CZ321699A3 - Use of human alpha1-acid glycoprotein for preparing pharmaceutical preparation - Google Patents

Abstract

The present invention relates to the use of human cti-acid glycoprotein (AGP) for the manufacture of a pharmaceutical composition for the treatment of disorders blood flow or rotocirculation of non-inflammatory type.

Description

Use of a human α-acid glycoprotein for the manufacture of a pharmaceutical composition

Technical field

The present invention relates to a novel use of orosumocoid in the medical field.

BACKGROUND OF THE INVENTION α-Acid glycoprotein (AGP), also called orosumocoid, is a plasma derived substance that has a molecular weight of 40,000 Daltons and a carbohydrate fraction of between 30 and 50%. AGP consists of a single polypeptide chain of 183 amino acids and has two disulfide bridges. It further comprises five carbohydrate chains, all of which are located in the first half of the peptide chain. These carbohydrate groups consist of approximately 14% of neutral hexos, 14% of hexosamines, 11% of sialic group and 1% of fructose. Depending on the source of the AGP preparation, the recovery method, or the characterization method, AGP occurs in different forms due to differences in the polypeptide chain as well as differences in the carbohydrate chain.

The properties of the biological functions of orosumocoid are described in review articles by Schmid (The Plasma Proteins Structure Function and Genetic Control, Vol. 1 (1975), Academic Press, Ed. Frank A. Putnam, second edition, pages 183228) and Kremer et al. (Pharmacological Reviews 40 (1988), pages 1-47).

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In the health sector, AGP has been considered to be an essential carrier substance for predominantly basic plasma drugs (see Kremer et al).

Furthermore, the positive effect of orosumocoid in inflammatory reactions was demonstrated. Thus, Denko et al. (Agents and Actions 15, 5/6 (1984), pages 539-540) discloses the anti-inflammatory effect of AGP in inflammation associated with uric acid and its salts in urine in rats. Libert et al. (J. Exp. Med. 180 (1984), pages 1571-1575) demonstrate that there is a similar indication in preventing septic shock in association with the action of TNF-α or lipopolysaccharides.

To improve perfusion disorders, especially microcirculation as well as reperfusion injuries, either vasoactive substances or special blood factors that affect hemostasis or fibrinolysis, especially anticoagulants or thrombolytically active factors, have been administered, or appropriate volume replacement is performed.

To date, volume replacement has been performed to treat hypovolemic shock conditions that occur independently of inflammatory reactions, using albumin solutions usually.

It is an object of the present invention to provide new medical indications for orosumocoid.

SUMMARY OF THE INVENTION

Surprisingly, it has been found that orosumocoid is suitable for the treatment of blood circulation or non-inflammatory-type microcirculation. It can therefore be used to improve perfusion disorders, especially microcirculation disorders, as well as perfusion disorders, and in particular in shock conditions to better supply vital organs such as the brain, lungs, heart, liver and kidney.

These indications are of general non-inflammatory type, that is, disorders are indicated after they do not occur in connection with systemic inflammatory response syndrome (SIRS). For a definition of SIRS, see Critical Care Medicine 20 (6), pages 864-874 (1992). . In inflammation, cells and fabrics are directly damaged, and subsequently the permeability of blood vessels and blood circulation is impaired. However, when using the AGP of the invention for the manufacture of a pharmaceutical composition for the treatment of blood circulation or microcirculation disorders, these disorders are non-inflammatory species and are thus due to other causes. These causes include altered pressure ratios, particularly in connection with decreased intravasal volumes. The blood flow disorders caused thereby lead to hypovolumic shock in untreated cases. The underlying mechanisms include acute haemorrhage, excessive fluid loss such as fractures, diarrhea, extreme sweating, dehydration, excessive brain failure, peritonitis, pancreatitis, ischemia in the splanchnic region, constipation of the intestines, gangrene, blunt injuries, damage to large muscle groups or burns.

These conditions have so far been usually treated with dextran solutions, hydroxyethyl starch, reingerlactate or albumin solutions. On the one hand, these substances do not exhibit any anti-inflammatory properties, and it was therefore surprising that orosumocoid, which was used to treat inflammation, assumed these functions in the indications according to the invention.

The orosumocoid of the invention can also be used in relative hypovolemia. This occurs when the absolute blood volume is not reduced but the organ supply is inadequate. This may be due to vasodilation changes, which may be of neurogenic, metabolic, toxic or humoral origin. Another reason is the increased permeability of the blood vessels, possibly an anaphylactic species or due to various snake venoms.

Pumping failure due to acute myocardial infarction, myocarditis or severely decreased volumetric performance, acute valve insufficiency, myocardial rupture, septal perforation, arrhythmias such as bradycardia, tachycardia or fibrillation, possibly by physical cardiac compression or physical cardiac compression may also be a cause of hypovolemic shock. thrombi or embolism.

Accordingly, the present invention also relates to the use of AGP for the manufacture of a composition for the treatment of hemorrhagic and / or hypovolemic shock and for the stabilization of intravasal volume, especially in acute bleeding, excessive fluid loss or vasodilation.

The AGP can also be used according to the invention to prevent reperfusion injury as a result of stroke, in particular to reduce cerebral edema. The reperfusion damage occurs primarily after removal of the obstruction of the flow, for example a vascular occlusion based on deposits or islets of blood clots. These damages are particularly observed as a result of stroke attacks, producing brain edema and neurological outages. Tissue damage in transplanted organs, which may result from re-induced perfusion, is also counted as a reperfusion injury.

Other possible microcirculation disorders that can be treated with AGP according to the invention are microcirculation disorders in vital organs, especially in the kidneys, which may potentially immediately induce proteinuria.

However, disorders of microcirculation in organs may also be conditioned by edema. Therefore, the present invention also relates to the use of orosumocoid in the manufacture of a composition for preventing or treating edema.

The method for producing AGP is known (for example as disclosed in WO 95/07703). The human AGP is preferably a human plasma or a plasma fraction, for example a COHN fraction, such as COHN IV or COHN V, as the source of human AGP.

The dosage employed will depend upon the indication and the severity of the patient's condition, for example, the rate of blood loss. Generally, a single dose in the range of 70 mg / kg body weight to 5 g / kg is used, with a range of 100 to 700 mg / kg being particularly preferred.

Administration according to the invention can be carried out in all ways, i.v., s.c., i, m and local administration being preferred.

Preferably, the pharmaceutical composition used comprises at least 50% orosumocoid, preferably more than 70%, especially more than 90%, based on the total protein. As a further component, the pharmaceutical composition may further comprise albumin A-.alpha. (AT2) .alpha.

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Advantageously, a stabilizer, in particular sodium caprylate and optionally surfactants, is added to the pharmaceutical composition before use in order to increase the storage and heat treatment stability.

It is also expedient to treat a pharmaceutical composition for inactivating or attenuating viruses, in particular by at least one physical treatment, such as heat treatment and / or filtration. A variety of physical, chemical or physicochemical methods are known for virus inactivation, such as heat treatment, for example according to EP 0 159 311 A or EP 0 637 451 A, hydrolase treatment according to EP 0 247 998 A or radiation treatment or treatment with organic solvents and / or surfactants, for example according to EP 0 131 740 A. Further suitable steps for virus inactivation in the preparation of the compositions according to the invention are described in E 0 506 651 or in WO 94/13329 A.

In certain indications, particularly in a hypovolaemic or neurogenic shock, administration of orosumocoid is preferably combined with administration of a vasoactive substance (constricting or dilating), which may be administered either together or in parallel.

Furthermore, the present invention according to a further aspect relates to an infusion formulation for the treatment of shock conditions comprising AGP and a vasoactive substance as active ingredients.

According to the invention, this infusion formulation is supplied in the form of a kit comprising

AGP in the pharmaceutical formulation and vasoactive substance, optionally in separate reservoirs7 • ·· ·· • 9 9 9 9 9 9

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The use of the AGP according to the invention can be carried out tactically, but preferably therapeutically.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be explained in more detail by the following examples and figures, which are not to be limited thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The pictures show:

Figure 1 to 5 results of hemorrhagic shock treatment in rat model, figures 6 to 8 results in defense against cerebral edema in Strokemodel rats, and Figure 9 results of proteinuria treatment in rat model.

In FIG

A: operated animals with 200 mg orosumocoid / kg / 20 ml (i.v.) with 5 ml blood perfusion n = 11

B: operated animals with 20 ml 0.9% NaCl / kg (i.v.) with 5 ml blood perfusion n = 8

C: Seemingly operated animals with 20 ml 0.9% NaCl / kg (i.v.) n '8

A: B p <0.05

A: C n.

B: C p <0.01 · 4 · 4 · 4 · 4 · 4 · 4 · 4

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In FIG

A: operated animals with 50 mg orosumocoid / kg / 20 ml (i.v.) with 5 ml blood perfusion n = 9

B: operated animals with 20 ml 0.9% NaCl / kg (i.v.) with 5 ml blood perfusion n = 10

C: Seemingly operated animals with 20 ml 0.9% NaCl / kg (i.v.) n = 11

n.

p < 0.05 p < 0.01

In FIG

A: operated animals with 200 mg orosumocoid / kg / 20 ml (i.v. n = 18 minutes after ischemia: operated animals with 20 ml 0.9% NaCl / kg (i.v.) 30 minutes after ischemia

C: Seemingly operated animals with 20 ml 0.9% NaCl / kg (i.v.) 60 minutes after O.P. - onset n = 17 p <0.01 n.

p <0.01 »

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DETAILED DESCRIPTION OF THE INVENTION

Example 1

Treatment of hemorrhagic shock in a rat model (at the time, in the applicant's opinion, the best way to carry out the invention)

This experiment was performed in an analogous manner to Vang and Chaudry (J. Surg. Res. 50, (1991), pages 163-169). The rats did not receive food overnight but had free access to water. To introduce anesthesia to animals i.m. injects 60 mg / kg of pentobarbital. Narcosis is maintained every 1.5 hours with 5 mg of pentobarbital s.c. per animal. Trachea is cannulated for emergency breathing. The polyethylene catheter is inserted into the left jugular vein for infusion (volume replacement) and injection. The second catheter is inserted through the right jugular vein into the right anterior chamber to inject cold isotonic sodium chloride solution (2020 ° C, thermodilution method). A thermoelement is inserted through the right carotid artery to measure blood temperature. Both femoral arteries are cannulated, one for blood pressure determination and the other for blood collection. Body temperature is maintained at 36.5 ° C throughout the experiment using a rectal thermometer connected to an infrared lamp. To prevent trauma from bleeding, a 5 cm laparotomy is performed in the Linea alba after depilation using an electrocauter. This section is then closed in layers.

Inject 1 IU heparin / g. Subsequently, by taking blood from the femoral artery within 10 minutes, the mean arterial blood pressure is reduced to 40 mm Hg. Blood pressure is maintained at 40 mm Hg either by further blood collection or by injecting Ringer's solution in a total volume not exceeding 40%> 9 9 9

999 999 blood collected for a maximum of 80 minutes. After these 80 minutes or earlier (if the blood pressure can no longer be maintained above 40 mm Hg), a volume substitution is started, replacing three times the volume of total blood loss with Ringer's solution within 60 minutes. After volume substitution, the observation period is 4 hours. Surviving animals are killed by an overdose of pentobarbital i.v.

Mean arterial blood pressure is continuously recorded by means of a polygraph using an electromechanical pressure transducer. Heart rate is continuously recorded based on pulse waves. The minute cardiac volume is determined by the thermodilution method, with 200 μΐ of cold common salt solution injected into the right anterior chamber. By measuring the blood temperature in the aortic arch, a thermodilution curve is integrated with Cardiomax II (model 85, Columbus Instruments) and the cardiac minute volume in ml / minute is reported. The percussion volume and total peripheral vascular resistance are calculated by dividing the cardiac output by the heart rate, or by dividing the blood pressure by the cardiac output by a minute, multiplying this ratio by 10 (mm Hg. Ml). min 10). Initial values are given as natural values. All other values are given as a percentage of the given default (delta%). The mean ± standard deviation is also calculated. The significance of the differences between baseline and all other values is verified by paired observation t-test. A two-tailed t-test was used to compare between groups.

It appears that in all animals (n = 30), the amount of blood collected is 7.0 ± 2.9 to reduce mean arterial blood pressure to 40 mm Hg. Blood pressure is maintained at this low level for 77.8 ± 1.5 minutes. Blood drop · ♦

• 9 9 9 9 9 «« pressure is accompanied by a decrease in cardiac output, beat volume, and any peripheral vascular resistance. Heart rate decreases in all three groups, with an initial increase in the AGP treated group.

Volumetric substitution in control animals (n = 13) with Ringer's solution i.v. (volumes: three times the amount of blood lost) is unable to regain mean atrial blood pressure, which decreases significantly over the entire observation period (range -50.5 ± 2.3% to -63.6 ± 10.3%). An insignificant decrease in heart rate is also observed, which is significant 180 to 240 minutes after volume replacement (maximum: -29.9 ± 8.8% at 240 minutes). The minute cardiac volume resets back to baseline immediately after the volume replacement, but decreases significantly after 30 to 240 minutes (range -25.9 ± 5.5% to - 51.4 ± 7.4%). The same time course is observed for the impact volume (drop area: -26.7 ± 8.8% to -31.1 ± 9.9%). Total peripheral vascular resistance decreases during the observation period (range -19.1 ± 18.2% to 39.7 ± 9.9%), with significant differences 30 to 120 minutes after resuscitation. Three animals died 150 minutes after volume replacement and were not included in the evaluation. Three more animals died after 180 minutes.

Two additional groups were treated using AGP or a placebo formulation instead of Ringer's solution. AGP solution (200 mg / kg) purified from human plasma COHN fraction by precipitation and further pasteurization at 60 ° C for 10 hours and an analogous amount of placebo formulation (human albumin albumin solution, IMMUNO, orosomucoid separation) is diluted with Ringer's solution to triple the amount of individual blood loss.

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AGP is tested on fourteen animals; one rat died during treatment (volume replacement), three rats died less than 150 minutes after treatment and one animal died 180 minutes after volume replacement. The placebo formulation is administered to eighteen animals, four of which died during treatment, four died within 150 minutes of treatment, four animals died more than 180 minutes after volume replacement. Rats that died within 150 minutes after treatment were not taken into account in the evaluation.

Comparing the results with Ringer's solution to those with the placebo formulation suggests that the same or less mean arterial blood pressure, heart rate, and total peripheral vascular resistance are achieved by treatment with placebo formulations. The cardiac minute volume and strike volume values are the same or higher in the placebo formulation group as in the Ringer's solution group.

In animals treated with AGP, blood pressure rises initially and then decreases gradually (see Figure 1).

However, it was not possible to achieve a complete return of blood pressure to baseline. All values over the observation period are lower than baseline (p ≥ 0.001). No change in heart rate from baseline (p> 0.05) was observed with respect to heart rate (Figure 2). Cardiac minute volume (Figure 3) is higher than baseline immediately after volume replacement (p <0.01), restored to baseline (+ 30 to + 90 minutes; p> 0.05) and finally falls below baseline (p <0.05 or <

0.01).

A similar situation develops for the impact volume (Figure 4) except that the values during the first 120 minutes are not • · · f>

Statistically different from baseline. The overall resistance of the peripheral vascular system (Figure 5) is lower than baseline during all observation periods; however, significance is not achieved at +30, +90 and + 120 minutes.

Figures 1 to 5 show comparisons between animals treated with AGP and a placebo formulation. Mean arterial blood pressure is significantly higher at all measurement points after volume substitution in the AGP-treated group (Figure 1). The heart rate is the same or significantly higher in the AGP group, but the differences are very small (Figure 2). The cardiac minute volume is significantly higher in the AGP treated group, except for the measured point value 240 minutes after volume replacement (Figure 3). The impact volume is significantly higher after AGP treatment at 30 to 150 minutes after volume replacement (Figure 4). Total peripheral vascular resistance is increased in the AGP group compared to the placebo formulation 60 to 120 minutes after infusion (Figure 5).

These experiments show the advantage of AGP treatment over a placebo formulation (containing the same amount of albumin-free protein that does not contain AGP) or Ringer's solution. It can be concluded that AGP can maintain the perfusion of vital organs in hypovolemic shock.

Example 2

Prevent brain edema in Strokemodel rats

Global cerebral ischemia (Stroke) is achieved by clamping both carotid arteries and collecting 5 ml of blood. After 30 minutes of ischemia, the carotid arteries reopen and the collected blood is infused again. 23.5 hours later the animals are sacrificed and the water content of both is determined. brain halves.

In preliminary experiments it was found that orosumocoid at 600 mg / kg i.v. is able to stabilize cerebral edema formation after global cerebral ischemia. The buffer formulation remains free of this effect. In the present example, the effect of dose and duration of action was found for the effect causing edema prevention by orosumocoid.

Orosumocoid, also used in Example 1, was tested at 200 mg / kg i.v. at the same time in blood perfusion in rats. The results are shown in Figure 6. It appears that the apparently operated animals (C, n = 12) have no cerebral edema, whereas ischemic animals treated with saline solution show massive brain edema (Β, n = 8). Ischemic animals that were treated with orosumocoid (A, n = 11) behave again as seemingly operated animals.

When the dose of orosumocoid was reduced to 50 mg / kg i.v. no protective effect was found (see column A in Fig. 7), thus demonstrating the dose-response effect.

Based on the therapeutic situation in humans, it was interesting to test whether orosumocoid can still be effective after the stroke. Therefore, the above dose was considered effective at 200 mg / kg i.v. given 30 minutes after the end of ischemia. As shown in Fig. 8 (column A), the orosumocoid is fully effective in this situation.

The protective effect of orosumocoid against cerebral edema resulting from stroke is thus demonstrated in animal experiments.

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Example 3

Proteinuria treatment in rat model

Rats are treated on day 0 with 100 mg / kg Puromycin aminonucleoside i.p. Control samples receive isotonic saline solution in the same manner (negative control).

Urine is collected in the metabolism cages in 24 hours for protein determination.

Puromycin-treated animals receive 200 mg / kg orosumocoid i.v. on day 6, 7, 8 and 9 of the experiment. or analogously isotonic sodium chloride solution (positive control).

On day 10, animals are weighed and sacrificed by cardiac puncture to obtain plasma. The weight of the kidney moisture is determined and creatinine and plasma urea are measured.

The following parameters are measured: total protein in urine (mg / 24 h), plasma creatinine (mg / dl), blood urea (mg / dl) and renal index (kidney weight in% body weight).

Fig. 9 shows the proteinuria values of the first round of experiments: animals that were treated with isotonic sodium chloride solution on day 0 show slight physiological proteinuria (solid rouges). In animals treated with Puromycin, the urinary protein content increased from day 3.

In animals that received isotonic sodium chloride solution on days 6-9, the total protein content was 500-600 mg / 24 h (open triangle). In animals treated with orosumocoid on days 6 to 9, protein secretion decreased to a control value (solid square).

Claims (19)

PATENT CLAIMS Use of a human α-acid glycoprotein for the manufacture of a pharmaceutical composition for the treatment of non-inflammatory type blood circulation or microcirculation. Use according to claim 1, characterized in that the composition is suitable for the treatment of hemorrhagic and / or hypovolemic shock. Use according to claim 2, characterized in that the composition is suitable for stabilizing intravasal volume, in particular in acute bleeding, excessive fluid loss or vasodilation. Use according to claim 1, characterized in that the composition is suitable for preventing reperfusion damages that occur in connection with reduced perfusion. Use according to claim 4, characterized in that the preparation is suitable for preventing reperfusion injury resulting from stroke attack, in particular for reducing cerebral edema. Use according to claim 1, characterized in that the preparation is suitable for the treatment of disorders of microcirculation in organs, in particular in the kidney. 4 · 4 4 4 «4 4 4 4 • 44 4 4 44 4 4 · 4 4 •« 4444 4444 4,444 · 444 4,444 444 44443 4 4 4 17 ............. Use according to claim 6, characterized in that the preparation is suitable for the treatment of proteinuria. Use according to claim 6, characterized in that the preparation is suitable for preventing eventually edema. Use according to any one of claims 1 to 8, characterized in that the pharmaceutical preparation is produced as a storage-stable infusion solution. Use according to any one of claims 1 to 9, characterized in that the pharmaceutical preparation is produced as a lyophilisate. Use according to any one of claims 1 to 10, characterized in that the pharmaceutical preparation is administered in a dose in the range of 70 mg / kg to 5 g / kg, in particular in the range of 100 to 700 mg / kg. Use according to any one of claims 1 to 11, characterized in that the pharmaceutical preparation contains at least 50% of the? -Acid glycoprotein, in particular more than 70%, preferably more than 90%, based on the total protein. Use according to any one of claims 1 to 12, characterized in that the pharmaceutical preparation further comprises albumin. • 9 * · 9 9 9 9 0 · 99 09 9099 0 · 9 0 99 0 090 9 0 999 9 9 9 9 9 9 909 9 ··· ··· 09999 1 · · 00090 09 09 99 · Use according to one of Claims 1 to 13, characterized in that the pharmaceutical preparation contains a stabilizer, in particular sodium caprylate. Use according to one of claims 1 to 14, characterized in that the pharmaceutical preparation is treated for inactivating or attenuating viruses, in particular by at least one physical treatment, such as heat treatment and / or filtration. Use according to claim 2, characterized in that the treatment comprises administration of a vasoactive substance, in particular catecholamine. Use according to claim 16, characterized in that the vasoactive substance is administered together or in parallel. An infusion preparation for the treatment of shock conditions comprising as an active ingredient an α-acid glycoprotein and a vasoactive agent. A shock treatment kit, comprising a) α-acid glycoprotein in the pharmaceutical preparation; and (b) a vasoactive substance.