This invention relates to a method for the production of streptokinase.

More particularly, the present invention is concerned with an improvement in the fermentative production of streptokinase by hemolytic streptococci. It has been known for some time that culture or culture filtrates of certain strains of beta hemolytic streptococci are able to cause rapid lysis of the fibrin clot. The active agent is a streptococcal fibrinolysin, so-called streptokinase, and the reaction has been termed fibrinolysis. The enzyme streptokinase has been most frequently associated with hemolytic streptococci of the Lancefield groups A, human C, and G, with the C group being preferred. In particular, the strain H46A (identified by the American Type Culture Collection, Rockville, Md. as No. 12449, Rebecca C. Lancefield, strain H46A, 1956) is the most generally employed strain. In the selection of a medium for growth of streptokinase, various materials have been suggested heretofore. For economic reasons, the medium should be capable of producing massive growth of the streptococci. The various factors necessary for such massive growth have been elucidated by Bernheimer, et al., J. Bact. 43, pp. 481-94 (1942). In general, the medium has comprised a nitrogen source such as gelatin hydrolysate or casein digest supplemented with various amino acids such as cystine, glycine, tryptophane, tyrosine, methionine and glutamine; uracil and adenine; various salts; glucose; and certain members of the Vitamin B group. U.S. Pat. No. 2,701,227 describes two of the primary nitrogenous materials employed heretofore in the fermentative production of streptokinase by hemolytic streptococci. They are an enzyme hydrolyzed casein digest available commercially from Sheffield Farms Co., Inc., New York, N.Y., and sold under the trademark "N-Z-Amine," and an animal protein digest available commercially from Difco Laboratories, Detroit, Mich., and marketed under the trademark "Neopeptone." Although both of the foregoing nitrogenous substances are capable of producing massive growth of the hemolytic streptococci, the yield of streptokinase when employing the animal protein digest as a nitrogen source is exceedingly low and the lot-to-lot variation of the casein hydrolysate causes extreme variations in the yield of streptokinase. Accordingly, it is an object of the present invention to provide an improved culture medium for the fermentative production of streptokinase by hemolytic streptococci. It is another object of this invention to provide an improved source of nitrogenous nutrients for the fermentation medium used in the production of streptokinase. Other objects and advantages of the invention will be apparent to those skilled in the art after reading the disclosure hereof. In accordance with the invention, the foregoing objects are achieved by employing corn steep liquor as the nitrogen source together with a metabolizable carbon source for the fermentative production of streptokinase by hemolytic streptococci. It has been found that when corn steep liquor is employed as the nitrogen source, a substantially more uniform and higher yield of streptokinase is obtained during fermentation than heretofore. Moreover, the total number of ingredients exogenously added to the fermentation medium can be reduced from about 16 to 2, thereby resulting in a substantial reduction in cost of production. Corn steep liquor is a well known by-product of the manufacture of corn starch. When corn starch is produced by the wet-milling process, the corn kernels are first soaked in a dilute solution of sulfurous acid at a pH of about 3 to 4, and a warm temperature of about 110 to 130 F for an extended period of time of about 2 to 4 days. In this soaking or steeping operation, the kernels are softened and swelled so that the subsequent milling operation results in a good separation of the starch from the germ, hull and gluten portions of the kernel. During this steeping process, soluble matter from the kernels passes into the steepwater. This liquor contains a complex of materials, including a variety of protein hydrolytic products, "B" complex vitamins, amines, and other organic and inorganic materials and entrained solids. The density of corn steep liquor ranges from about 3 to 7 Baume. Although corn steep liquor has been described as useful in the fermentative production of certain antibiotics and enzymes, it has not been known heretofore to be suitable for use in the production of streptokinase. Since the

previously required components for fermentative production of streptokinase have been defined as critical, it was surprising and unexpected to find that significant and substantial improvements in yield could be obtained even though accompanied by a reduction in 14 of the conventional 16 components when corn steep liquor is used in the medium as the nitrogen source. In use of the corn steep liquor, it is preferred to remove entrained solids or insoluble matter therefrom prior to addition of the liquor to the fermentation medium. It has been observed that occasional lots of corn steep liquor, while supporting growth of the microorganism, do not allow significant synthesis of streptokinase. It has been found that clarification of the corn steep liquor converts these poor lots to lots that allow a high degree of synthesis of streptokinase and that adding back the removed solids to the clarified liquor again inhibits the streptokinase synthesis. Thus, it is believed that the entrained solids or insoluble matter in certain lots of corn steep liquor contain streptokinase synthesis inhibitors which can be removed by clarification.

Tell your doctor of any over-the-counter or prescription medication you may take, including: blood thinners (e.g., warfarin), NSAID (e.g., ibuprofen, naproxen), aspirin. Drugs that can reverse effects of streptokinase include: aminocaproic acid, aprotinin, tranexamic acid. Do not start or stop any medicine without doctor or pharmacist approval.

This medication is an enzyme which works to break up and dissolve blood clots which can block arteries. It is used in the treatment of heart attack or lung blood clots (pulmonary embolism) as well as leg blood clots (deep venous thrombosis-DVT). HOW TO USE: This medication is given by injection by a health care professional. It is important this medication be used as prescribed. It is most effective when administered as soon as possible (within 6 hours after symptoms (e.g., chest pain) appear. SIDE EFFECTS: Nausea, dizziness, low blood pressure or mild fever may occur. It can also cause nerve damage. If any of these effects persist or worsen, notify your doctor promptly. Notify your doctor promptly if you develop: easy bruising,headache, flushing, rapid or abnormal heartbeat, chest pain. A serious allergic reaction to this drug is unlikely, but seek immediate medical attention if it occurs. Symptoms of a serious allergic reaction include: rash, itching, swelling, severe dizziness, trouble breathing. Immediately report any signs of bleeding to your doctor. If you notice other effects not listed above, contact your doctor or pharmacist. PRECAUTIONS: Tell your doctor if you have: bleeding disorders, high blood pressure, endocarditis, recent biopsy or surgery, recent injury, any allergies. Use extra caution to avoid injury and trauma (e.g., carefully brush teeth) while using this medication due to the increased risk of bleeding. This medication should be used only if clearly needed during pregnancy. Discuss the risks and benefits with your doctor. It is not known if this medication appears in breast milk. Consult your doctor before breast-feeding.

Acute Evolving Transmural Myocardial Infarction: Administer Streptokinase as soon as possible after onset of symptoms. The greatest benefit in mortality reduction was observed when Streptokinase was administered within four hours, but statistically significant benefit has been reported up to 24 hours (see CLINICAL PHARMACOLOGY ).

Route Intravenous infusion

Total Dose 1,500,000 IU

Dosage/Duration 1,500,000 IU within 60 min. 20,000 IU by bolus followed by 2,000 IU/min. for 60 min.

Intracoronary infusion

140,000 IU

Pulmonary Embolism, Deep Vein Thrombosis, Arterial Thrombosis or Embolism: Streptase, Streptokinase, treatment should be instituted as soon as possible after onset of the thrombotic event, preferably within 7 days. Any delay in instituting lytic therapy to evaluate the effect of heparin therapy decreases the potential for optimal efficacy. Since human exposure to streptococci is common, antibodies to Streptokinase are prevalent. Thus, a loading dose of Streptokinase sufficient to neutralize these antibodies is required. A dose of 250,000 IU of Streptokinase infused into a peripheral vein over 30 minutes has been found appropriate in over 90% of patients. Furthermore, if the thrombin time or any other parameter of lysis after 4 hours of therapy is not significantly different from the normal control level, discontinue Streptokinase because excessive resistance is present.
IV Infusion Dosage/Duration 100,000 IU/hr for 24 hr (72 hrs if concurrent DVT is suspected). 100,000 IU/hr for 72 hr

Indication

Loading Dose

Pulmonary Embolism

250,000 IU/30 min.

Deep Vein Thrombosis Arterial Thrombosis or Embolism

250,000 IU/30 min.

250,000 IU/30min.

100,000 IU/hr for 24-72 hr

Arteriovenous Cannulae Occlusion: Before using Streptase, Streptokinase, an attempt should be made to clear the cannula by careful syringe technique, using heparinized saline solution. If adequate flow is not re-established, Streptokinase may be employed. Allow the effect of any pretreatment anticoagulants to diminish. Instill 250,000 IU Streptokinase in 2 mL of solution into each occluded limb of the cannula slowly. Clamp off cannula limb(s) for 2 hours. Observe the patient closely for possible adverse effects. After treatment,aspirate contents of infused cannula limb(s), flush with saline, reconnect cannula. Pediatric Patients: Specific dosage and administration recommendations cannot be made based on the limited data available. However, published experience generally used loading and continuous infusion doses administered on a weight-adjusted basis. See Precautions, Pediatric Use. Reconstitution and Dilution: The protein nature and lyophilized form of Streptase, Streptokinase, require careful reconstitution and dilution. Slight flocculation (described as thin translucent fibers) of reconstituted Streptokinase occurred occasionally during clinical trials but did not interfere with the safe use of the solution. The following reconstitution and dilution procedures are recommended: Vials and Infusion Bottles

1. Slowly add 5 mL Sodium Chloride Injection, USP or 5% Dextrose Injection, USP to the Streptase, Streptokinase, vial, directing the diluent at the side of the vacuum-packed vial rather than into the drug powder. 2. Roll and tilt the vial gently to reconstitute. Avoid shaking. (Shaking may cause foaming.) (If necessary, total volume may be increased to a maximum of 500 mL in glass or 50 mL in plastic containers, and the infusion pump rate in Table 1 should be adjusted accordingly.) To facilitate setting the infusion pump rate, a total volume of 45 mL, or a multiple thereof, is recommended. 3. Withdraw the entire reconstituted contents of the vial; slowly and carefully dilute further to a total volume as recommended in Table 1. Avoid shaking and agitation on dilution. 4. When diluting the 1,500,000 IU infusion bottle (50 mL), slowly add 5 mL Sodium Chloride Injection, USP, or 5% Dextrose Injection, USP, directing it at the side of the bottle rather than into the drug powder. Roll and tilt the bottle gently to reconstitute. Avoid shaking as it may cause foaming. Add an additional 40 mL of diluent to the bottle, avoiding shaking and agitation. (Total volume = 45 mL). Administer by infusion pump at the rate indicated in Table 1. 5. Parenteral drug products should be inspected visually for particulate matter and discoloration prior to administration. (The Albumin (Human) may impart a slightly yellow color to the solution.) 6. The reconstituted solution can be filtered through a0.8 m or larger pore size filter. 7. Because Streptase, Streptokinase, contains no preservatives, it should be reconstituted immediately before use. The solution may be used for direct intravenous administration within eight hours following reconstitution if stored at 2-8C (36-46F). 8. Do not add other medication to the container of Streptase, Streptokinase. 9. Unused reconstituted drug should be discarded. TABLE 1: SUGGESTED DILUTIONS AND INFUSION RATES
Total Vial Size Solution (IU) Volume

Dosage

Infusion Rate

I. Acute Myocardial Infarction A. Intravenous Infusion B. Intracoronary Infusion 1. 20,000 IU bolus 2. 2,000 IU/minute for 60 minutes 1,500,000 45 mL 250,000 125 mL >1. Loading Dose of 10 mL >2. Then 60 mL/hour Infuse 45 mL within 60 min.

II. Pulmonary Embolism, Deep Vein Thrombosis, Arterial Thrombosis or Embolism Intravenous Infusion A. 1. 250,000 IU loading dose over 30 1,500,000 90 mL minutes 2. 100,000 IU/hour maintenance dose >1. Infuse 30 mL/hour for 30 minutes

2. Infuse 6 mL per hour

B. SAME

1,500,000 infusion 45 mL bottle

1. 15 mL/hour for 30 minutes 2. Infuse 3 mL per hour

For Use In Arteriovenous Cannulae: Slowly reconstitute the contents of 250,000 IU Streptase, Streptokinase, vacuum-packed vial with 2 mL Sodium Chloride Injection, USP or 5% Dextrose Injection, USP. HOW SUPPLIED Streptase, Streptokinase, is supplied as a lyophilized white powder in 50 mL infusion bottles (1,500,000 IU) or in 6.5 mL vials with a color-coded label corresponding to the amount of purified Streptokinase in each vial as follows: green blue red red 250,000 IU 750,000 IU 1,500,000 IU 1,500,000 IU NDC 0186-1770-01 NDC 0186-1771-01 NDC 0186-1773-01 NDC 0186-1774-01 box of 1 box of 1 box of 1 (vials) box of 1 (infusion bottles)

Store unopened vials at controlled room temperature (15-30C or 59-86F).

125342- call no , pankaj rana