg using an injection volume of 1.3 to 5 mL.

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