Anti-diabetic compounds
BACKGROUND OF THE INVENTION 
     This invention is directed to novel compounds having anti-diabetic activity. Each compound of this invention is available by conversion of human proinsulin or an intermediate derived from human proinsulin. 
     Human proinsulin is recognized to exhibit anti-diabetic activity, albeit at a much lower level, relative to human insulin itself. The compounds of this invention exhibit anti-diabetic activity at a level much greater than that demonstrated by human proinsulin. 
     SUMMARY OF THE INVENTION 
     Thus, this invention is directed to a class of compounds having the formula ##STR2## and pharmaceutically acceptable non-toxic salts thereof in which X is --OH, -Arg-Arg-OH, or -Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-OH. 
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention is directed to three peptides, any of which may be in the form of its pharmaceutically acceptable non-toxic salt. 
     The peptide sequences and corresponding short-hand descriptions used herein in which the term HPI denotes human proinsulin are as follows: ##STR3## 
     Included in the compounds of this invention are their pharmaceutically acceptable non-toxic acid addition salts and their pharmaceutically acceptable non-toxic carboxylic acid salts. 
     The term &#34;pharmaceutically acceptable non-toxic acid addition salts&#34; encompasses both organic and inorganic acid addition salts including, for example, those prepared from acids such as hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, propionic, carbonic, and the like, or salts, such as, for example, ammonium bicarbonate. Preferably, the acid addition salts are those prepared from hydrochloric acid, acetic acid, or carbonic acid. Any of the above salts can be prepared by conventional methods. 
     The term &#34;carboxylic acid salts&#34; includes, for example, zinc, ammonium, alkali metal salts such as sodium, potassium, and lithium, and the like. Preferred carboxylic acid salts are the zinc and sodium salts. 
     For the sake of convenience, the amino acids of the peptides referred to herein may be described by their approved single-letter or three-letter shorthand designations. 
     These designations are as follows: 
     
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Single-Letter                                                             
           Three-Letter    Amino Acid                                     
______________________________________                                    
A          Ala             Alanine                                        
R          Arg             Arginine                                       
N          Asn             Asparagine                                     
D          Asp             Aspartic Acid                                  
C          Cys             Cysteine                                       
E          Glu             Glutamic Acid                                  
Q          Gln             Glutamine                                      
G          Gly             Glycine                                        
H          His             Histidine                                      
I          Ile             Isoleucine                                     
L          Leu             Leucine                                        
K          Lys             Lysine                                         
F          Phe             Phenylalanine                                  
P          Pro             Proline                                        
S          Ser             Serine                                         
T          Thr             Threonine                                      
Y          Tyr             Tyrosine                                       
V          Val             Valine                                         
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     The compounds of this invention can be prepared by routine peptide synthesis methods. 
     Alternatively, and preferably, the compounds of this invention can be prepared from human proinsulin. Human proinsulin is available via a variety of routes, including organic synthesis, isolation from human pancreas by conventional methodology, and, more recently, recombinant DNA methodology. 
     In broad outline, the production of proinsulin using recombinant DNA methodology involves obtaining, whether by isolation, construction, or a combination of both, a sequence of DNA coding for the amino acid sequence of human proinsulin. The DNA coding for human proinsulin then is inserted in reading phase into a suitable cloning and expression vehicle. The vehicle is used to transform a suitable microorganism after which the transformed microorganism is subjected to fermentation conditions leading to (a) the production of additional copies of the human proinsulin gene-containing vector and (b) the expression of human proinsulin or a human proinsulin precursor product. 
     In the event the expression product is a human proinsulin precursor, it generally will comprise the human proinsulin amino acid sequence joined at its amino terminal to another protein, whether foreign or that normally expressed by the gene sequence into which the human proinsulin gene has been inserted. The human proinsulin amino acid sequence is joined to the protein fragment through a specifically cleavable site, typically methionine. This product is customarily referred to as a fused gene product. 
     The human proinsulin amino acid sequence is cleaved from the fused gene product using cyanogen bromide after which the cysteine sulfhydryl moieties of the human proinsulin amino acid sequence are stabilized by conversion to their corresponding S-sulfonates. 
     The resulting human proinsulin S-sulfonate is purified, and the purified human proinsulin S-sulfonate then is converted to human proinsulin by formation of the three properly located disulfide bonds, using, for example, the method of U.S. Pat. No. 4,430,266. The resulting human proinsulin product then is purified using recognized methodology. 
     The compounds of this invention can be prepared by enzymatic digestion of human proinsulin. Thus, treatment of human proinsulin with chymotrypsin leads to the production of (56-57 split)HPI. Treatment of the product mixture by reverse phase high performance liquid chromatography (HPLC) permits recovery of purified (56-57 split)HPI. 
     The des(33-56)HPI and des(31-56)HPI of this invention are prepared via (32-33 split)HPI (human proinsulin split between the 32 and 33 amino acid residues). The latter is prepared from human proinsulin. Thus, treatment of HPI with trypsin leads to the production of, among others, (32-33 split)HPI. Treatment of (32-33 split)HPI with chymotrypsin yields des(33-56)HPI, and the latter, upon treatment with carboxypeptidase B, yields des(31-56)HPI. 
     As noted, the compounds of this invention have an insulin-like, anti-diabetic effect substantially greater than that recognized for human proinsulin. 
     The compounds of this invention, due to their insulin-like activity, are useful in the treatment of diabetes. As such, they can be used in a variety of pharmaceutical compositions and formulations and can be administered by a variety of conventional routes, such as intramuscular, intravenous, subcutaneous, and intraperitoneal. 
     In administering the compounds of this invention, the pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. 
     Sterile injectable solutions can be prepared by incorporating the compounds of this invention in the calculated amount of the appropriate solvent along with various of the other ingredients, as desired. 
    
    
     The following examples are provided to illustrate this invention. They are not intended to be limiting on the scope thereof. 
     EXAMPLE 1--Preparation of (56-57 split)HPI 
     Human proinsulin (111 mg wet wt.) was dissolved in 40 ml of 0.1M CaCl 2  -0.08M Tris (pH 7.9) and digested with 81 μg of chymotrypsin at pH 7.9 for 15 minutes at 25° C. The digestion was terminated by acidifying the resulting solution to pH 2.5 with concentrated HCl. The solution was chromatographed on a 2.5×60 cm C-18 HPLC column using 29% CH 3  CN in 0.2M ammonium formate (pH 4.2) as the column solvent. 
     Conservative pooling of the column fractions provided 15.6 mg (u.v. wt.) of purified (56-57 split)HPI (19.3% yield). The product was chromatographed on 1.5×100 cm G25 C Sephadex column packed in 2% acetic acid to obtain 14.5 mg of very pure (56-57 split)HPI. 
     EXAMPLE 2--Preparation of des(33-56)HPI 
     Human proinsulin (312 mg wet wt.) was dissolved in 57 ml of 0.1M Tris buffer (final pH 7.0), and the solution was warmed to 25° C. in a water bath. Trypsin (25.1 micrograms) dissolved in 57 microliters of 0.05M Tris-0.02M CaCl 2  (pH 7.0) was added. The reaction was terminated after 68 minutes by lowering the pH of the solution to 2.5 by addition of acetic acid. 
     The acidified solution (57 ml) was chromatographed on a 5×200 cm G50 Superfine Sephadex column at 6° C. using 1M molar acetic acid as the column solvent. A large broad peak containing unreacted HPI, (32-33 split)HPI, and (65-Al split)HPI eluted first and was fairly well separated from a second peak containing di-Arg 31 ,32 human insulin. 
     Column fractions were pooled as follows: 
     
         ______________________________________                                    
                       Protein content                                    
        Beginning and end                                                 
                       of pool as                                         
Pool    points of pool determined by u.v.*                                
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A       1768-2146 ml   83.3 mg of a mixture of                            
                       HPI and monosplit HPI&#39;s                            
B       2146 ml-2464 ml                                                   
                       98.1 mg of a mixture of                            
                       HPI and monosplit HPI&#39;s                            
C       2464 ml-2843 ml                                                   
                       34 mg of di-Arg.sup.31,32                          
                       human insulin                                      
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 *Amounts calculated based on O.D..sup.276 and a calculated extinction    
 coefficient.                                                             
 
    
     The above pools were lyophilized. 
     The components in the three lyophilized pools were further resolved by chromatographing the materials in three portions on a 2.5×60 cm C-18 HPLC column using 34% CH 3  CN in 0.5% trifluoroacetic acid as the column solvent. 
     A portion (79 mg) of Pool A dissolved in dilute HCl (final pH=2) was applied to the column and yielded two pools of interest (D and E). 
     
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                         Protein content                                  
         Beginning and end                                                
                         of pool as                                       
Pool     points of pool  determined by u.v.                               
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D        519-627 ml      34.4 mg of pure (32-33                           
                         split)HPI [43%                                   
                         of 79 mg]                                        
E        700-808 ml      14.7 mg of 70% pure                              
                         (65-Al split)HPI                                 
                         [18.5% of 79 mg]                                 
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     Pool D was lyophilized to obtain pure (32-33 split)HPI. (32-33 split)HPI (27.5 mg) was dissolved in 9.91 ml of 0.08M Tris-0.1M CaCl 2  (final pH 7.8), and the mixture was treated with 19.82 μg of chymotrypsin for 26 minutes at 25° C. The reaction was terminated by addition of 1M HCl to pH 3.0. 
     The resulting crude des(33-56)HPI was chromatographed at 6° C. on a 2.5×125 cm G50 SF Sephadex column packed in 1M acetic acid. The major portion of the remaining unreacted (32-33 split)HPI eluted before the des(33-56)HPI. Fractions containing des(33-56)HPI were pooled and lyophilized to obtain product containing 11.08 mg des(33-56)HPI as determined by u.v. 
     The product from the G50 SF Sephadex column was purified on a 5×50 mm Pharmacia Mono S cation exchange column packed in 7M urea-0.1M acetic acid. A linear salt gradient from 0 to 0.533M NaCl over a 40 minute period was used to elute the product. The main peak material was chromatographed on a 0.9×40 cm G25M Sephadex column to obtain 5.37 mg (u.v. wt.) of des(33-56)HPI. 
     EXAMPLE 3--Preparation of des(31-56)HPI 
     Des(33-56)HPI in 0.1M Tris (final pH 7.1) is treated with Carboxypeptidase B at 25° C. The reaction is terminated by acidifying the mixture to pH 3.4 with 7M urea-0.1M acetic acid and 1M HCl. 
     The resulting des(31-56)HPI is applied to a G50 SF Sephadex column and eluted at 6° C. with 1M acetic acid. Fractions containing des(31-56)HPI are pooled and lyophilized. 
     The lyophilized material is chromatographed on a Pharmacia Mono S cation exchange column equilibrated with 7M urea-0.1M acetic acid. The protein is eluted using a sodium chloride linear gradient. The main peak material then is chromatographed on a G25M Sephadex column equilibrated with 2% acetic acid to obtain pure des(31-56)HPI. 
     Biological Activity 
     A. IM-9 Radioreceptor Assay 
     IM-9 cells were grown in RPMI media containing 2 mM glutamine, 25 mM HEPES, and 10% fetal bovine serum. Cells were harvested by centrifugation, washed, and resuspended in HEPES assay buffer, pH 7.6 [DeMeyts, P., Insulin and Growth Hormone Receptors in Human Cultured Lymphocytes and Peripheral Monocytes, Blecher, M., Ed., New York, Marcel Dekker, Inc, 301-330 (1976)]. Cell viability, determined by exclusion of trypan blue, was greater than 90% in each experiment. Triplicate tubes were prepared, each set containing 100 μl of assay buffer, human insulin, human proinsulin, or a compound of this invention, 200 μl  125  I-insulin (final concentration 1-2×10 -11  M), and 200 μl cells (about 500,000 cells). Incubations were carried out in 1.5 ml microfuge tubes at 15° C. for two hours. Concentrations of stock solutions containing insulin, proinsulin, or compound of this invention used in the binding studies were established by amino acid analysis and by their absorbance at 276 nm. The cells were resuspended during the assay every 30 minutes by inverting the tubes several times. At the end of the incubation, the tubes were centrifuged for one minute in a Beckman Microfuge, the supernatant was aspirated, the tips of the tubes containing the cell pellet were excised, and the radioactivity was measured. 
     The results from the foregoing are provided in Table I following. 
     
                       TABLE I                                                     
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IM-9 Radioreceptor Assay                                                  
                             Relative                                     
Compound       ED.sub.50 ( --M)                                           
                             Potency                                      
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Human Insulin  4.27 ± 0.3 × 10.sup.-10                           
                             70                                           
Human Proinsulin                                                          
               2.97 ± 0.2 × 10.sup.-8                            
                             1                                            
(56-57 split)HPI                                                          
               1.39 ± 0.1 × 10.sup.-8                            
                             2                                            
des(33-56)HPI  2.65 ± 0.5 × 10.sup.-9                            
                             11                                           
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     B. Isolated Fat Cell Radioreceptor Assay Isolated fat cells were prepared by a modification [Huber, C. T., Solomon, S. S., and Duckworth, W. C., J. Clin. Invest. 65, 461-468 (1980)] of the method described in Rodbell, M., J. Biol. Chem. 239, 375-380 (1964). All incubations were in Krebs-Ringer-Hepes (KRH) buffer, pH 7.4, with 4% bovine serum albumin (BSA) in a total volume of 2 ml. The fat cells were incubated at 15° C. for two hours with  125  I-labeled insulin (1-2×10 -11  M) and, at a selected concentration, with buffer or human insulin or human proinsulin or a compound of this invention. At selected times, triplicate 300 μl aliquots were removed and added to microfuge tubes containing 100 μl dinonyl phthalate [see Gliemann, J., Osterlind, K., Vinten, J., and Gammeltoft, S., Biochem. Biophys. Acta. 286, 1-9 (1972)]. After centrifugation for one minute in a microfuge, the tubes were cut through the oil layer, and the cell pellet was counted using a gamma counter to determine binding. Degradation of the  125  I-labeled insulin was determined by adding the buffer layer from the microfuge tube to ice-cold KRH buffer followed immediately by sufficient trichloroacetic acid to give a final concentration of 5%. 
     The results from the foregoing are provided in Table II following. 
     
                       TABLE II                                                    
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Isolated Fat Cell Radioreceptor Assay                                     
                             Relative                                     
Compound       ED.sub.50 ( --M)                                           
                             Potency                                      
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Human Insulin  1.43 ± 0.2 × 10.sup.-9                            
                             178                                          
Human Proinsulin                                                          
               2.55 ± 0.6 × 10.sup.-7                            
                             1                                            
(56-57 split)HPI                                                          
               7.87 ± 1.0 × 10.sup.-8                            
                             3                                            
des(33-56)HPI  9.50 ± 0.4 × 10.sup.-9                            
                             27                                           
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     C. Biological Activity in Isolated Rat Adipocytes 
     Adipocytes were prepared from epididymal fat pads by a modification (Huber, supra) of the collagenase digestion procedure of Rodbell, supra. Krebs-Ringer-HEPES (KRH) buffer, pH 7.4, containing 4% bovine serum albumin and 0.55 mM glucose was used in all isolation and incubation steps. 
     Approximately 2×10 5  adipocytes were incubated in one ml of buffer with  125  I-(A14) port insulin and varying concentrations of unlabeled human insulin, human proinsulin, or a compound of this invention. Incubations were conducted at 15° C. for 4.5 hrs. At the end of the incubation period, triplicate samples were withdrawn for determination of binding (cell-associated radioactivity) as described in Frank, B. H., Peavy, D. E., Hooker, C. S., and Duckworth, W. C., Diabetes 32, 705-711 (1983). At the 15° C. temperature of the incubation, no degradation of the tracer insulin was detectable. Samples were counted with a Tracor Analytic Model 1285 gamma scintillation spectrometer with a counting efficiency of 85%. 
     Biological activity was assessed according to the method of Moody, A. J., Stan, M. A., Stan, M., and Glieman, J., Horm. Metab. Res. 6, 12-16 (1974) by monitoring the incorporation of 2- 3  H-glucose in total fat cell lipid. Cells were incubated with varying concentrations of cold human insulin, human proinsulin, or a compound of this invention at 37° for 1 hr, and the reaction then was terminated by the addition of 10 ml of Liquifluor (New England Nuclear). Radioactivity was determined in a Searle Isocap 300 liquid scintillation counter at an efficiency of approximately 30%. Blanks were prepared in which the scintillation fluid was added to the vials prior to the addition of cells. The average counts obtained from these vials were subtracted from those observed in all other samples. 
     Competitive binding curves and biological activity dose-response curves were analyzed using the PREFIT and ALLFIT programs [DeLean, A., Munson, P. J., and Rodbard, D., Am. J. Physiol. 235, E97-E102 (1978)] based on a four-parameter logistic model. These analyses indicated the concentration of insulin or proinsulin necessary to produce a half-maximal response, as well as the maximal and minimal values. All values are presented as the mean±SEM. 
     The results from the foregoing are provided in Table III following. 
     
                       TABLE III                                                   
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Biological Activity in Isolated Rat Adipocytes                            
                             Relative                                     
Compound       ED.sub.50 ( --M)                                           
                             Potency                                      
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Human Insulin  5.32 ± 0.7 × 10.sup.-11                           
                             233                                          
Human Proinsulin                                                          
               1.24 ± 0.2 × 10.sup.-8                            
                             1                                            
(56-57 split)HPI                                                          
               5.76 ± 1.0 × 10.sup.-9                            
                             2                                            
des(33-56)HPI  1.39 ± 0.3 × 10.sup.-9                            
                             9                                            
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