MXPA96004802A - Treatment of cardiac failure congest - Google Patents

Abstract

A mammal with congestive heart failure is treated by administering to the mammal an effective amount of growth hormone. Treatment results in increased left ventricular cystolic pressure, increased left ventricular maximum, increased cardiac output, and increased cystolic volume index. The treatment also results in a reduced left ventricular end-systolic pressure and reduced systemic vascular resistance. These measurements indicate improvements in cardiac function due to increased ventricular contraction capacity and decreased peripheral ventricular resistance.

Description

t. 1. FIELD OF THE INVENTION This invention relates to the field of treatment of patients with growth hormone (GH) for patients with congestive heart failure. 10 DESCRIPTION OF BACKGROUND AND RELATED TECHNIQUE In vitro studies have shown that chronic hypersecretion of growth hormone by The implantation of a growth hormone - secreting tumor is associated with an increase in the maximum isometric strength of the normalized left ventricular papillary muscle by area in. cross section and without changes in the shortening speed without loading of isolated muscle and calcium in, and actin-activated myosin in normal rats. This is observed despite a marked shift of the isomyosin pattern towards the V3 isoform of low activity with ATPase. These results suggest that growth hormone can induce a unique pattern of contraction myocardium: a normal shortening speed and a REF: 23229 increased force generation are associated with changes in the iosin phenotype that allows the heart muscle to function more economically. Timsit, J. et al. , J. Clin. Invest. 86: 507-515 (1990); Timsit, J. et al., Acta Paediatrics. Suppl. 383: 32-34 (1992). In vitro studies of the same researchers have been carried out on rat cardiac staple fibers. These studies have shown that the contraction performance of rat myocardial staple fibers subjected to chronically elevated circulating growth hormone concentrations increases. The increase in contraction performance has been shown to be due to alterations in the properties of the contractile apparatus, which include an increase in both maximum tension and myofibrillar sensitivity to calcium. Mayoux, E. et al., Circulatian Research 72 (1): 57-64 (1993). It has been found that the left ventricular dP / dt, the systolic index and the cistolic work increase significantly in rats anesthetized with chloralose urethane, with a growth hormone-secreting tumor that grows in a transplantable manner. The data suggest that chronic growth hormone hypersecretion increases cardiac output by increasing contraction capacity in anesthetized rats. Penney, D.G. et al., Cardiovascular Research 19: 270-277 (1985).

They have also formed an opposite result. Rubin, S.A. et al., J. Mol. Cell Cardiol. 22: 429-438 (1990) has reported that chronic similar growth hormone hypersecretion induced by a growth hormone-secreting tumor causes significant decrease in the capacity of left ventricular contraction (dP / dt maximum) and increases in LVEDP in rats anesthetized with ketamine. In a clinical study it has been demonstrated that the administration of human growth hormone to normal subjects during a week increases the capacity of ventricular contraction and cardiac output when evaluated by echocardiography. Thuesen, L. et al., Dah. Med. Bull. 35 (2): 193-196 (1988). In adults with growth hormone deficiency, treatment with growth hormone produces significant increases in stroke volume and exercise capacity. These results suggest that growth hormone may improve cardiac function at rest and during exercise in adult patients with growth hormone deficiency. Jorgensen, J. et al., The Lancet i: 1221-1225 (1989); Cuneo, R. et al., J. Appl. Physiol. 70: 695-700 (1991); Christiansen, J. s. et al., Acta Paedi a tr. Suppl. 383: 40-42 (1992).

Cuneo et. al., Lancet i: 838-839 (1989) has reported a very important case showing effects of growth hormone therapy in a patient with extremely poor cardiac function. The patient developed severe heart failure eight months before hypophysectomy due to Cushings syndrome. The conventional therapy includes diuretics and angiotensin-converting enzyme inhibitors which had little effect and were considered to be heart transplantation. As a last resort, treatment with growth hormone 12 IU / day s.c. It was attempted, with a remarkable beneficial effect. Clinical improvement and increases in myocardial contraction capacity and cardiac output were observed. So far, the effects of human growth hormone in patients with heart failure without growth hormone deficiency has not been reported, as far as the applicants know. Heart failure affects approximately three million Americans, and develops in approximately 400,000 people each year. The current therapy for heart failure is insufficient. Although angiotensin-converting enzyme (ACE) inhibitors have been shown to have beneficial effects in patients with heart failure, they consistently appear unable to relieve symptoms in more than 60% of patients with heart failure. In addition, they reduce mortality from heart failure by only about 15-20%. Therefore, there is a need for an improvement in cardiac failure therapy. Accordingly, it is an object of this invention to provide a method of treatment for a patient with congestive heart failure.

BREVE D_BC_-EPC! ICM DE LA IMVENCJQKf The present invention achieves these objectives by providing a method of the treatment of congestive heart failure, the method is characterized by the administration of an effective amount of growth hormone (GH). The administration of GH results in an improvement in cardiac function due to an increase in ventricular contraction capacity and a decrease in peripheral vascular resistance.

BRIEF DESCRIPTION OF THE FIGURES The figure shows the body weight before treatment in controls linked and operated in false ** P < 0.01, compared to the respective vehicle group. Figure Ib shows an increase in body weight after treatment in bound rats and controls operated in false. ** P < 0.01, compared to the respective group treated with vehicle. The figure shows a comparison of the increase in the proportion of ventricular weight with respect to body weight in bound rats and controls operated in false. ** P < 0.01, compared to the respective group treated with vehicle. Figure 2a shows the effect of the administration of GH on the serum concentrations of GH in bound rats and controls operated in false. ** P < 0.01, compared to the respective group treated with vehicle. Figure 2b shows the effects of administration of GH on the serum concentrations of IGF-I in bound rats and controls operated in false. ** P < 0.01, compared to the respective group treated with vehicle. Figure 3a shows the effects of GH and a vehicle on mean arterial pressure (MAP) in bound rats and controls operated in false. #P < 9.05, ## P < 0.01, compared to the respective group falsely treated. * P < 0.05, compared to the respective group treated with vehicle. Figure 3b shows an effect of GH on cystolic blood pressure (SAP) in bound rats and controls operated in false. #P < 0.05, ## P < 0.01, compared to the respective group operated in false.

* P < 0.05, compared to the respective group treated with vehicle. Figure 3c shows the effects of GH on heart rate (HR) in bound rats and controls operated in false. #P < 0.05, ## P < 0.01, compared to the respective group falsely treated. * P < 0.05, compared to the respective group treated with vehicle. Figure 4a shows the effects of GH on the left ventricular maximum dP / dt. * P < 0.05, ** P < 0.01, compared to the respective group treated with vehicle.

#P < 0.05, ## P < 0.01, compared to the respective group treated with vehicle. Figure 4b shows the effects of growth hormone (GH) on left ventricular cystolic pressure (LVSP). * P < 0.05, ** P < 0.01 compared to the respective group treated with vehicle. # P < 0.05, ## P < 0.01, compared to the respective group operated in false. Figure 4c shows the effects of growth hormone (GH) on left ventricular end diastolic pressure (LVEDP). * P < 0.05, ** P < 0.01 compared to the respective group treated with vehicle. # P < 0.05, ## P < 0.01, compared to the respective group operated in false. Figure 5a shows the effects of growth hormone (GH) on the cardiac index (Cl) in bound rats and in controls treated in false. * P < 0.01, compared to the respective group treated with vehicle.

# P < 0.05, 'compared to the respective group falsely treated. Figure 5b shows the effects of growth hormone (GH) on the cystolic volume index (SVI) in bound rats and controls operated in false. * P < 0.05, compared to the respective group treated with vehicle.

# P < 0.05, compared to the respective group operated in false. Figure 5c shows the effects of growth hormone (GH) on systemic vascular resistance (SVR) in bound rats and controls operated in false.

# P < 0.05, compared to the respective group treated with vehicle. # P < 0.05, compared to the respective group operated in false.

PFfl prmH prRAtItaPA PB TA TMVEHTION to. Definitions In general, the following words, phrases or abbreviations have the indicated definition when they are used in the description, examples and claims: As used herein, "BW" refers to body weight. As used herein, "CO" refers to cardiac output. As used herein, "Cl" refers to the cardiac index. The cardiac index can be measured as the cardiac output divided by body weight (CO / BW). As used herein "dP / dt" refers to the left ventricular maximum. As used herein, "HR" refers to the heart rate. As used herein, "LCA" refers to the left coronary artery. As used herein, "LVEDP" refers to the left ventricular end diastolic pressure. As used herein, "LVMP" refers to the left ventricular mean pressure. As used herein, "LVSP" refers to the left ventricular cystolic pressure. As used herein, "MAP" refers to mean arterial blood pressure. As used herein, "RAP" refers to the right atrial pressure. As used herein, "SAP" refers to systolic blood pressure.

As used herein, "SV" refers to stroke volume. The stroke volume is measured as CO / HR. As used herein, "SVI" refers to the systolic volume index. The systolic volume index is measured as SV / BW. As used herein, "SVR" refers to systemic vascular resistance. The SVR is measured as MAP / CI. As used herein, "VW" refers to ventricular weight. As used herein, "infarction" refers to an area of necrosis that results from a lack of blood supply. As used herein, "myocardial infarction" refers to myocardial necrosis resulting from insufficient coronary blood supply. As used herein, "treatment" refers to the reduction of the congestive heart failure condition. As used herein, "congestive heart failure" refers to .... As used herein, the term "mammal" refers to any animal classified as a mammal, which includes humans, domestic and farm animals, and zoo animals, sports or pets such as dogs, horses, cats, cows, and so on. Preferably the mammal in the present is a human. As used herein, "growth hormone" or "GH" refers to growth hormone in either a native or variant sequence form, and which comes from any source whether natural, synthetic or recombinant.

Examples include human growth hormone (hGH), which is natural or recombinant GH, with a human native sequence (somatotropin or somatropin), and recombinant growth hormone (rGH), which is related to any GH or variant produced by means of recombinant DNA technology, which include somatrem, somatotropin and somatropin. In the present, mature GH of recombinant human native sequence, with or without methionine in its N-terminal part, is preferred for human use. Methionylated human growth hormone (met-hGH) produced in E. coli is further preferred, for example, by the process described in U.S. Patent No. 4,755,465 issued July 5, 1988 and Goeddel et al., Nature 282: 544 (1979). Met-hGH, which is sold under the trademark Protropin "by Genentech, Inc., is identical to the natural polypeptide, with the exception of the presence of a methionine residue in the N-terminal part.This added amino acid is a result of the bacterial protein synthesis process, recombinant hGH available from Genentech is also preferred, Inc., under the trade name Nutropin. "The latter hGH lacks this methionine residue and has an amino acid sequence identical to that of the natural hormone, see Gray et al., Biotechnology 2: 161 (1984). as hGH have equivalent pharmacokinetic potencies and values Moore et al., Endocrinology, 122: 2920-2926 (1988) Another suitable hGH candidate is a variant of hGH which is a placental form of GH with pure somatogenic activity and no lactogenic activity , as described in U.S. Patent No. 4,670,393, issued June 2, 1987. GH variants are also included as described in WO 90/04788 published May 3, 1990 and WO 92/09690 published January 11, 1990. June 1992 b. Modes for carrying out the invention Ccppoaicionea terapéu i can and af-? IiniBtracicaí da GH Therapeutic formulations of GH are prepared by storage by mixing GH having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers (Remington 's Pharmaceutical Sciences) supra), in the form of a lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants that include ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). For administration, GH can complex or bind to a polymer to increase its circulatory half-life. Examples of polyethylene polyols and polyoxyethylene polyols useful for this purpose include polyoxyethylene glycerol, polyethylene glycol, polyoxyethylene sorbitol, polyoxyethylene glycol or the like. The skeleton or glycerol core of polyoxyethylene glycerol is the same skeleton as is present in, for example, animals and humans in mono-, di- and triglycerides. The polymer does not need to have any particular molecular weight, but it is preferred that the molecular weight is between about 3500 and 100,000, more preferably between 5000 and 40,000. Preferably, the PEG homopolymer is unsubstituted, but may also be substituted at one end with an alkyl group. Preferably, the alkyl group is an alkyl group of 1 to 4 carbon atoms, and more preferably a methyl group. More preferably, the polymer is an unsubstituted homopolymer of PEG (mPEG), a homopolymer substituted with monomethyl PEG (mPEG) or polyoxyethylene glycerol (POG) and has a molecular weight of about 5000 to 40,000. The GH is covalently linked via one or more amino acid residues of the GH to a terminal reactive group in the polymer, based mainly on the reaction conditions, the molecular weight of the polymer, etc. The polymer with reactive group or groups is referred to herein as an activated polymer. The reactive group reacts selectively with free amino groups or other reactive groups on GH. However, it will be understood that the type and amount of the reactive group chosen, as well as the type of polymer used to obtain optimal results, will depend on the particular GH used to avoid having the reactive group that reacts with many particularly active groups on the GH. . As it is not possible to avoid this completely, it is recommended that they are generally used, from about 0.1 to 1000 moles, preferably from 2 to 200 moles of activated polymer per mole of protein, based on the concentration of protein. The final amount of activated polymer per mole of protein is a balance to maintain optimal activity, and at the same time optimize, if possible, the circulating half-life of the protein. Although the residues may be reactive amino acids on the protein, such as one or two cysteines at the N-terminal amino acid group, the reactive amino acid is lysine, which is attached to the reactive group of the activated polymer through its free epsilon amino group, or glutamic or aspartic acid, which is attached to the polymer through an amide bond. The covalent modification reaction can be carried out by an appropriate method generally used to react biologically active materials with inert polymers, preferably, at about pH 5-9, more preferably 5-9 if the groups reactive on GH are lysine groups. Generally, the process involves preparing an activated polymer (with at least one terminal hydroxyl group), preparing an active substrate from this polymer, and subsequently reacting GH with the active substance to produce the GH suitable for formulation. The above modification reaction can be carried out by various methods, which may involve one or more steps. Examples of modifying agents that can be used to produce the activated polymer in a one-step reaction include cyanuric acid chloride (2,4,6-trichloro-S-triazine) and cyanuric acid fluoride. In one embodiment, the modification reaction is carried out in two stages, wherein the polymer is first reacted with an acid anhydride such as succinic or glutaric anhydride, to form a carboxylic acid, and subsequently, the carboxylic acid is made reacting with a compound capable of reacting with the carboxylic acid to form an activated polymer with a reactive ester group that is capable of reacting with GH. Examples of such compounds include N-hydroxysuccinimide, 4-hydroxy-3-nitrobenzenesulfonic acid and the like, and preferably, N-hydroxysuccinimide or 4-hydroxy-3-nitrobenzenesulfonic acid is used. For example, the monomethyl substituted PEG can be reacted at elevated temperatures, preferably at about 100-110 ° C for four hours, with glutaric anhydride. The monomethyl PEG-glutaric acid thus produced is then reacted with N-hydroxysuccinimide in the presence of a carbodiimide reagent such as dicyclohexyl or isopropyl carbodiimide to produce the activated polymer, methoxypolyethylene glycolyl-N-succinimidyl glutarate, which can then be reacted with the GH. This method is described in detail in Abuchowski et al., Cancer Biochem. Biophys. 7: 175-186 (1984). In another example, the monomethyl substituted PEG can be reacted with glutaric anhydride followed by reaction with 4-hydroxy-3-nitrobenzenesulfonic acid (HNSA) in the presence of dicyclohexylcarbodiimide to produce the activated polymer. HNSA is described by Bhatnagar et al., Peptides: Synthesis-Structure-Function, Proceedings of the Seventh American Peptide Symposium, Rich et al. (eds.) (Pierce Chemical Co., Rockford IL, 1981), p. 97-100, and in Nitecki et al., High-Technology Route to Virus Vaccines (American Society for Microbiology: 1986) entitled "Novel Agent for Coupling Synthetic Peptides to Carriers and Its Applications" ("Novel Agents for Coupling Synthetic Peptides to Carriers and its applications "). Specific methods for producing GH conjugated to PEG include the methods described in U.S. Patent No. 4,179,337 on PEG-GH and in U.S. Patent No. 4,935,465, which discloses reversibly but covalently linked PEG to GH. Other specific methods for producing PEG-GH include the following: PEGylation (binding of a PEG molecule) with methoxypolyethylene glycol aldehyde (Me-PEG aldehyde) by reductive alkylation and purification which is carried out when adding to 2 mg / ml of GH in PBS pH 7.0, 5 mM of Me-PEG aldehyde-5000 (molecular weight, 5000 daltons) and 20 mM of NaCNBH3 and mix gently at room temperature for 3 hours. Ethanolamine is then added at 50 mM to reductively amidate the unreacted Me-PEG, remaining, the mixture is separated on an anion exchange column, FPLC Mono Q. The excess of unreacted Me-PEG does not bind to the column and then it can be separated from the mixture. Two fractions of PEGylated GH (conjugated with PEG) are obtained, with apparent molecular weights of 30K and 40K on reduced SDS-PAGE, compared to 20K which has not reacted. The GH-GHBP complex is PEGylated (conjugated to PEG) in the same manner to provide a 150K derivative by gel filtration. PEGylation with N-hydroxysuccinimidyl PEG (NHS-PEG) and purification is carried out by adding NH-PEG to a 5-fold molar excess relative to the total lysine concentration of GH to a solution containing 2 mg / ml of GH in 50 mM sodium borate buffer at pH 8.5 0 PBS at pH 7, and mix at room temperature during 1 hour. The products are separated in a column for dimensioning Superóse 12 and / or Mono Q of FPLC. PEGylated GH varies in size based on the pH of the reaction, from about 300 K for the reaction carried out at pH 8.5, to 40 K for pH 7.0, as measured by gel filtration. The GH-GHBP complex is also PEGylated (coupled) with PEG in the same manner, with a resulting molecular weight of 400 to 600 Kd from the gel filtration. PEGylation of GH cysteine mutants with PEG-maleimide is carried out by preparing a single GH cysteine mutant by site-directed mutagenesis, then secreting E. coli strain 16C9 (W3110? TanA phoA .E15? { argF-lac) 169 deoC2 that does not produce the deoC protein), and purify it in an anion exchange column. Strain 16C9 is genetically constructed by transferring the deoC allele? from strain CGSC # 6092 (No. 6092, available from the E. coli Genetic Stock Center, New Haven, Conn. and described in Mark et al., Molec. Gen. Genet .. 155: 145-152 (1977), with the trxAl recAl genotype HvE720:: tn5 metE70 deoC2 lacZ53 rha5 malB45 rpsLldl) in a strain designated 7C1. Strain 7C1 [with genotype W3110? TanA phoA? E15? (ArgF-lac) 169] is constructed in several stages using techniques involving transductions with phage PlKc, derived from Pl (J. Miller, Experiments in Molecular Genetics [Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory, 1972]), and transposon genetics (Kleckner et al., J. Mol.

Biol .. _J__: 125-159 [1977]). E. coli K12 W3110, which is a strain K12 that is F-, 8- (the wild type is F +, 8+) (Bachmann, Bact. Rev .. 3__: 525-557 [1972]), is used as the initial host. First, the tonA gene (fhuA) (Kadner et al., J. Bact .. ____: 256-264 [1980]; Bachmann, Microbiol. Rev. 4: 180-230 [1983]) is deleted by the insertion and subsequent imprecise cut of the TnlO transposon in the tonA gene. In the first stage of this procedure, E. coli W3110 is transduced with? :: Tnl0 to generate an accumulated TnlO hop of E. coli W3110 (Kcleckner et al., J. Mol. Biol., Supra). The accumulated E. coli W3110:: Tnl0 hop is grown in L broth at 37 ° C to a cell density of approximately 1 x 109 / ml- A total of 0.5 ml of the culture is subjected to centrifugation and the pellet is resuspended in 0. 2 ml of 8phi80 lysate containing 7.0 x 109 pfu. The phage is allowed to be absorbed for 30 minutes at 37 ° C.

The suspension is subsequently dispersed in EMB plates supplemented with tetracycline (15 μg / ml). After overnight incubation at 37 ° C, the colonies are harvested in 3 ml of L broth, grown overnight at 37 ° C, washed twice, and resuspended in L. broth. produces a lysate of bacteriophage Plkc in this culture (Miller, JH, Experiments in Molecular Biology. above, page 304). E. coli AT982 (No. 4546, JE ?, coli Genetic Stock Center, New Haven, Conn.) Undergoes transduction for tetracycline resistance by this Plkc lysate. The transductants (cells subjected to transduction) are selected in L broth plates supplemented with tetracycline (15 μg / ml) and 40 μg / ml diaminopimelic acid (dap). The resulting transductants are examined to determine their resistance to tetracycline and the regeneration of the dap gene. { dap *, tet?). The transducers dap *, tet? Later they are tested to determine their resistance to? PhißO. Subsequently, the Plkc lysates are carried out in several strains, dap *, tet? 8phi80. The lysates were used to transduce E. coli W3110 with resistance to tetracycline. The transductants are examined and selected to determine their? Phi? 0 resistance. The tetracycline sensitive isolates of the W3110 tonA:: TN10-? PhißOR transductants are selected. Maloy and Nunn,. Bacteriol .. 1___: 1110 (1981).

These isolates are verified to determine their? PhißOR resistance and their sensitivity to tetracycline after the purification of single colonies. DNA is isolated from several mutants sensitive to tetracycline and resistant to? Phi? O, and digest with Sstll. DNA digested with Sstll is characterized by the Southern staining procedure by the use of radioactive labeling and DNA? :: TnlO digested with .SstlI as a probe in order to determine if TnlO has been cut. Davis et al. , Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, New York, 1980). We show that one of the tetracycline sensitive isolates has lost two of the TnlO hybridization bands compared to the hybridization between? DNA? :: TnlO and the original strain W3110 tonA:: Tnl0? Phi80R. A third band of hybridization that has altered mobility, which indicates that a suppression caused by the imprecise cut of TnlO has occurred. The SDS gel electrophoresis of the outer membrane preparations from the strain with an inaccurate TnlO cut, show that the band is assumed to be in the TonA protein and has an altered electrophoretic mobility compared to the TonA protein of type wild. The resulting protein is non-functional as a phage receptor protein? Phi? 0. A second independent strain that has also experienced inaccurate TonlO cleavage also does not show the TonA protein in the SDS gel.

None of these strains shows reversion to tetracycline resistance or PhißO susceptibility, indicating that there is an inaccurate cut for all or part of the TnlO transposon together with the partial or complete deletion of the tanA gene. Therefore, the TonA protein (MW 78,000) is removed from an outer membrane, which makes the W3110 tonA strain resistant to several bacteriophages. Subsequently, two are transferred simultaneously deletion mutations, phoA? E15 (Sarthy et al., J. Bact. ____: 288-292 [1981]) and. { argF-lac) -169 (Schweizer et al., Mol. Gen. Genet .. .____: 293-294 [1983]), in W3110 topA by genetic linkage to a transposon of kanamycin resistance inserted into a proline biosynthetic gene fifteen . { proC:: Tn5). The transposon is eliminated by selecting a spontaneous prototrophic revertant. { pro *) on plates , agar. minimum glucose. The introduction of the paA mutation is recognized as transductants that form white colonies on minimal glucose agar plates with 0.2 mM phosphate and 20 mg / l of 5-bromo-4-chloro-3-indolyl phosphate. In the same way, the mutation). { argF-lac) 169 causes the loss of the beta-galactosidase enzyme and results in cells that form white colonies on MaConkey agar plates with 1% lactose. The result is strain 7C1.

Finally, the deoC mutation is introduced (Bachmann, supra), by eliminating aldolase, in 7C1 by means of multi-step transduction processes using the phage Plkc. Standard methods are used for transduction. First, threonine auxotropia is introduced at 7C1 to provide a means for positive selection of transduced chromosomal segments in the region of the deoC gene as follows. Plkc is grown in threonine auxotroph such as the auxotrophs described in Clare N. Berg and Douglas E. Berg, Microbiology-1981. "Bacterial Transposons", pp 107-116 (Amer. Soc. For Microbiology, Washington, DC, 1981) . The resulting lysate is used to transduce the strain 7C1 with tetracycline resistance, select the transductants in LB plates containing 25 μg / ml of tetracycline. The resulting strain, designated 14A9 (tanA ?, phoME15,?. {ArgF-lac) 169 thr:: tnlO), spontaneously reverts to prototrophy at a high frequency, so plates of fusaric acid are used (J. Bact. r 145: 1110 [1981]) to select a stable, tetracycline-sensitive threonine auxotroph, designated strain 16C4. Plkc is grown in strain CGSC # 6092, described supra.

The resulting lysate is used to transduce strain 16C4 to prototrophy, by selecting it for growth on minimal glucose agar plates. To obtain a high frequency transducer lysate of strain 2D4, the phage Plkc must be cycled for growth twice in this host. Five prototrophic transductants of strain 16C4 are isolated, purified and tested for growth on minimal thymidine agar plates. Four of the five of these isolates could not grow in thymidine and therefore received the deoC2 mutation that eliminates the synthesis of the deoC protein. One of these four isolates is saved and designated as strain 16C9 (? TonA, phoA,? E25,? { ArgF-lac) 169, deoC2). PEG-maleimide is produced by reacting monomethoxyPEG amine with sulfo-MBs in 0.1 M sodium phosphate, pH 7.5, for one hour at room temperature and exchanging the buffer to phosphate buffer, pH 6.2. Subsequently, GH is mixed with an additional free cysteine for one hour, and the final mixture is separated into a mono Q column as in GH PEGylated with Me-PEG aldehyde. Since ester linkages are chemically and physiologically labile, it is preferable to use a PEG reagent in the conjugation reaction that does not contain the ester functionality. For example, a carbamate link can be made by reacting PEG-monomethyl ether with phosgene to provide PEG-chloroformate. This reagent can then be used in the same manner as the NHS ester to functionalize the amines of the lysine side chain. In another example, the urea bond is produced by reacting the amino-PEG monomethyl ether with phosgene. This would produce a PEG-isocyanate that would react with amines of lysine. A preferred way of producing PEG-GH, which does not contain a releasable ester in the PEG reagent, is described in the following: methoxypoly is converted(ethylene glycol) to a carboxylic acid by titration with sodium naphthalene to generate the alkoxide, followed by treatment with bromomethyl acetate to form the ethyl ester, followed by hydrolysis to the corresponding carboxylic acid by treatment with sodium hydroxide and water. As reported by Bückmann et al., Macromol. Chem. 182: 1379-1384 (1981). Subsequently, the resulting carboxylic acid is converted to the PEG-N-hydroxysuccinimidyl ester suitable for acylation of GH by reaction of the resulting carboxylic acid with dicyclohexylcarbodiimide and NHS in ethyl acetate. The resulting NHS-PEG reagent is then reacted with 12 mg / ml of GH by using a 30-fold molar excess relative to GH in a sodium borate buffer, pH 8.5, at room temperature for 1 hour and applied to a column of Q Sepharose in Tris buffer and eluted with a salt gradient. It is then applied to a second column (phenyl Toyopearl) equilibrated with 0.3 M sodium citrate buffer, pH 7.8. The PEGylated GH is subsequently eluted with an inverse saline gradient, harvested and exchanged with buffer using a G25 desalting column in mannitol, glycine, and sodium phosphate buffer at pH 7.4 to obtain PEG7-GH suitably formulated . The PEGylated GH molecules and the GH-GHBP complex can be characterized by SDS-PAGE, gel filtration, NMR, tryptic mapping, liquid chromatography-mass spectrophotometry and in vitro biological assay. The degree of PEGylation is first shown adequately by SDS-PAGE and gel filtration, and subsequently analyzed by NMR, which has a specific resonance peak for the methylene hydrogens of PEG. The number of PEG groups in each molecule can be calculated from the NMR spectrum or mass spectrometry. Polyacrylamide gel electrophoresis in 10% SDS is carried out appropriately in 10 mM Tris-HCl, pH 8.0, 100 mM NaCl as an elution buffer. To demonstrate which residue is PEGylated, tryptic mapping can be performed. Therefore, the PEGylated GH is digested with trypsin in a protein / enzyme ratio of 100 to 1 in a base in mg, at 37 ° C for 4 hours in 100 mM sodium acetate, 10 mM Tris-HCl, chloride 1 mM calcium, pH 8.3, and acidified at pH < 4 to stop digestion before separation in CLAP with Nucleosil C-18 (4.6 mm X 150 mm, 5 μ, 100 A). The chromatogram is compared with that of the initial non-PEGylated material. Subsequently, each peak can be analyzed by mass spectrometry to verify the size of the fragment in the peak. The fragment or fragments that present PEG groups are often not retained in the CLAP column after injection and disappear from the chromatograph. Such disappearance of the chromatograph is an indication of PEGylation on that particular fragment which could contain at least one lysine residue. GH PEGila is subsequently tested for its ability to bind to GHBP by conventional methods. The various PEGylation methods used produce various classes of PEGylated wild type GH, with apparent molecular weights of 35K, 51K, 250K and 300K by size exclusion chromatography, which may be closer to the native hydrodynamic volume. These are designated PEGl-GH and PEG2-GH, PEG3-GH, and PEG7-GH, respectively. From the results of the tryptic mapping, both PEGl-GH and PEG2-GH have a fragment of 9 amino acids in the N-terminal part of the chromatogram and possibly PEGylated, which could be confirmed by mass spectrometry of the large molecular species that is found in the flow through the liquid chromatograph. From the molecular weight on SDS-PAGE, PEGl-GH can have a PEG group on the N-terminal amine part, and PEG2-GH can have two PEG molecules on the N-terminal amine part, which would form a tertiary amide. PEG3-GH has approximately 5 PEG groups per molecule, based on the results by NMR, and by the triptych mapping, at least five peptide fragments are lost, suggesting that they are PEGylated. The molecule PEG7-GH is considered to have 6-7 groups per molecule, based on mass spectrometry. The sites for adding the PEG groups to GH, and those which are the preferred residues for such conjugations, are methionine or phenylalanine in the N-terminal part, lysine 38, lysine 41, lysine 70, lysine 140, lysine 145, lysine 158 and lysine 168. Two lysines that appear to be non-pegylated are lysine 115 and lysine 172. The GH to be used for in vivo administration must be sterile. This is easily carried out by filtration through sterilization filtration membranes, before or after lyophilization and reconstitution. Usually, GH can be stored in lyophilized form or in solution. The therapeutic GH compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or a vial having a plug that can be pierced by a needle for hypodermic injection. The route of administration of GH is in accordance with known methods. Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial and intraperitoneal administration, or by sustained release systems as indicated below. Injection or subcutaneous and intravenous infusion is preferred. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, for example films or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919, EP 58,881), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556 [ 1983]), poly (2-hydroxyethyl methacrylate) (Langer et al., "Bicmed, Mater. Res. 15: 167-277 [1981], and Langer, Chem. Tedch., 12: 98-105 [1982] ), ethylene vinyl acetate (Langer et al., J. Bicmed, Mater. Res. 15: 167-277 [1981]) or poly-D- (-) -3-hydroxybutyric acid (EP 133,988). sustained release also include liposomally entrapped GH Liposomes containing GH are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Nati Acad. Sci. USA 77: 4030-434 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; North American patents 4,485,045 and 4, 544, 545; and EP la 102, 324. Typically, the liposomes are small (about 200-800 Angstroms) unilamellar in which the lipid content is greater than about 30 mol% cholesterol, the selected ratio is adjusted for optimal therapy with GH. An "effective amount" of GH that can be used therapeutically will be based, for example, on the route of administration and the condition of the patient. Accordingly, it will be necessary for the attending physician to dose and modify the route of administration as required to obtain the optimal therapeutic effect. Usually, the doctor will administer GH until a dosage is reached that achieves the desired effect. The progress in this therapy can be easily verified by conventional tests.

In the treatment of congestive heart failure by GH, the composition of GH will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular mammal in question, the disorder or clinical condition of the individual patient, the GH delivery site, the particular type of GH, the method of administration, the administration protocol, and others. factors known to those who practice medicine. The "therapeutically effective amount" of GH to be administered will be indicated by such considerations, and is the minimum amount necessary to reduce or treat congestive heart failure, so as to increase ventricular contraction capacity and decrease peripheral vascular resistance. , or to decrease other conditions of similar importance in patients with congestive heart failure. Preferably, such amount is below the amount that is toxic to the host or that makes the host significantly more susceptible to infections. As a general proposition, the total pharmaceutically effective amount of GH administered parenterally per dose will be in the range of about 1 μg / kg / day to 10 mg / kg / day of the patient's body weight, although, as indicated in above, this will be subject to a great extent to therapeutic discretion. More preferably, this dose is at least 0.01 mg / kg / day, and more preferably for humans between approximately 0.01 and 1 mg / kg / day. If administered continuously, GH is usually administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either for 1-4 injections per day or by continuous subcutaneous infusions, per example, by using a mini-pump. An intravenous bag solution can also be used. Preferably, in human patients, a pharmaceutically effective amount of the GH administered parenterally per dose will be in the range of about 10 to 100 micrograms per kilogram of patient body weight per day. It should be noted that physicians designing doses of both IGF-I and GH should take into account the known side effects of treatment with these hormones. However, as indicated above, these suggested amounts of GH are largely subject to therapeutic discretion. The key factor in selecting an appropriate dose and protocol is the result obtained, as indicated in the above.

EXAMPLES Use of GH / IGF-I to treat congestive heart failure Introduction The objective of this study is to examine the cardiac effects of human GH treatment in an animal model of congestive heart failure.

Mé-OdCS Male Sprague-Dawley rats (SD) are acclimated (Charles River Breeding Laboratories, Inc., 8 weeks old) to the facility for at least 1 week before surgery. The rats are fed a food prepared for rats and with water ad libitum and are caged in a room with light and temperature control.

Ligation of the coronary artery Myocardial infarction occurs by ligation of the left coronary artery as previously described.

Geenen, D.L. et al., J. Appl. Physiol. 63: 92-96 (1987); Buttrick, P. et al., Am. J. Physiol. 260: H473-H479 (1991).

/ * ~ Rats are anesthetized with sodium pentobarbital (60 mg / kg, ip), intubated by tracheotomy and ventilated with a respirator (Harvard Apparatus Model 683). After thoracotomy of the left side, left coronary artery 5 is ligated approximately 2 mm from its origin with a 7-0 silk suture. False surgery animals undergo the same procedure except that the suture is passed under the coronary artery and then removed. All rats are maintained in accordance with the "Position of the American Heart Association on Research Animal Use" ("Position of the American Heart Association Regarding the Use of Research Animals") adopted on November 11, 1984 by the American Heart Association. At 4-6 weeks after ligation, it develops myocardial infarction and heart failure in rats. In clinical patients, myocardial infarction or coronary artery disease is the most common cause of heart failure. Congestive heart failure in this model reasonably mimics congestive heart failure in most patients. human patients.

Electrocardiograms One week after surgery, 25 electrocardiograms are obtained under anesthesia with light methophane to document the development of infarcts. The rats bound in this study are grouped into subgroups according to the depth and persistence of pathological Q waves through the precordial electrodes. Buttrick, P. et al., A. J. Physiol .260: H473-H479 (1991); Kloner, R.A. et al., Am. Hart J. 106 (5): 1009-1013 (1983). This provides a general estimate of the size of the infarct and ensures that large and small infarcts are not distributed differently in the bound rats treated with GH and with a vehicle. The confirmation is made by accurate measurement of the infarct size.

GH Administration Four weeks after surgery, recombinant human GH (1 mg / kg, twice daily for 15 days) is injected subcutaneously (Genentech, Inc., South San Francisco, CA) or vehicle with saline, both in the bound rats as in rats operated in false. Previous studies have shown that this dose of human GH can produce significant anabolic effects in rats. Moore et al., Endocrinology 122: 2920-2926 (1988); R. Clark and M. Cronin, U.S. Patent No. 5,126,324 (1992). Body weight (BW) is measured twice a week during treatment. See Figure 1. GH can be administered in saline or in water as vehicles.

Catheterization After 13 days of treatment with GH or with vehicle, the rats were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneally). A catheter (PE-10 fused with PEG 50) filled with saline-heparin (50 U / ml) was implanted in the abdominal aorta through the right femoral artery for measurement of blood pressure and heart rate. A second catheter (PE 50) was implanted in the right atrium through the right jugular vein for measurement of right atrial pressure and for saline injection. For the measurement of left ventricular pressure and contraction capacity (dP / dt), a third (PE 50) was implanted in. the left ventricle through the right carotid artery. For measurement of cardiac output by a thermodilution method, a thermistor catheter (Lyons Medical Instrument Co., Sylmar, CA) was inserted into the aortic root. The catheters are exteriorized at the back of the neck with the help of a stainless steel wire that forms a subcutaneous tunnel and then is fixed. After the implantation of the catheters, all rats are housed individually.

Hßnocynamic measurements One day after catheterization, the thermistor catheter was processed in a microcomputer system (Lyons Medical Instruments Co.) for the determination of cardiac output, and the other three catheters were connected to a pressure transducer model CP-10 (Century Technology Company, Inglewood, CA, USA) coupled to a Grass polygraph Model 7 (Grass Instruments, Quincy, MA, USA). Mean arterial pressure (MAP), systolic blood pressure were measured (SAP), heart rate (HR), right atrial pressure (RAP), left ventricular systolic pressure (LVSP), left ventricular mean pressure (LVMP), left ventricular end disodic pressure (LVEDP) and maximum left ventricular difference (dP / dt) in conscious rats and without tensions. For the measurement of cardiac output, isotonic saline solution (0.1 ml) is injected at room temperature, as a bolus by means of the jugular vein catheter. The thermodilution curve is monitored by multiple trace records VR-16 (Honeywell Co., NY) and cardiac output (CO) is obtained digitally by the microcomputer. The stroke volume (SV) = CO / HR; as well as the cardiac index (CI) = CO / BW; and systemic vascular resistance (SVR) = MAP / CI. After measuring these hemodynamic parameters, 1 ml of blood is collected through the arterial catheter. The serum is separated and stored at -70 ° C for measurements of GH and IGF-I. At the conclusion of the experiments, the rats are anesthetized with sodium pentobarbital (60 mg / kg) and the heart is suppressed in diastole with an intraarterial injection of KCl (1 M). The heart was removed, and the atrium and large vessels were cut away from the ventricle. The ventricle was weighed and fixed in 10% buffered formalin. See figure 1, bottom part. All the experimental procedures were approved by the Institutional Committee of Care and Use of Animals of Genentech before the start of the study.

Infarct size measurements The right ventricular free wall of the left ventricle was dissected. The left ventricle was cut into four cross sections from the apex or vertex to the base. Five micron sections were cut, mounted and stained with Massons trichrome stain and mounted. The endocardial and epicardial circumferences of the infarcted and non-infarcted left ventricle were determined with a Digital Image Analyzer planimeter. The infarcted circumference and the left ventricular circumference of the four sections were added separately for each of the epicardial and endocardial surfaces, and the sums were expressed as the ratio of the infarcted circumference to the left ventricular circumference for each surface. These two proportions were then averaged and expressed as a percentage for infarct size.

Hormone assays Serum human GH was measured by sensitive ELISA. Celniker, A., J. Clin. Endocrinol Metab.68: 469-476 (1989); Greenen, D.L. et al., J. Appl. Physiol. 63: 92-96 (1987). This assay does not detect rat GH. Total serum IGF-I is measured after extraction with acid-ethanol by radioimmunoassay eg RIA described by Furlanetto et al., J. Clin. Invest.60: 648-657 (1977); Bala and Bhaumick, J. Clin. Endocrinol and Metab. 49: 770-777 (1979); Zapf et al., J. "Clin. Invest. 68: 1321-1330, (1981), Hall et al.,". Clin. Endo. Metab. 48: 271-278 (1979); EP 292,656., By using human IGF-I (Genentech M3-RD1) as the standard, and antiserum against rabbit polyclonal IGF-I. The acceptable range was 1.25-40 ng / ml, whereas the intra-assay and inter-assay variabilities are 5-9% and 6-15%, respectively. See figure 2.

Statistic analysis The results are expressed as mean ± MEE. Two-way and one-way analysis of variance was performed to determine differences in parameters between the groups. Subsequently, the significant differences were subjected to post hoc analysis by using the Newman-Keuls method. P < 0.05.

The mean BW before the GH treatment or the vehicle was not different between the experimental groups (Figure la). There was a significantly higher increase in BW after GH treatment for the both sham-treated and ligated rats (Figure IB). The LCA ligation causes a significant increase in the proportion of ventricular weight (VW) with respect to BW, while the treatment with GH does not alter this proportion significantly (Figure 1C).

Treatment with GH significantly increases the serum concentrations of human GH and IGF-1 in both groups of false-linked ligated rats (Figures 2A and 2b). The increase induced by GH in the serum concentrations of human GH and IGF-1 is not significantly different between the rats operated in false and those bound. The size of the infarct in bound rats is not different between the group treated with vehicle (33.2 ± 2.2% of the left ventricle) and the group treated with GH ((31.4 ± 2.6% of the left ventricle.) The ligation with LCA results in significant decreases MAP in rats treated with vehicle, but not in rats treated with GH (Figure 3A) Treatment with GH significantly decreases MAP in rats operated in false, but not in bound rats. associated with a significant reduction in SAP in rats both treated in vehicle and treated with GH (Figure 3B), however, the decrease in SAP induced by ligation is significantly higher in rats treated in vehicle than in rats treated with GH. GH administration does not alter SAP significantly in the false-operated rats, neither the LCA ligation nor the GH treatment significantly alter the HR (Figure 3C).

LCA ligation significantly decreases left ventricular dP / dt and LVSP in vehicle-treated rats, but not in the rats treated with GH (Figure 4A and B). Treatment with GH increased dP / dt and LVSP in the bound rats, but not in the rats operated in false. In the animals treated with vehicle, LVEDP rises significantly in the bound gr compared to the controls operated in false (Figure 4C). However, in animals treated with GH there is no significant difference in LVEDP between the bound grand the false operator. The administration of GH decreases LVEDP significantly in the bound rats, but not in the rats operated in false. LCA ligation results in significant reductions in Cl and SVI in rats treated with vehicle, but not in rats treated with GH (Figure 5A and B). The administration of GH significantly increases Cl and SVI in the bound rats, but not in the rats operated in false. There is no significant difference in SVR between the bound and the false-operated rats, whereas the treatment with GH significantly decreases the SVR in bound rats and tends to decrease SVR in the rats operated in false (Figure 5C).

In the current study, 6 weeks after the left coronary artery (LCA) ligation, vehicle-treated rats showed significant decreases in LVSP, dP / dt, Cl and SVI, and increases in LVEDP, compared to controls operated on false. These results indicate that congestive heart failure occurs in this animal model of myocardial infarction mainly due to the decrease in ventricular contraction capacity. Treatment with GH at a dose of 2 mg / kg / day for 15 days significantly increases LVSP, dP / dt, CO and SVI, and reduces LVEDP and SVR in rats bound by LCA. This result demonstrates that the administration of GH improves cardiac function by increasing ventricular contraction capacity and decreasing peripheral vascular resistance in congestive heart failure. However, in rats operated in false, the administration of GH at this dose does not alter so. significant cardiac function except by slightly decreasing blood pressure and peripheral vascular resistance. It can reasonably be expected that the rat data, hereby, can be extrapolated to horses, cows, humans and other mammals, making the appropriate corrections for the mammalian body weight according to recognized veterinary and clinical procedures.

By using standard protocols and procedures, a veterinarian or a physician will be able to adjust the doses, procedure and mode of administration of GH and its variants in order to obtain maximum effects in the desired mammal in question. Likewise, it is considered that humans respond in this way. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (9)

RE? P- ICACIQNES

1. A method for treating a mammal that shows congestive heart failure, the method is characterized in that it comprises administering to the mammal an effective amount of growth hormone.

2. The method according to claim 1, characterized in that the growth hormone is human growth hormone.

3. The method according to claim 1, characterized in that the mammal is a human.

4. The method according to claim 3, characterized in that the effective amount is in the range of 10-100 micrograms per kilogram of body weight per day.

5. The method according to claim 3, characterized in that the administration is subcutaneous or intravenous.

6. The method according to claim 1, characterized in that congestive heart failure results from myocardial infarction.

7. The use of growth hormone in the preparation of a drug to treat a mammal that shows congestive heart failure.

8. The use according to claim 7, characterized in that the growth hormone is human growth hormone.

9. The use according to claim 7 or claim 8, characterized in that the medicament comprises growth hormone in an amount between 10-100 micrograms per kilogram of body weight per day.