Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
  • A61K9/0021—Intradermal administration, e.g. through microneedle arrays or needleless injectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present invention is related to the branches of Biotechnology and Medicine, especially with the obtaining of a highly concentrated and stable formulation of the humanized monoclonal antibody nimotuzumab to be administered subcutaneously (SC) or intramuscularly (IM) in the treatment of cancer. .
• the humanized monoclonal antibody (mAb) nimotuzumab recognizes the receptor for human epidermal growth factor Her ⁇ is of the lgG1 isotype and was obtained by cloning the DNA of the hypervariable regions of the mouse mAb ior egf / r3 and the frameworks of the variable regions and of the constant regions of the heavy and light chains of human origin (REI and NEWN, 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.
• mAb nimotuzumab is administered by intravenous (IV) infusion using a liquid formulation where said mAb is in a concentration of 5 mg / mL (https://www.cecmed.cu/registro/rcp/cimaher-nimotuzumab). This administration is carried out by dissolving four bulbs of nimotuzumab at 5 mg / mL in a bag of 0.9% NaCI solution.
• the composition of the commercial formulation of nimotuzumab also comprises sodium phosphate buffer at 15 mM and pH approximately 7, sodium chloride at 150 mM, and polysorbate 80 at 0.02% (Revista Cubana de Farmacia. 2012; 46 (3): 379-380J.
• Nimotuzumab is administered in the clinic at a dose of 200 mg (400 mg in pancreatic cancer) once a week for six weeks, accompanied by radiotherapy and chemotherapy. Then maintenance doses of 200 mg are administered every 15 days. until the patient allows it, hence the importance of having an administration route that facilitates this chronic use of nimotuzumab without the need for the patient to go to a hospital.
• IV administration brings some disadvantages such as long administration periods, the need for qualified personnel for its administration, the risk of infection and violent reactions (Michael F. Haller (2007) Pharmaceutical Technology, 31 (10): 118-132).
• trastuzumab US 9,345,661
• rituximab US 10,280,2257
• the enzyme hyaluronidase is used to achieve injecting a volume greater than 1.5 ml_ with good biodistribution of Ab and reaching the necessary doses to achieve therapeutic effect (Shpilberg, O. and C. Jackisch. (2013) British Journal of Cancer. 109: 1556-1561).
• Ac administered IM is Ac palivuzumab.
• This Ac is presented as a lyophilized formulation that, after being reconstituted in water, has palivuzumab Ac at 100 mg / ml_ in addition to excipients such as histidine, glycine and mannitol and is used in doses of 15 mg / kg in the treatment of respiratory syncytial virus .
• the inventors of the present invention found formulations for Ac nimotuzumab different from those already reported and with a specific combination of excipients, which guarantees its stability, low turbidity and low viscosity, in addition to being simpler by dispensing with the hyaluronidase enzyme to guarantee a stability and permeability suitable for the case of the SC formulation.
• these specific formulations it is also possible to reach concentrations of between 100 and 180 mg / mL of nimotuzumab, thus allowing the Ab dose to be increased without increasing the volume of administration.
• the use of these formulations by the SC or IM route facilitates their use when chronic administration is needed with doses between 200 mg and 360 mg for the SC route and between 200 - 720 mg for the IM route, with the advantage that they can be self- administered by the patient.
• the object of the present invention is highly concentrated and stable pharmaceutical formulations of monoclonal Ab nimotuzumab that comprise mAb nimotuzumab in a concentration range between 100 and 210 mg / mL, a buffer substance in a range between 5 and 30 mM, providing a pH value of 6.5 ⁇ 0.5, a surfactant in a range between 0.02 and 0.06%, an amino acid or a mixture of them in a range between 30 and 150mM and optionally a carbohydrate as stabilizer in a range between 2 and 6%.
• the buffer substance of said formulations is selected from histidine buffer and sodium phosphate.
• the surfactant is selected from polysorbate 20 and polysorbate 80.
• the amino acids are selected from L-methionine and glycine.
• the carbohydrate used in the formulation is sucrose.
• the above formulations can be in liquid or lyophilized form.
• the liquid formulation in a concentration of 150 mg / mL has a viscosity of £ 5cP.
• the object of the present invention is the use of the pharmaceutical formulations described above in the treatment of cancer.
• a method of treating a patient in need comprises the administration of the pharmaceutical formulations described in the present invention in a dose between 200 mg / 70kg - 750 mg / 70kg by the SC route or the IM route.
• SC administration of this Ac is achieved.
• this inoculation can be distributed to one or more injection sites when higher doses of Ac are needed.
• the injection volume ranges from 1.3 to 5 mL for a concentration of 150 mg / mL and doses from 200 to 750 mg / 70kg. In the case of using a concentration of 200 mg / mL and intramuscular volumes of 1.3 - 5 mL, doses of 260-1000 mg / 70kg would be achieved.
• the present invention is related to formulations in high concentrations of the mAb nimotuzumab which we will also refer to herein as hR3.
• concentration of nimotuzumab in the formulations described in the present invention is in a range between 100 and 210 mg / ml, preferably between 100 and 180 mg / ml.
• the nimotuzumab formulations described in this application comprise a buffer substance, a surfactant, amino acids, and optionally a carbohydrate.
• the buffer substance can be histidine buffer or sodium phosphate both in a range between 5 and 30 mM, which makes it possible to maintain the pH of the formulation in a pH range of 6.5 ⁇ 0.5.
• the formulations of the present invention can comprise polysorbate 20 or polysorbate 80 as surfactant in a concentration range between 0.02 and 0.06%.
• Carbohydrates can be sucrose and trehalose with a concentration between 2 and 6%.
• amino acids are selected from L-methionine or glycine, or both in a concentration range between 30 and 150mM.
• UF / DF ultrafiltration-diafiltration
• a centrifugation speed in the range of 900-1100 g can be used at temperatures between 2-8 ° C.
• the sample is subjected to a sterilizing filtration where, depending on its volume, this will be the type of filter to be used.
• a sterilizing filtration where, depending on its volume, this will be the type of filter to be used.
• Another way in which the formulations described in the present invention can be prepared is by means of a tangential flow filtration system on a laboratory or pilot scale. Membranes with pores of 30 and 50kD can be used for this.
• the diafiltration of the samples to change the buffer is done using between 10-15 diavolumes of final buffer. To concentrate the samples, the initial volume must be reduced to a volume smaller than the calculated volume that corresponds to the desired concentration.
• formulations can be obtained by a step-by-step screening strategy by first selecting the best buffers and pH values. Then a second stage selecting the best salts, surfactants and carbohydrates and later a third stage to select the best amino acids for the formulation.
• Another variant to be used can be by means of a simultaneous screening where the different types of excipients are used with their different combinations according to a factorial design. In this case, there is in the same formulation the possibility of interaction between more than 2 or 3 excipients.
• Another aspect of the invention contemplates the lyophilization of the above-described formulations and their subsequent reconstitution as well as a method for preparing a stable, isotonic reconstituted formulation.
• Said method comprises the reconstitution of the lyophilized mixture of Ac and stabilizers in the buffers described above in such a way that the concentration of Ac in the reconstituted formulation is at least 100 mg / mL and a maximum of 210 mg / mL, this being 3- 5 times higher than the concentration of Ac in the mixture before lyophilization.
• DLS dynamic light scattering
• Another method used to determine the best formulations is the stress study, where the samples are subjected to a temperature of 50 ° C in a thermal bath for a period of 10 to 20 days and then analyzed at different time intervals. by physicochemical and biological determinations.
• turbidity measurement through the absorbance of the samples in the UV spectrum between 340 and 450 nm, which allows detecting the formation of large aggregates.
• Samples are also analyzed by SDS-PAGE to determine fragment formation during stress. In this case, non-reducing conditions with 7.5% gels and Coomassie stain or silver nitrate were used. The determination of monomer purity is also carried out by means of high performance molecular exclusion chromatography (SEC-HPLC).
• SEC-HPLC high performance molecular exclusion chromatography
• the biological activity of Ab determines the relative potency with respect to the reference material of nimotuzumab and can be performed by flow cytometry or by inhibiting cell proliferation in a cell line expressing human Heri.
• the formulations object of the present invention may have application in the treatment of patients with tumors of the head and neck, glioma, esophagus, lung and pancreas.
• said formulations must be administered to a subject carrying the disease independently or in combination with conventional therapies for the treatment of cancer such as radiotherapy or chemotherapy, in order to enhance their therapeutic action.
• the administration of the same is done by SC or IM route.
• SC route a minimum sample volume of 1.33 mL is used for the 200 mg / 70kg dose.
• the injection volume can be increased to 2 mL and a dose of 300 / 70kg can be reached.
• a concentration of Ab greater than 150 mg / mL can be used or two injection sites can be used, these sites could be any of the deltoid regions, abdominal region and front region of the thighs. , without being limited to these.
• nimotuzumab For example, for the dose that is used in most treatments of 200 mg per week, only a concentration of 134 mg / ml_ of nimotuzumab would suffice to effect the treatment. In the case of a higher dose, a higher concentration of nimotuzumab up to 210 mg / mL can be used. For the treatment of pancreatic cancer with doses of 400 mg, two injection sites would be used.
• the maximum allowed IM administration volume is 5mL, so when this route of administration is used, the injection volume can vary between 1.3 - 5 ml_ for a concentration of 150 mg / mL and doses between 200 and 750 mg. When concentrations of 200 mg / mL are used, the dose can reach up to 1000 mg.
• Figure 2 kD values for the different formulations of mAb hR3 with the presence of amino acids determined by DLS.
• Figure 3 Particle size graph of the hR3 mAb formulations with the presence of amino acids determined by DLS.
• Figure 4 Particle size for formulations of mAb hR3 at 150 mg / mL and stored at 4 ° C for 3 months measured by DLS.
• Figure 5 Viscosity of formulations of mAb hR3 at 150 mg / mL and stored at 4 ° C for 3 months measured by DLS.
• Figure 6 Monomer purity of formulations of mAb hR3 at 150 mg / mL and stored at 4 ° C for 3 months measured by DLS.
• Figure 7 Antitumor effect of nimotuzumab mAb administered IV and SC at doses of 50 mg / kg in athymic Balb / c mice.
• Example 1 Screening of formulation excipients for mAb nimotuzumab.
• the buffer was changed using UF / DF in Amicon Ultra tubes of 50kD pore size, regenerated cellulose membrane and 15mL volume. The samples were centrifuged at 936 g at a temperature of 4 ° C.
• the hR3 mAb was used in 15 mM sodium phosphate buffer, 150 mM sodium chloride and Tween 80 at 0.2 mg / mL at pH 7. After preparing the formulations at a concentration of Ac of 10 mg / mL Diffusion interaction parameter (kD) was measured by DLS.
• the most stable mAb hR3 formulation variants are those that have a carbohydrate such as sucrose and trehalose in their composition in the absence of sodium chloride. Furthermore, the combination of sucrose with histidine favors stability much more than the combination of phosphate with trehalose.
• Example 2 Screening of amino acids for the formulation of mAb hR3.
• Table 4 Normalized monomer purity values for formulations of mAb hR3 at 5 mg / mL with the presence of amino acids after stress at 50 ° C determined by SEC-HPLC. Table 4 shows that the variants with the highest monomer purity after stress are hR3sol82 and then hR3sol80. This indicates that the variants that have histidine buffer are more stable than those with phosphate buffer. It also suggests that the presence of methionine improves stability compared to the formulation without amino acids. On the contrary, the formulations of lower purity were hR3so! 81 and hR3so! 90. This suggests that there is a destabilizing effect of both L-arginine and sodium chloride in the hR3 Mab formulations. According to the previous results, the hR3 mAb formulation that has histidine buffer at pH6, sucrose, polysorbate 80 and L-methionine is the most stable.
• Figure 4 shows the particle diameter obtained by DLS for the different variants.
• the diameter for the hR3sol90 variant from the initial time is around 25nm, being greater than that of the other formulations. This suggests a greater tendency for self-association between molecules in this variant with respect to the hR3sol80 and hR3sol82 variants.
• Figure 6 shows the graph of monomer purity by SEC-HPLC, as observed the purity of the monomer does not have considerable changes and therefore suggests that the formulations of AcM hR3 at 150 mg / mL are stable stored at 4 ° C. Additionally, the biological activity of the hR3 mAb formulations was determined at 150 mg / ml_ without stressing at time 0 and 3 months. Biological activity was measured by the recognition of mAb hR3 to the human epidermal growth factor receptor Her ⁇ by flow cytometry and by inhibition of cell proliferation.
• Table 5 shows that the relative potency for the hR3sol80, hR3sol82 and hR3sol90 variants was between 79 and 83% by the flow cytometry assay, which indicates that they maintain their biological activity even at high concentrations.
• the potency values for the inhibition test were between 93 and 104%, which confirms the cytometry result and indicates that the samples in high concentration retain their biological activity. It should be taken into account that each value has a confidence interval due to the intrinsic variability of the experiment in both tests.
• Table 6 shows the biological activity for the hR3 samples at 150 mg / mL after 3 months of stability at 4 ° C.
• the samples with the highest relative potency value were hR3sol80 and hR3sol90 with values between 78 and 81% for recognition by cytometry and between 77 and 86% for proliferation inhibition.
• Example 4 Stress study at 50 ° C of better formulation variants of mAb hR3 at 150 mg / mL.
• the concentration of hR3 was carried out up to 150 mg / mL by UF / DF in Amicon Ultra tubes of 50kD pore size, regenerated cellulose membrane and 15 mL of volume. The samples were centrifuged at 936 g at a temperature of 4 ° C. Once the concentration of 150 mg / mL was reached, the samples were filtered on 0.2 pm filters and placed in a thermal water bath at 50 ° C. A sampling of the different formulations was carried out on days 0, 4, 10 and 15 for physicochemical parameters. Table 7 shows the results of the monomer purity measurement on the different sampling days.
• the hR3sol90 variant had completely precipitated forming a white solid while the hR3sol80 variant had a monomer purity of 45.3% and the hR3sol82 variant one of 58.9%.
• the above indicates that the most stable formulation in high concentrations is hR3sol82 while the least stable is hR3sol90. This result coincides with the results obtained at low concentration.
• the hR3sol80 variant forms a yellow gel while the hR3sol82 variant continues in solution with 43.9% monomer purity. On sampling at 15 days, the hR3sol82 variant also forms a yellow gel.
• the above results indicate that the The hR3sol82 formulation that has L-methionine in the composition is more stable at high concentrations than the variants that lack this amino acid. These results suggest that in the long term the most stable formulations will be hR3sol80 and hR3sol82, as opposed to the hR3sol90 formulation.
• Table 8 shows the percentage of monomer and dimers in the samples at 150 mg / mL without stress.
• the hR3sol82 formulation is the one with the highest percentage of monomer with 94.1% and the one with the lowest dimer with 5.2%, which shows the tendency of this formulation to decrease the association between molecules favoring stability.
• the hR3sol80 and hR3sol90 variants have 93.0% and 92.0% monomer respectively.
• the biological activity of the hR3 mAb formulations was also determined at 150 mg / mL before being subjected to the stress of 50 ° C by flow cytometry and by inhibiting cell proliferation with the same methodology used in Example 3 for these determinations. These results coincide with those shown in Table 5.
• the bioavailability is considered as 100%.
• Example 6 Antitumor effect of nimotuzumab mAb administered IV and SC route at doses of 50 mg / kg in atonic Balb / c mice.
• mice Female athymic Balb / c mice weighing approximately 20 g were divided into three groups of five animals each. All groups received 2x10 6 A431 cells on day zero and on days 10, 12, 14 and 16 Group 1 was administered 1 mg of total mass of the hR3sol90 formulation by the SC route, Group 2 received 1 mg of mass total nimotuzumab mAb by IV route and Group 3 was the control and received phosphate buffered saline solution.
• Figure 7 shows how the tumor volume for the mice that were administered nimotuzumab by the SC route was similar to that of the IV route. This shows that there was no differences in the efficacy of treatment by both routes despite the fact that the absorption rate and ethical pharmacokinetic characteristics of each route are different. For its part, the control group had a much higher tumor growth even with significant differences. This indicates the efficacy of the administration of the mAb nimotuzumab via the SC route.
• Example 7 Purity of lyophilized nimotuzumab mAb at 150 mg / mL.
• the hR3sol80, hR3sol82 and hR3sol90 formulations were lyophilized at 150 mg / mL and a volume of 500 pL.
• a laboratory lyophilizer was used at a temperature of -30 ° C and it was evacuated for 30 hours until a white powder was left. Then it was reconstituted with 500mI of ultrapure water up to the initial concentration, leaving a clear solution free of particles.
• Table 10 presents the purity data of the different formulations of the mAb nimotuzumab after being lyophilized and then reconstituted.
• the hR3sol82 formulation had a monomer purity of more than 98% while the hR3sol80 variant had around 94% and the hR3sol90 variant more than 93%. It should also be noted how the percentage of dimers changes depending on the type of formulation. Additionally, the Table shows the total purity of monomer plus dimer of these lyophilized and reconstituted formulations, which in all cases was above 99%, which indicates that the mAb nimotuzumab does not lose its stability despite stress during lyophilization process. It should also be understood that nimotuzumab Ac has a balance between monomer and dimer due to the characteristics of this Ac, which can vary depending on the type of interactions in the solution.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Organic Chemistry (AREA)
• Immunology (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Dermatology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Inorganic Chemistry (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (10)

Applications Claiming Priority (2)

Publications (2)

Family

ID=76478863

Family Applications (1)

Country Status (12)

Citations (4)

Family Cites Families (6)

• 2019
  • 2019-12-17 CU CU2019000104A patent/CU20190104A7/es unknown
• 2020
  • 2020-12-10 BR BR112022010488A patent/BR112022010488A2/pt unknown
  • 2020-12-10 EP EP20903282.0A patent/EP4079294A2/en active Pending
  • 2020-12-10 WO PCT/CU2020/050007 patent/WO2021121444A2/es not_active Ceased
  • 2020-12-10 JP JP2022536924A patent/JP7796646B2/ja active Active
  • 2020-12-10 CA CA3163630A patent/CA3163630A1/en active Pending
  • 2020-12-10 CN CN202080088100.8A patent/CN114867468A/zh active Pending
  • 2020-12-10 US US17/786,094 patent/US20230018364A1/en active Pending
  • 2020-12-10 MX MX2022007509A patent/MX2022007509A/es unknown
  • 2020-12-10 KR KR1020227024674A patent/KR20220119091A/ko active Pending
  • 2020-12-10 AU AU2020409887A patent/AU2020409887B2/en active Active
• 2022
  • 2022-07-11 CO CONC2022/0009693A patent/CO2022009693A2/es unknown

Patent Citations (4)

Non-Patent Citations (13)

Also Published As

Similar Documents

Legal Events