Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA

Definitions

• This invention relates to stable plasma proteins, substantially free of blood pressure depressant components, and to a method for producing such depressantfree plasma proteins.
• solutions of plasma protein fractions are infused into the patient at a slow rate and heretofore no side effects of any significance have been experienced with such solutions.
• solutions of plasma protein fractions have been used in numerous heart-lung bypass procedures for open-heart surgery where the infusion rate is necessarily much greater, i.e., on the order of 100 ml. of 5 percent heattreated plasma protein solution within about five minutes.
• the arterial pressure has been observed to drop markedly in a number of such patients whereas this undesirable effect could be controlled if the infusion rates were reduced.
• the use of such plasma solutions in situations requiring rapid infusion could be seriously detrimental to the patient when such a depressor effect occurs.
• the depressor substance which causes a decrease in arterial pressure during the rapid infusion of solutions of heat-treated plasma protein fractions can be substantially removed by contacting a solution of a heat-treated plasma protein fraction with a fractionating substance, including surface active adsorbents, cation exchangers, ultrafiltration membranes and gel filtration particles.
• the starting materials for the process of this invention are stable human blood plasma protein fractions which contain a depressor, i.e., a substance which lowers blood pressure significantly when plasma containing it is infused rapidly.
• Stable plasma protein fractions are those which have been rendered heat stable by heating, up to 60 C., for up to hours.
• Such stable plasma protein fraction typically consist predominantly of albumin plus small amounts of alpha and beta globulins.
• a preferred class of starting material is a nonhomogeneous plasma protein fraction, e.g., that obtained from Supernatant IV-l by precipitation with ethanol (Cohn Method 6 process), which protein fraction has been reconstituted to a 5 percent solution containing NaCl and a stabilizer, e.g., acetyl tryptophan and/or sodium caprylate, and then heated to 60 C. for 10 hours to destroy hepatitis virus.
• This stable plasma protein fraction is described in Japanese Patent Publication No. 5297/60 which issued as Japanese Pat. No. 265704, the counterpart of U.S. Pat. No. 2,958,628.
• Another group of starting materials are solutions of plasma protein fractions which have been heated at about 60 C.
• a solution of a stable plasma protein fraction is heated at about 60 C. for a few hours, for example, about 1 to 4 hours. Any precipitate which forms is removed, e.g., by filtration or centrifugation, and the clear plasma solution is then treated with any one of the means of this invention for removing depressor substances. Subsequently, heating at 60 C. for 10 hours or longer may then be performed for the purpose of destroying any hepatitis virus in the plasma.
• Another starting material is the heat-treated stable plasma protein fraction obtained from human placenta.
• the exact nature of the material employed for removing the depressor is not critical and can readily be determined according to methods well known in the art, given the knowledge that the depressor is a relatively low molecular weight, readily separable material. For example, using an ultrafiltration membrane which allows materials of a molecular weight below 10,QO0 to pass through, the depressor can be separated from any starting plasma protein solution. The operability of any fractionating material which is inert to plasma proteins 0 for separating the depressor substance from the starting plasma solution can then be determinedusing this separated solution of the depressor to determine experimentally the optimum conditions using that fractionating material.
• Preferred fractionating materials for removing the depressor are surface active adsorbents, cation exchangers, preferably cationic ion exchange resins, ultrafiltration membranes and gel filtration molecular sieves, and accordingly, the preferred methods of removal of the depressor are surface active adsorbent chromatography, cation exchanger chromatography, ultrafiltration and gel molecular sieve filtration.
• a preferred treatment of the protein solution for removal of the depressor substance or substances comprises treating the solution with a surface active adsorbent, for example, silica gel, hydrated alumina gel, magnesium hydroxide gel or barium sulfate, followed by separation of the adsorbent from the mixture to give a plasma protein solution substantially free of depressor substances.
• a surface active adsorbent for example, silica gel, hydrated alumina gel, magnesium hydroxide gel or barium sulfate
• a concentration of about 5 percent is preferred.
• optimum amount of adsorbent used will vary, depending upon the particle size of the adsorbent, the relative concentration of the protein, and the desired level to which the depressor substance is to be reduced, i.e., either complete removal or substantially complete removal, i.e., to a point where a slight depressor effect by rapid infusion of the solution would not be harmful to the recipient.
• silica gel having a particle size of about 10-40 microns is a preferred adsorbent although particle sizes greater or smaller than 10-40 microns are effective.
• Typical of the preferred silica gels are Aerosil 200 (Degussa Inc.) and Merck Silica Gel H.
• the starting protein solution can be mixed with the adsorbent batchwise with gentle agitation or it can be passed through a suitable column of the adsorbent.
• Treatment time is not critical, and, in fact, in the batchwise procedure, mixing for several minutes or for several hours appears to have substantially the same effect in the successful removal of the depressor substances.
• the temperature at which the operation is performed also is not critical so long as it is maintained below that which is detrimental to the plasma proteins and generally is in the range of about to about 60 C.
• the adsorbent Following treatment of the protein solution with the adsorbent, the latter can be removed by conventional means, e.g., centrifugation and/0r filtration.
• the solution of plasma protein can then be sterile-filtered into suitable containers and preferably then heated at about 60 C. for at least hours to inactivate any hepatitis virus that may be present, if the solution has not previously been subjected to prolonged heating.
• cation exchangers for example, carboxymethyl cellulose; carboxymethyl Sephadex (Pharmacia Fine Chemicals), which is a cross-linked dextran with terminal carboxymethyl groups; Amberlite CG-50, which is a sulfonated polystyrene cross-linked with divinylbenzene sold by Rohm and Haas and Co.; and Dowex 50-X2 which is a similar cation exchanger sold by Dow Chemical Co.
• the protein solutions may be allowed to pass through columns of the ion exchangers such as those previously equilibrated with 0.25 percent sodium chloride solution, at a rate of about 50-150 ml./hr./cm and at a temperature of about 4 to 60 C.
• the eluates are then substantially free of depressor substances.
• depressor substances from stable plasma protein fractions are by ultrafiltration.
• the starting protein solutions containing depressor substance can be subjected to ultrafiltration using a suitable membrane of a porosity which allows passage essentially only of low molecular weight species below about 10,000 (which includes the depressor substance) under conditions which are well known to those practicing ultrafiltration procedures.
• UM10 Diafio ultrafiltration membrane which is a non-cellulosic polymer with ionic groups on the surface, available from Amicon Corporation.
• Other acceptable membranes are PMlO Diaflo membranes (also from Amicon), similar to UM10 membrane but nonionic; Pellicon type PSED (Millipore Corporation) and Nitrocellulose membrane 8-1 2136 (Sartorius Division of Brinkman Instruments).
• Still another means for removal of depressor substances from stable plasma protein fractions according to the present invention is by gel filtration. Similar in some respects to ultrafiltration, such a system depends on the ability of lower molecular weight species, including the depressor substance, to pass through the interstices of bead-formed gel particles and become more or less entrapped thereby.
• the procedure for the use of molecular sieve gels in gel filtration is well known in the art and is exemplified in numerous references, including Whitaker, J.R., Determination of molecular weights of proteins by gel filtration on Sephadex, Anal.
• Sephadex G- 25 Sephadex G-50, Sephadex G and Sephadex G- which are a variety of cross-linked dextran available from Pharmacia Fine Chemicals.
• Sepharose 68 a bead-formed agarose gel available from Pharmacia.
• the depressor substance which is removed from stable plasma protein fractions is a polypeptide having a molecular weight between 1,000 and 10,000. the depressor substance is believed to be generated primarily during the heating of the protein solution. However, we have found that this purified stable plasma protein fraction may thereafter be heated up to 60 C. for extended periods without generating additional depressor substance.
• the resulting purified, stable plasma protein fraction which is substantially free of depressor substance can be rapidly infused intravenously into a patient without the danger of depressing the blood pressure, particularly in cases of heart-lung by-pass.
• a purified stable plasma protein fraction is obtained, whose scope of clinical application is extended over that of previously obtainable plasma protein fractions.
• substantially free of depressor means that the plasma protein fraction lacks true depressor activity, as distinguished from the volume effect which causes a nominal drop in blood pressure, when any liquid, including saline or isotonic solution, is injected. This effect can be distinguished by the dog isolated hind limb test, which detects only true depressor effect.
• the novel depressor-free plasma protein fraction products of this invention give a negative response in this test and in the smooth muscle rat uterus test for kinin and kinin-like substances.
• the'depressor substance may be bradykinin or a kinin-like material, since it is well established that blood plasma contains kininogens capable of being converted to kinins when activated by a certain specific enzyme or enzymes.
• Treatment of plasma protein fractions with either surface active adsorbents or cation exchangers or by ultrafiltration or gel filtration results in the removal of any kinin or kinin-like substance, as evidenced by lack of significant depressor effect when the treated protein is subjected to testing on smooth muscle or perfused in the isolated hind limb of a dog or administered by systemic infusion in dogs.
• Contractions of the smooth muscle of a rat uterus is a highly sensitive test and quite specific for indicating the presence of kinin or kinin-like substances.
• the isolated hind limb test is a highly sensitive test for detecting depressor substances which cause dilation of the peripheral circulatory system thus producing a fall in blood pressure.
• Systemic infusion is an in vivo test which simulates effects on blood pressure following rapid infusion in a patient.
• the dried plasma protein powder reconstituted to a 5 percent protein solution in water and stabilized with 0.004 M quantities of sodium acetyltryptophanate and sodium caprylate, and also containing sodium chloride in an amount to make the solution slightly less than isotonic (as described in US. Pat. No. 2,958,628) was heated at 60 C. for about 1 to 4 hours, in this instance about 2 hours, rather than for 10 hours as in the case of Starting Material A. A small amount of flocculent precipitate generally formed. After cooling to about room temperature, the precipitate was removed by filtration and the clear solution was then ready for use.
• Smooth muscle contractibility was measured by the method of Magnus (Trautshold, K., Handbook of Experimental Pharmacology, Vol. XXV, p. 55, Springer- Verlag, New York, 1970).
• a 1.5 cm. strip of uterus muscle was isolated from a virgin Wistar rat weighing about 150 g. The strip was suspended in 8.6 ml. de Jalon solution saturated with air and containing 0.1 mg. percent of atropine sulfate. Kymograph recordings of the contractile forces were made before and for seconds following the addition of 0.4 ml. of the test solution.
• Mean arterial pressure before infusion Mean arterial pressure after infusion 7: Decrease X 0 Mean arterial pressure before infusion 10 way of polyethylene catheters at each end to the tied l artery, one catheter being placed well into the portion of the artery leadidng to the heart and the other catheter being placed about 6-10 cm. in the direction leading toward the limb extremity.
• a Sigma motor pump was connected directly into the loop below the first named catheter and a pressure transducer was connected into the loop on the peripheral side of the pump. Heparin (l0 mg./kg.) was injected I.V. and after 30 minutes, the pump was started and adjusted so that the pressure of the blood leaving the pump to flow into the limb essentially matched the arterial pressure recorded from the right femoral artery.
• a Sigma motor pump which provides a substantially constant pulsatile flow was used so that base lines in the pressure recordings could be determined more accurately.
• a base line is that figure obtained by adding to the diastolic pressure one-third of the difference betweenthe systolic and diastolic pressures. Decreases following the infusion of the test solutions were expressed as the actual difference in mm. Hg. between the baseline before infusion and the base line after infusion.
• This isolated hind limb procedure is more sensitive than the systemic infusion method (above) and will show direct effects of depressor substances on arterial pressure as a result of changes in peripheral resistance.
• Example 1 A 5 percent solution of heat-treated plasma protein Depressor Substance Method A Method B Starting Material A Strong Contraction" l 1.5% Product of Example I No Contraction 0.0%
• Contraction was slightly greater than that obtained by 10 ng. of Bradykinin. Dose of I87 mg. protein/kg. at a rate of IS ml./min.
• Silica gel effectively removed depressor substance from non-homogeneous plasma protein.
• Example 2 100 ml. of heat-treated plasma protein solution (Starting Material A) was stirred gently at about 25 C. with 2.0 g. silica gel (Aerosil 200) for 5 hours, then centrifuged and the clear supernatant solution was tested for depressor substance.
• Example 3 100 ml. each of Starting Material A was stirred with 1.0 g. and 2.0 g. silica gel (Aerosil 200) for 4 hours at room temperature. The treated solutions were then tested for depressor substance.
• Depressor Substance Method C fraction (Starting Material A) was passed through a column of Merck H silica gel previously equilibrated with 0.25 percent sodium chloride. The flow rate of the protein solution was -150 ml./hr./cm About percent of the protein was recovered in the effluent as determined by optical density at 280 nm. in a Hitachi 6 Spectrophotometer. By electrophoretic analysis, the final product comprised 88.5 percent albumin, 7.5 percent alpha globulin and 4.0 percent beta globulin.
• Example 4 A ml. portion of heat-treated plasma protein solution (Starting Material B) was stirred gently with 2 g. of silica gel (Aerosil 200) at room temperature for 3 hours. The silica gel was removed and a portion of the solution was heated at 60 C. for l 1 hours.
• Example Depressor Substance Method C Av. Decrease Starting Material A Product of Example 5 The results show the effectiveness of aluminum hytion were combined. About 90 percent of the protein was recovered as determined by optical density at 280 nm. on this mixture.
• Example 8 100 ml. of a 5 percent plasma protein solution Starting Material A) was stirred gently with 5 g. of moist Dowex 50-X2 (freshly regenerated with dil. NaOH, then dil. HCl) for 3 hours at room temperature at pH 7.2. The resin was filtered off and the solution showed 85 percent recovery of protein as determined by optical density at 280 nm.
• Depressor Substance Method C droxide in removing depressor substance from plasma Before After o. Deter- Av. protein 7 4O Infusion Infusion mmations Decrease Starting Example 6 Material A I61 78 7 83 100 ml. portions of plasma protein solution (Starting gggr 8 I 61 HO 3 51 Material A) were treated as follows with the indicated saline comm] 9 I 16 results:
• Example 7 A 5 percent protein solution (Starting Material C) was allowed to pass through a column of carboxymethyl Sephadex which had previously been equilibrated with 0.05 M phosphate buffer containing 0.1 M sodium chloride. The flow rate was between -150 ml./hr./cm The column was then washed with the same buffer-saline solution and the effluent and wash solu- The results show depressor substance is removed from plasma protein by treatment with a strong cation exchange resin.
• the plasma protein fraction consists essentially of a mixture of 85-92 percent albumin, 4-10 percent globulin and 2-7 percent beta globulin.
• a process for the production of an electrophoretically non-homogeneous plasma protein solution which is substantially free of depressor substance so that the solution may be rapidly infused intravenously without causing a significant fall in blood pressure which comprises the step of contacting a solution of an electrophoretically non-homogeneous human plasma protein containing depressor substance with a material selected from the group consisting of surface active adsorbents, cation exchangers, ultrafiltration membranes and gel filtration molecular sieves which removes the depressor substance from the protein. membranes and gel molecular sieves.
• proteins of the starting protein solution are a mixture consisting of at least 83 percent albumin and not more than 17 percent alpha-globulin and beta-globulin.
• the process of claim 10 comprising the step of heating the protein solution after the removal of the depressor therefrom at about 60 C. for a period of time of at least 10 hours effective to destroy any hepatitis virus therein.
• the starting plasma solution containing the depressor is a heattreated mixture of at least 83 percent albumin and not more than 17 percent alpha-globulin and beta-globulin.
• the process of claim 15 further including the step of separating the surface active adsorbent from the mixture by centrifugation or filtration.
• the cation exchanger is a member of the group consisting of sulfonated polystyrene cross-linked with divinylbenzene, carboxymethyl cellulose, and cross-linked dextran having terminal carboxymethyl groups.
• gel filtration particles are selected from the group consisting of cross-linked dextrans and agarose gels having the capacity for entrapping substances with molecular weights below about 10,000.
• contacting the heated solution with a material selected from the group consisting of surface active adsorbents, cation exchangers, ultrafiltration membranes and gel filtration molecular sieves.
• Step b) the material is a surface active agent selected from the group consisting of silica gel and aluminum hydroxide gel.

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Citations (3)

• 1972
  • 1972-06-19 JP JP6121072A patent/JPS5620287B2/ja not_active Expired
• 1973
  • 1973-01-09 SE SE7300244A patent/SE420049B/xx unknown
  • 1973-01-12 AT AT25973A patent/AT320853B/de not_active IP Right Cessation
  • 1973-01-12 CA CA161,150A patent/CA1004598A/en not_active Expired
  • 1973-01-12 DE DE2301501A patent/DE2301501C3/de not_active Expired
  • 1973-01-13 CH CH43473A patent/CH596839A5/xx not_active IP Right Cessation
  • 1973-06-13 US US369478A patent/US3876775A/en not_active Expired - Lifetime

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