Abstract:
The invention relates to a soluble preparation for injection in humans, characterized in that the preparation (1) contains human insulin or a monomeric insulin analog, (2) has a lower concentration of insulin-bound zinc than about 1.8 ions per hexamer of insulin, (3) contains nicotinamide, (4) has a lower conductivity than 5 mSi/cm. The concentration of insulin-bound zinc ions is less than 0.02 ions per hexamer of insulin and the conductivity less than 0.2 mSi/cm. Further disclosed are that the preparation contains a non-ionic detergent, a preservative preferable m-cresol, an injectable preparation, said preparation being for use as a medicament for treating or preventing diabetes mellitus, use of the said preparation for the manufacture of a medicament for treating or preventing diabetes mellitus and a method of treating or preventing diabetes mellitus comprising of administering subcutaneously the said preparation to an individual in need of such treatment or prevention.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to the PCT Application PCT/DK2015/000010 filed on 6 Mar. 2015 which further claims the priority of Patent Application No. PA20140000171 filed on 26 Mar. 2014 filed in the Denmark Patent and Trademark Office which further claims the benefit of Danish Application PA20140000125 filed on 7 Mar. 2014. The specification of the above referenced patent applications is incorporated herein by reference in its entirety 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    A. Technical Field 
         [0003]    The present invention relates to stable, extra rapid-acting human or monomeric insulin analog preparations for subcutaneous injection. 
         [0004]    B. Description of Related Art 
         [0005]    Insulin treatment of diabetic patients comprises the use of rapid-acting, intermediate-acting, long-acting and biphasic insulin preparations. The rapid-acting insulin preparations have undergone a development towards preparations with still earlier onset of action; from acid solutions to neutral solutions of animal insulin, further to neutral solutions of human insulin (e.g. Actrapid® HM, Humulin® Regular and Insuman® Rapid), and finally to neutral solutions of monomeric insulin analogs. Examples of the latter are NovoRapid® based on B28Asp human insulin (“insulin aspart”), Humalog® based on B28LysB29Pro human insulin and Apidra® based on B3LysB29Glu human insulin. The term “monomeric” alludes to the prevailing formation of monomers from aggregates in solutions of the analog and thereby faster absorption after subcutaneous injection, compared to human insulin, due to the construction of the analog molecule. 
         [0006]    Rapid-acting insulin preparations are widely used in connection with a multiple subcutaneous injection (basal/bolus) regimen in which an intermediate-acting insulin is injected twice a day or a long-acting insulin once a day, in order to provide a basal level of plasma insulin, while a rapid-acting insulin is injected at a time most suitable for, as far as possible, normalizing the blood sugar after a meal. In order to mimic the quick release of insulin from pancreas in normal persons by a meal, human insulin has to be injected about ½ hour before the meal in diabetic patients, because of the retarded absorption of the insulin. Due to the faster absorption of monomeric insulin, injection can be made closer to the meal, which is more convenient for the patient. Furthermore, the appearance of monomeric insulin in plasma follows to a higher degree the pattern of plasma insulin appearance in healthy persons after a meal, thus providing an overall improved blood glucose control, including a lower rate of hypoglycaemic events. The above-mentioned commercially available monomeric insulin preparations are therefore preferred for use as the rapid-acting components in a basal/bolus regimen today. 
         [0007]    However, there is still a need for yet more rapidly acting insulin preparations. From the point of view of the diabetic patients, it would be most desirably to minimize the time length from the injection to the insulin effect. This would mean more freedom and a better metabolic control. Nicotinamide has been shown to accelerate the absorption of subcutaneously injected human or monomeric insulin, vide U.S. Pat. No. 5,382,574, WO/1996/010417 and WO/2010/149772. Preparations with nicotinamide have not yet been commercialized. 
     
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
       [0008]    The present invention reveals that nicotinamide-containing insulin preparations with extra rapid action after subcutaneous injection can be made sufficiently stable when combined with low concentrations of zinc and low conductivities. 
         [0009]    The preparations of the invention are soluble and characterized in that they contain human insulin or a monomeric human insulin analog, have a lower concentration of insulin-bound zinc than about 1.8 ions per hexamer of insulin, (3) contain nicotinamide. 
         [0010]    The preferred content of insulin is in the range from about 0.2 mM to about 6 mM, preferably from about 0.4 mM to about 3 mM, more preferred from about 0.5 to about 0.7 mM. 
         [0011]    The preferred content of zinc is less than about 1.2, preferably less than about 0.4, more preferred less than about 0.1, most preferred less than about 0.02 insulin-bound zinc ions per hexamer of human insulin or analog. A low content of insulin-bound zinc, or virtual absence of zinc, is mandatory for obtaining the extra high rate of absorption after injection of the preparations according to the invention; the lower the content of insulin-bound zinc, the better the effect. It is within the scope of the present invention, that the total concentration of zinc can be higher than about 1.8 ions per hexamer of insulin, provided that the insulin-bound part of total zinc content is lower than about 1.8 zinc ions per hexamer of insulin. This can be the case, if a zinc-binding, non-insulin substance such as EDTA is present. 
         [0012]    The preferred content of nicotinamide is in the range from about 10 to about 500 mM, preferably from about 25 to about 400 mM, more preferred from about 100 to about 350 mM, most preferred from about 150 to about 300 mM. 
         [0013]    The preferred conductivity is less than 5 mSi/cm. preferably less than 1 mSi/cm, more preferred lower than 0.5 mSi/cm and most preferred less than 0.2 mSi/cm, all at 22° C. 
         [0014]    A non-ionic detergent such as polysorbate 20 (Tween 20) may be added to the preparations in order to improve physical stability. The preferred concentration of non-ionic detergent range from about 5 to about 50 ppm. Non-ionic detergents are well known as stabilizers of protein solutions. 
         [0015]    The range of pH is from about 6.0 to about 8.0, preferably from about 6.3 to about 7.5. 
         [0016]    The preferred preservative is m-cresol, since solutions of insulins with low contents of zinc have a tendency to precipitate with addition of phenol, but not with addition of m-cresol. In cases where the choice of concentrations of the components, required according to the invention, makes the preparations hypotonic, non-ionic excipients must be added in order to make the preparations isotonic. 
       EXPERIMENTS 
       [0017]    Ultrapure Milli-Q® water was used for making and storage of the preparations throughout. Nicotinamide in solution was deionized by passing an anion exchanger (Sep-Pak Accell Plus QMA) on hydroxyl ion form and a cation exchanger (Sep:Pak Accell Plus CM) on hydrogen ion form. 
         [0018]    Glassware was used for making and storage of the preparations. 
         [0019]    Centrifugation was used for isolation of precipitates. 
         [0020]    Conductivity measurements were performed with a CDM 230 instrument (Radiometer). 
         [0021]    Rotation of samples were performed by a 820 SWELAB mixer. 
         [0022]    The final preparations were run through 0.22 μm filters, 
         [0023]    Reverse phase HPLC on a C4 column (VYDAC 214TP54) was used in the assessment of chemical stability of preparations. 
         [0024]    The mobile phase consisted of A: Water, 0.1% v/v trifluoroacetic acid and B: Acetonitrile, 0.07% v/v trifluoroacetic acid. Elution was performed at a rate of 1 ml/min and at 40° C. after the following schedule: 
         [0000]    0-5 min: isocratic with 20% B
 
5-7 min: linear gradient to 24% B
 
7-27 min: isocratic with 24% B
 
27-33 min: linear gradient to 27% B
 
33-42 min: isocratic with 27% B
 
42-48 min: linear gradient to 40% B
 
48-50 min: linear gradient to 80% B
 
50-52 min: isocratic with 80% B
 
52-53 min: linear gradient to 20% B
 
53-60 min: isocratic with 20% B
 
         [0025]    Absorbance of eluate was recorded at 250 nm and 276 nm. In the chromatogram, peaks of insulin-like transformation products, e.g. mono-desaminoinsulins, appeared closely in front of and after the main insulin peak. Then followed an interval with no material appearing. The most hydrophobic insulin-related products, e.g. covalently bound insulin dimer and other high molecular weight products, appear in the linear gradient from 27% to 40% B. 
         [0026]    The chemical stability of preparations by storage in closed vials for 14 days at 37° C., in relation to stability by storage at 4° C., was assessed by determination of the percentage distribution between areas of the insulin-like peaks, the main insulin peak and the most hydrophobic insulin-related products, for both temperatures of storage. The percentage formation of products by raising the temperature of storage from 4° C. to 37° C. could then be assessed. 
         [0027]    The physical stability of preparations was assessed by rotation at 37° C. of 400 μl of preparations in 100×10 mm test tubes, closed by rubber stoppers, and inspecting the physical state at different days after start. 
       Example I 
       [0028]    A sample of Novo Rapid® was deprived of zinc by adjustment of pH to 3.0, followed by twice salting-out of insulin with NaCl, using centrifugation for isolation of precipitates. The salt-cake was suspended in water and pH adjusted to 5.1, followed by centrifugation. The precipitate was suspended in water and the pH adjusted to 6.0, followed by centrifugation. The precipitate was suspended in water followed by centrifugation. The final precipitate was suspended in water and dissolved by adjusting pH to about 7.4. This solution of virtually zinc-free insulin aspart, was then formulated to Prep. 1 and with added salt to Prep. 2: 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Insulin aspart, 
                 m- 
                 Nicotin- 
                   
                   
                 Conductivity 
               
               
                 Prep. 
                 Zinc free. 
                 cresol 
                 amide 
                 NaCl 
                   
                 mSi/cm at 
               
               
                 No 
                 mM 
                 M 
                 M 
                 M 
                 pH 
                 22° C. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 0.6 
                 0.028 
                 0.27 
                 0 
                 7.4 
                 0.18 
               
               
                 2 
                 0.6 
                 0.028 
                 0.17 
                 0.05 
                 7.4 
                 5.1 
               
               
                   
               
             
          
         
       
     
       Example II 
       [0029]    A sample of NovoRapid, 100 IU/ml, was deprived of zinc by adjustment of pH to 3.0, and addition of Na2 EDTA in a mole amount tenfold that of zinc in the sample of NovoRapid, followed by adjustment of pH to 5.1 and centrifugation. The precipitate was suspended in 0.01 M NaCl, followed by centrifugation. The precipitate was suspended in 0.01 M NaCl and the pH adjusted to 6.0, followed by centrifugation. The precipitate was suspended in 0.01 M NaCl, followed by centrifugation. The final precipitate was suspended in water and dissolved by adjusting pH to about 7.4. This solution of virtually zinc-free insulin aspart, was then formulated to Prep. 3 and with added salt to Prep. 4: 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Insulin aspart, 
                 m- 
                 Nicotin- 
                   
                   
                 Conductivity 
               
               
                 Prep. 
                 Zinc free. 
                 cresol 
                 amide 
                 NaCl 
                   
                 mSi/cm at 
               
               
                 No 
                 mM 
                 M 
                 M 
                 M 
                 pH 
                 22° C. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 3 
                 0.6 
                 0.028 
                 0.27 
                 0 
                 7.4 
                 0.17 
               
               
                 4 
                 0.6 
                 0.028 
                 0.17 
                 0.05 
                 7.4 
                 5.1 
               
               
                   
               
             
          
         
       
     
       Example III 
       [0030]    A sample of Actrapid HM, 100 IU/ml, was processed in a similar way as the sample of NovoRapid in Example II in order to obtain a final solution of virtually zinc-free human insulin at pH 7.4. The solution was then formulated to Prep. 5 and with added salt to Prep. 6: 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Human Insulin, 
                 m- 
                 Nicotin- 
                   
                   
                 Conductivity 
               
               
                 Prep. 
                 Zinc free. 
                 cresol 
                 amide 
                 NaCl 
                   
                 mSi/cm at 
               
               
                 No 
                 mM 
                 M 
                 M 
                 M 
                 pH 
                 22° C. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 5 
                 0.6 
                 0.028 
                 0.27 
                 0 
                 7.4 
                 0.12 
               
               
                 6 
                 0.6 
                 0.028 
                 0.22 
                 0.05 
                 7.4 
                 5.0 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Chemical stability. Formation of insulin impurities and 
               
               
                 survival of insulin by 14 days storage of preparations at 37° C. 
               
             
          
           
               
                   
                   
                   
                 Formation of 
                   
               
               
                   
                   
                 Formation of 
                 most 
               
               
                   
                 Conductivity 
                 insulin-like 
                 hydrophobic 
                 Survival 
               
               
                 Prep. No 
                 mSi/cm 
                 products, % 
                 products, % 
                 of insulin, % 
               
               
                   
               
             
          
           
               
                 1 
                 0.18 
                 1.8 
                 0.9 
                 97.3 
               
               
                 2 
                 5.1 
                 2.9 
                 1.3 
                 95.8 
               
               
                 3 
                 0.17 
                 3.8 
                 0.9 
                 95.3 
               
               
                 4 
                 5.1 
                 4.5 
                 2.0 
                 93.5 
               
               
                 5 
                 0.12 
                 4.4 
                 0.8 
                 94.8 
               
               
                 6 
                 5.0 
                 4.1 
                 1.3 
                 94.6 
               
               
                 NovoRapid 
                 — 
                 1.9 
                 0.3 
                 97.8 
               
               
                 Actrapid 
                 — 
                 2.4 
                 0.4 
                 97.2 
               
               
                 Apidra 
                 — 
                 1.1 
                 1.1 
                 97.8 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Physical stability. Formation of precipitates (fibrils) by rotation of 
               
               
                 preparations at 37° C. Assessment by observing the appearance of the 
               
               
                 samples in normal light (NL) and in a concentrated beam of light (CBL) at 
               
               
                 different days of ratation. Preparations 1 and 2 was only observed in 
               
               
                 normal light. C: clear O: opal T: turbid 
               
             
          
           
               
                   
                 Days after 
                   
                   
                   
               
               
                 Appearance 
                 start 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 1 
                 NL 
                 C 
                 C 
                 C 
               
               
                 2 
                 NL 
                 T 
                 T 
                 T 
               
               
                 3 
                 NL 
                 C 
                   
                 C 
               
               
                   
                 CBL 
                 C 
                   
                 O 
               
               
                 4 
                 NL 
                 T 
                   
                 T 
               
               
                   
                 CBL 
                 T 
                   
                 T 
               
               
                 5 
                 NL 
                 C 
                 C 
                 C 
               
               
                   
                 CBL 
                 C 
                 C 
                 C 
               
               
                 6 
                 NL 
                 O 
                 T 
                 T 
               
               
                   
                 CBL 
                 T 
                 T 
                 T 
               
               
                 NovoRapid 
                 NL 
                 T 
                 T 
                 T 
               
               
                   
                 CBL 
                 T 
                 T 
                 T 
               
               
                 Actrapid 
                 NL 
                 C 
                 C 
                 C 
               
               
                   
                 CBL 
                 C 
                 C 
                 C 
               
               
                 Apidra 
                 NL 
                 C 
                 O 
                 T 
               
               
                   
                 CBL 
                 C 
                 T 
                 T 
               
               
                   
               
             
          
         
       
     
       Example IV 
       [0031]    40 mg of human insulin with about 2.2 Zn ++  per hexamer (Novo Nordisk) was dissolved in 8 ml water with HCl added to pH 3. A trace amount of the radioactive isotope Zn-65 and 10 μmol Na2EDTA were added to the solution. The mixture was then run through a column of about 100 mg of the anion exchanger QAE Sephadex A-25 equilibrated with 0.05 M NaCl, followed by washing with 2 ml water. All Zn-65 was retained in the column. The pH of the zinc free eluate was adjusted to 7.4 with NaOH. Equal parts of the solution were transferred to each of two Amicon® Ultra centrifugal-10000 Dalton filter units placed in tubes for collection of filtrate. The volumes were reduced seven times by centrifugation allowing salts to pass through the filters. The retained solutions were mixed with water to the original volumes. The volumes were again reduced 7 times. The retained volumes were pooled and the insulin concentration measured by HPLC. The pool was used for making a preparation with 0.6 mM zinc free human insulin, 0.27 M nicotinamide and 0.028 M m-cresol. The conductivity at 22° C. was 0.085 mSi/cm. HPLC of the filtrates showed that less than 1% of the total amount of insulin had passed the filters. 
       DISCUSSION OF THE INVENTION 
       [0032]    It appears from Table 1, that the experimental preparations 1, 3 and 5 with the lowest conductivities (0.18, 0.19 and 0.12 mSi/cm, resp.) form hydrophobic products to a lower degree (0.9, 0.9 and 0.8% versus 1.3, 2.0 and 1.3%) than the experimental preparations 2, 4 and 6 with the highest conductivities (5.1, 5.1 and 5.0 mSi/cm). The formation of hydrophobic products in 1, 3 and 5 is within the range (0.3-1.1%) of formation in NovoRapid (3 Zn ++  per hexamer), Actrapid {3Zn ++  per hexamer) and Apidra (zinc-free). The range of formation of the more insulin-like products, (1.8-4.4%) for 1, 3 and 5 is at a somewhat higher level than the corresponding range (1.1-2.4%) for NovoRapid, Actrapid and Apidra. 
         [0033]    Based on the method chosen for assessment of physical stability the results in Table 2 show, that the experimental preparations 1, 3 and 5 with the lowest conductivities are more physically stable than the experimental preparations 2, 4 and 6 with the highest conductivities, and not less physically stable than NovoRapid, Actrapid and Apidra, respectively. 
         [0034]    These findings are surprising, since preparations containing zinc free insulin are considered unstable, even when the solution is made in a salt free medium of a zinc free insulin material from an insulin supplier. Such material contains salt in a varying degree, that can cause a higher conductivity in the preparations than that of preparations 1, 3 and 5, and hence a higher degree of instability at elevated temperatures and rotation. The upper limits of conductivity pertaining to this invention are therefore important. 
         [0035]    While preparations 1-6 described in Examples I-III are made from the commercial preparations Actrapid and NovoRapid, the preparation described in Example IV is made from dry insulin powder being a technically relevant starting material for large scale production of preparations according to the present invention. Example IV also describes ultrafiltration as an alternative method for desalting compared to the precipitation technique described in Examples I-III. 
         [0036]    The following considers, how the present invention relates to published patent applications. 
         [0037]    The earlier mentioned U.S. Pat. No. 5,382,574 deals with preparations comprising insulin or an insulin derivative and nicotinamide or nicotinic acid or a salt thereof. Examples 9 and 10 in this Application describe preparations containing nicotinamide and zinc free B10Asp human insulin, a monomeric analog. In both examples the absorption of analog after subcutaneous injection of the preparation in pigs was found to be considerably faster than that of a reference preparation containing zinc free analog without nicotinamide, thus demonstrating the absorption promoting effect of nicotinamide, which is the essence of U.S. Pat. No. 5,382,574. 
         [0038]    Examples 9 and 10 did not reveal the impact of zinc concentration on the absorption rate of the analog in preparations with nicotinamide. Neither the salt content of the applied zinc-free analog, nor the conductivities of the preparation, nor the stability of the preparations were disclosed. Example 7 in the same application describes a preparation containing nicotinamide and human insulin with 3 Zn ++  per hexamer The time until half of the insulin had disappeared after subcutaneous injection of the preparation in pigs was found to be 22% lower than that of a reference preparation with 3 Zn ++  per hexamer (composed as Actrapid) and without nicotinamide. 
         [0039]    Example 4 in the same application describes a preparation containing nicotinamide and zinc free human insulin. The time until half of the insulin had disappeared after subcutaneous injection of the preparation in pigs was found to be about 46% lower than that of a reference preparation with 3 Zn ++  per hexamer (composed as Actrapid) without nicotinamide. Neither the salt content of the applied zinc free insulin, nor the conductivities of the preparation, nor the stability of the test preparation were disclosed. 
         [0040]    The earlier mentioned WO/1996/010417 deals with preparations containing insulin aspart and nicotinamide. The only example in the Application describes a test preparation containing 0.6 mmol analog, 3 zinc ions/hexamer, 0.26 mM nicotinamide and 3 g/l phenol (pH 7.4) and a reference preparation with nicotinamide substituted by 16 g/l glycerol. In pigs the test preparation revealed a significantly earlier decrease in plasma glucose and a significantly faster absorption than the reference preparation after subcutaneous injection. The time until half of the insulin had disappeared after subcutaneous injection of the preparations in pigs was found to be about 24% lower than that of a reference preparation, The invention of the present patent application deviates from that of WO/1996/010417 in that it contains less zinc (&lt;I,8 Zn ++ /hexamer of insulin). Therefore injected insulin aspart is faster acting when composed according to the present application, vide the discussion above in connection with U.S. Pat. No. 5,382,574. 
         [0041]    The earlier mentioned WO/2010/149772 deals with preparations containing an insulin compound, a nicotinic compound and arginine. All of the described preparations contain 3 Zn ++  per hexamer. Thus the matter of the present application is not anticipated. The combination of characteristics pertaining to the present invention has not been disclosed before.