DE2617493B2 - Use of haptoglobin or hemoglobin-haptoglobin complexes in the prophylaxis and therapy of cerebral vasospasms - Google Patents

Description

B. The arterial strip is suspended ^{at 37 °} C. in a plastic chamber which contains artificial CSF solution saturated with oxygen. The reagent is added to this chamber and mixed using a magnetic stirrer.

Fig. 2

Typical response of the basilar artery to
aged serum (0,1,4 and 7 days at 37 ° C)

A. The varoactivity of serum decreases with increasing Aging quickly. Samples incubated for 4 days are essentially non-vasoactive. the The most pronounced contraction is caused by fresh serum. It is almost the same as the contraction when 5 μg / ml serotonin was administered.

B. Methysergide (serotonin antagonist) reduces the contraction caused by serum completely prevented. The antagonist is added to the bath 3 minutes before the agonist.

Fig. 3

Typical response of the basilar artery to the
Supernatant of a blood-CSF mixture

Fig. 4

15th

25th

A. The vasoactivity of a blood-CSF mixture increases progressively with age to. The contraction caused by a sample incubated for 7 days is most pronounced and almost corresponds to the contraction caused by serotonin.

B. The contraction produced by the supernatant of an aged blood-CSF mixture not antagonistically prevented by methysergide The antagonist is the bath 3 minutes before the Agonists added.

40

45

Contraction of the basilar artery by the
Supernatant from an aged blood-CSF mixture

A. A particularly distinctive contraction occurs. A tonic tension is the phasic Superimposed contraction. The duration is generally several hours.

B. The contraction cannot be achieved simply by washing the arterial strip with artificial Allow the CSF solution to subside.

C. The contraction is markedly exacerbated by the hypoxic state caused by interruption the continuous supply of oxygen to the bath.

With renewed saturation with oxygen, the previous state quickly occurs again.

F i g. 5

A. Column chromatography of a blood-CSF mixture on Sephadex G-100. Left: fresh sample; right: sample incubated for 7 days. The third maximum increases significantly with aging.

B. Vasoactivity of the three maxima in a sample incubated for 7 days: the third maximum indicates strong vasoactivity.

55 Fig. 6

One by Hb or its related polypeptide

evoked contraction is caused by application

inhibited by haptoglobin

A. riei combination of Hb with Hp can be clearly seen find that the vasoactivity of Hb disappears.

B. By adding Hb or its related polypeptide, after application of the Hb-Hp complex no further contraction caused.

C. When haptoglobin or Hp-Hb on the arterial strip that is due to free hemoglobin maximum is contracted, the contraction is decreased to some extent.

A haptoglobin preparation from the applicant is used for the experiments, which is produced in accordance with JA-OS 77 516/75 and 77 527/50 or in accordance with DT-OS 24 09 650 by purifying <x ₂ -globulin. The a2 globulin is produced from human plasma by fractionating the Cohn fraction IV. Composition: 2000 units of haptoglobin from human plasma (1 unit of haptoglobin combines with 1 mg of hemoglobin) and 0.90 g of sodium chloride in a 100 ml ampoule. Purity: at least 90 percent haptoglobin (* ₂ -globulin) with a small amount of j3- Globulin (determined electrophoretically).

The example and the clinical trials illustrate the invention.

A. B is ρ ie 1 e for in-vitro experiments
preparation

Adult dogs weighing 10 to 15 kg are killed. Immediately thereafter, the basilar artery is removed and in accordance with RN S ρ ede η, J. Physiol. Vol. 154 (1960), pp. 15 to 25, cut into a spiral-shaped strip (see. Fig. IA). This strip is attached to a plastic chamber containing 5 ml of artificial CSF (concentrations [millimolar]: NaCl 128.6, NaHCO ₃ 15, KCl 1.8, KH ₂ PO ₄ 1.3, MgSO ₄ · 7 H ₂ O 1 , 3, CaCl ₂ · 2 H ₂ O 1.3, glucose 1.5). One end of the arterial strip is fixed while the other end is attached to a sliding scale connected to a strain gauge. The isometric tension of the arterial strip is recorded with an electronic recorder (see FIG. 1B). A mixture of 95 percent O ₂ and 5 percent CO ₂ with oxygen is added to the solution throughout the experiment. The pH is maintained at 7.4 and the temperature of the liquid at 37 ° C. The vasoactivity of fresh or aged serum samples, whole blood or blood-CSF mixtures (1: 1, 1-.4) on the isolated artery is examined by adding 0.1 to 0.5 ml of the sample to the medium. The samples are aged by incubation for 0 to 7 days under aseptic conditions at 37 ° C.

To purify the vasoactive substances, the samples are first subjected to gel filtration and are then used one with Dextran G-100 (cf. company publication from Phai rrwcia Fine Chemicals, Dextran Fractions, Dextran Sulphate, DEAE-Dextran, 1974-1975, p. 8, Tab. 2) loaded column of dimensions 2.5 × 90 cm subject. This is done with 0.1 M Tris-HCl buffer from pH 8.0. which is 5-m in NaCl, eluted. To

Carrying out the ninhydrin reaction, the absorption is measured at 280 and 415 πιμ. The vasoactive Fractions are passed on to a carboxymethyl cellulose ion exchange resin The loaded column measuring 2.5 40 cm was further purified. The elution will with 0.01 m phosphate buffer of pH 7.0, the 0.5 m on NaCl is carried out. The analysis of the polypeptide contained in the vasoactive fraction is carried out electrophoretically on polyacrylamide gel according to LOrstein, Ann. N.Y. Acad. Sciences, 121, 321-349 (1969).

The contraction caused by serum is most pronounced in a fresh sample and takes with it aging of the sample quickly. Serum aged 7 days is essentially non-vasoactive (see Fig. 2A). Furthermore, the contraction caused by serum is controlled by methysergide, a serotonin antagonist, practically completely eliminated (see FIG. 2B). The one evoked by serum or serotonin Contraction drops immediately when the original medium of artificial CSF is in the vicinity of the Arterial strip is restored. Thus, it is found that serotonin or one related to serotonin Compound in the serum is practically solely responsible for the development of the early spasm.

On the other hand, the vasoactivity increases in the supernatant of incubated whole blood and of blood-CSF mixtures progressively increases with aging of the samples and reaches the maximum after about 7 days of incubation (see Fig.3A). In addition, the vasoactivity remains in the Supernatant from aged blood-CSF mixtures or from aged whole blood after addition of methysergide unchanged (see Fig. 3B). The arterial contraction caused by the addition of an aged blood-CSF mixture is significantly greater than the concentration produced by aged blood alone. This concentration is characterized by one with the phasic contraction of the isolated artery basilaris superimposed tonic tension. The contraction generally lasts several hours (cf. F i g. 4A). After a contraction caused by these aged samples has occurred washing the artery with artificial CSF (see FIG. 4B) does not simply reduce the contraction. This contraction is also continued under hypoxic conditions, caused by termination of the O2 supply into the reaction medium can be increased significantly (cf. FIG. 4C).

To isolate the vasoactive substance, the supernatant of a blood-CSF mixture (1: 1), which is 7 days has been incubated at 37 ° C, passed through a column loaded with Dextran G-100.

The vasoactivity can only be determined in the fractions which correspond to the third maximum of the chromatogram correspond (see. Fig. 5A and 5B). This vasoactive fraction (maximum III) is collected and concentrated by diafiltration. The concentrated vasoactive substance is given an appropriate amount of Fibrinogen and thrombin added. Applying a thin slice of the lump obtained in this way to fco the caudal pons of an adult dog through the trephine opening located on the midline of the clivus Applied, a pronounced vasospasm develops on the basilar artery and on the Willis circle. This generally lasts for 2 weeks. This is what b5 Angiograms reveal.

For further purification of the vasoactive substance, the material of the maximum III is over a with Column loaded with CM cellulose ion exchange resin given. The vasoactivity of the individual fractions is determined by an in vitro test. the Vasoactivity is found in the third maximum (maximum III CM-C). When recording a spectrum of the This material corresponding to this maximum III shows pronounced absorption bands at 415, 540 and 575 ιημ. These bands are typical of oxyhemoglobin. Even when electrophoresed on polyacrylamide gel, this material shows properties such as oxyhemoglobin. It it can therefore be assumed with a high degree of probability that the isolated from the blood-CSF mixture vasoactive substance is either oxyhemoglobin itself or a closely related polypeptide.

Haptoglobin, «2-globulin is a normal component of serum. It is known that haptoglobin is slightly stoichiometric with hemoglobin to form a stable haptoglobin-hemoglobin complex.

Haptoglobin and haptoglobin-hemoglobin complexes (Hp-Hb) are used for further experiments. Hp-Hb is produced by adding haptoglobin to either the chromatographically eluted material, which corresponds to the maximum III CM-C, or to hemoglobin produced by the Drabkin process is, there and then performs an isolation by gel filtration.

execution

The in vitro experiments described below were carried out according to the method explained above confirm the following:

1) The contraction caused by Hp-Hb is compared to the contraction caused by hemoglobin alone much lower (see Fig. 6A). Furthermore, with further addition of free hemoglobin occurs no contraction occurs if the arterial strip has first been treated with Hp-Hb (cf. Fig. 6B).

2) Allowing haptoglobin or Hp-Hb to act on a strip of arteries caused by treatment with free hemoglobin at its maximum contraction, the contraction becomes somewhat decreased (see Fig. 6C).

Thus, it can be assumed that upon release of hemoglobin or related polypeptides a combination through the spontaneous hemolysis of erythrocytes that have leaked out of the vessels of the hemoglobin or the polypeptides with haptoglobir occurs until the haptoglobin! is consumed. Even if the amount of hemoglobin exceeds the binding capacity of the available haptoglobin If hemoglobin accumulates to a certain extent, vasoactivity is reduced of hemoglobin blocked by the presence of Hp-Ht. This may be the explanation for that The fact that the vasoactivity of aged whole blood is much less evident than the one! Blood-CSF mixture of equal volume. This effect occurs regardless of the fact that the Hemoglobin content in whole blood is greater than in the blood-CSF mixture. It is also assumed that in some Aneurysm rupture through extravasated blood which is first diluted with CSF and then with serum the CSF circulation is washed out and a comparatively large proportion of the blood cells ir remains in the basal cistern or in the Sylvian furrow. These conditions are for the origin

favorable from vasospasms, as the amount of free hemoglobin increases continuously due to hemolysis, while the total amount of haptoglobin drops sharply.

In the in vivo experiment, an appropriate amount of hemoglobin is injected into the Cisterna Magna. It results there is pronounced vasospasm of the basilar artery and the Willis circle.

Compared to control animals, this vasospasm can be observed for 4 or 5 days. On the third Day after injection of free hemoglobin is diffuse after angiographic confirmation Vasopasm injected 1 ml of a haptoglobin solution (20 mg HbBC / ml) into the basal cistern. About 5 hours After the haptoglobin injection, the angiogram shows a decrease in the vasospasm, and the Arterial diameter returns to normal.

B. Clinical Observations

Vasospasm has been treated with haptoglobin in several patients.

a) A 60 year old male patient is hospitalized 7 days after a subarachnoid hemorrhage delivered. The next day, angiography reveals a sac-like aneurysm on the anterior connecting artery and marked vasospasm at Ci, Ai, and Mi. 2 days after When the patient is admitted, the aneurysm neck is severed. The spastic arteries are filled with 3 ml a solution containing 20 mg HbBC / ml. In the two days after the operation haptoglobin infusions are performed repeatedly through a catheter in the basal cistern. A Angiogram taken on the third day after surgery shows relaxation of the vasospasm at the Willis Circle.

b) The test subject is a 46-year-old female patient who has the middle cerebral artery two consecutive aneurysm ruptures have occurred. One 14 days after the second bleeding recorded cerebral angiogram shows a sac-like aneurysm on the right median cerebral artery and marked vasospasm, particularly of the middle cerebral artery. The patient is operated on the following day. After severing the neck of the aneurysm, the Sylvian groove supplied haptoglobin to the arteries. The arteries that are severely spasmodic relax immediately after the operation and stay relaxed for several days, as if from one results in angiogram recorded on the third day after the operation.

For this purpose 4 sheets of drawings

Claims (3)

Claim: Use of haptoglobin or hemoglobin-haptoglobin complexes in the local prophylaxis and therapy of cerebral vasospasms. Subarachnoid hemorrhage is a bleeding in the subarachnoid space between the arachnoid and pia mater. This bleeding can cause apoplexy. The main cause of subarachnoid hemorrhage is an aneurysm rupture. In the case of a stroke, aneurysm rupture is relatively common in younger or middle-aged people. With appropriate treatment, successful rehabilitation can often be achieved without noticeable symptoms of neuro-vasospasm, so that re-bleeding can be prevented by early surgery. According to the invention, it was found that cerebral vasospasms can be prevented by administering haptoglobin or hemoglobin-haptoglobin complexes to the vessels. Aqueous solutions of human serum haptoglobin are already known from DT-OS 24 09 650, which are administered intravenously and used for the treatment of kidney disorders caused by hemolysis, but the use according to the invention could not be suggested as a result. The invention thus relates to the subject matter characterized in the patent claim. The mechanism by which cerebral vasospasms develop is still largely unclear. Angiography has recently shown in animal experiments that cerebral vasospasms can be artificially induced if blood components are allowed to act directly on the basilar artery or if blood is injected directly into the subarachnoid space. Based on these findings, it can be assumed that blood that has leaked from a blood vessel plays an important role in the development of cerebral vasospasm. To date, there are virtually no effective agents for treating cerebral vasospasm. For research purposes, attempts have been made to administer vasodilators such as papaverine, procaine and lidocaine, sympathetic agents such as phenoxybenzamine, phentolamine and isoproterenol, serotonin antagonists, antihistamines, dibutyryl-cyclo-AMP or ethanol, locally or systemically. Attempts have also been made to prevent fibrinolysis by administering inhibitors of fibrinolysis and by inhaling carbon dioxide. However, all of these drugs showed insufficient or temporary effects. So far it has been assumed that mechanical stimuli, vasoactive substances, stimuli on the vasomotor nerves and changes in vascular smooth muscle cells are the main causes of cerebral vasospasm. On this basis, with special consideration of vasoactive substances, biochemical analyzes of mixtures of blood and cerebrospinal fluid (blood-CSF mixture) were carried out in order to determine which role this mixture plays in cerebral vasospasms. It was found according to the invention that serotonin or serotonin-analogous compounds are responsible for the early spasm and that cerebral vasospasms attributable to these substances are completely controlled by serotonin antagonists. It has also been found that late spasms, i.e. H. cerebral vasospasm, caused by oxyhemoglobin or a polypeptide with biochemical properties very close to those of oxyhemoglobin, which is released by hemolysis of red blood cells. As already mentioned, these cerebral vasospasms can be effectively treated by the local administration of haptoglobin or a hemoglobin-haptoglobin complex. It has been found that the vasospasm effect of oxyhemoglobin occurs only on the cerebral artery and not, for example, on the mesenteric artery at all. To treat cerebral vasospasm, haptoglobin or hemoglobin-haptoglobin complexes are used as follows: 1. Postponed surgical interventions
for aneurysm ruptures Local application of haptoglobin to the spastic cerebral artery is the most effective Method for resolving persistent vasospasm. Each local dose consists of 2 or 3 ml of one Haptoglobin solution (20 mg HbBC / ml). The spastic arteries must be soaked with the hemoglobin solution and then washed with normal saline. This procedure is used during the Operation repeated several times. Before closing the dura mater, the same dose of haptoglobin is applied to the spastic, cerebral artery applied. After the operation will be on the following two days through a catheter in the basal cistern repeated haptoglobin infusions (2 ml of solution - 10 mg HbBC / ml). 2. Early surgery for aneurysm rupture 100 ml of haptoglobin solution (20 mg HbBC / ml) are diluted with 500 ml of normal saline solution. To When the neck of the aneurysm is severed, the arteries proximal and distal to the aneurysm become with it rinsed diluted haptoglobin solution. 3. Other uses I) Intraventricular use A haptoglobin solution is infused several times through a shunting tube or an external drainage tube (2 ml solution - 10 mg HbBC / ml). II) Intrathecal use A haptoglobin solution (2 ml solution - 10 mg HbBC / ml). III) Haptoglobin is also suitable for preventing persistent spasm after severe hemorrhage in the basal cistern for any operations. The individual figures, which are also referred to in the examples, have the following meanings: Fig. 1 A. Apparatus for producing arterial strips. While rotating the needle, the artery is drawn in spiral-shaped strip cut.