WO1993015778A1

WO1993015778A1 - Method of inhibiting the aggregation of blood platelets

Info

Publication number: WO1993015778A1
Application number: PCT/GB1993/000258
Authority: WO
Inventors: Anthony Ernest Bolton
Original assignee: Anthony Ernest Bolton
Priority date: 1992-02-07
Filing date: 1993-02-08
Publication date: 1993-08-19
Also published as: AU3506293A

Abstract

A method of treating blood which comprises contacting blood with a blood platelet-aggregation inhibiting effect of amount of ozone gas and ultraviolet radiation. The blood may be administered to a patient such as a human for inhibiting the aggregation of blood platelets.

Description

METHOD OF INHIBITING THE AGGREGATION OF BLOOD PLATELETS

Platelets are the smallest of the formed elements^' of the blood. Every cubic millimeter of blood contains about 250 million platelets, as compared with only a few thousand white cells. There are about a trillion platelets in the blood of an average human adult. Platelets are not cells, but are fragments of- the giant bone-marrow cells called megakaryocytes. When a megakaryocyte matures, its cytoplasm breaks up, forming several thousand platelets. Platelets lack DNA and have little ability to synthesize proteins. When released into the blood, they circulate and die in about ten days. However, platelets do possess an active metabolism to supply their energy needs.

Because platelets contain a generous amount of contractile protein (actomyosin) , they are prone to contract much as muscles do. This phenomenon explains the shrinkage of a fresh blood clot after it stands for only a few minutes. The shrinkage plays a role in forming a hemostatic plug when a blood vessel is cut. The primary function of platelets is that of forming blood clots. ^' When a wound occurs, platelets are attracted to the site where they activate a substance (thrombin) which starts the clotting process. Thrombin, in addition to converting fibrinogen into fibrin, also makes the platelets sticky. Thus, when exposed to collagen and thrombin, the platelets aggregate to form a plug in the hole of an injured blood vessel.

Platelets not only tend to stick to one another, but to the walls of blood vessels as well. Because they promote clotting, platelets have a key role in the formation of thrombi. The dangerous consequences of thrombi are evident in many cardiovascular and cerebrovascular disorders.

In this regard, the precise function of blood platelets in various human disease states has recently become increasingly understood as advances in biochemistry permit the etiologies of diseases to be better understood. For example, many attempts have been made to explain the process of atherogenesis, that is, the creation of plaque which narrows arteries and, of particular concern, the coronary arteries. Recently, there has been increasing interest in the possible role of platelets in atherosclerosis.

In addition, a number of disease states in humans are believed to be associated with an aggregation of platelets in the blood. These platelet aggregation associated conditions include: peripheral vascular disease; thrombotic diseases such as coronary thrombosis and pulmonary thrombosis; stroke; eclampsia and pre-ecla psia; and hypertension. A study completed by the University of Oxford, England, and published in the British Medical Journal, Vol. 296, January 30 1988, pages 320-331, entitled "Secondary Prevention of Vascular Disease by Prolonged Antiplatelet Treatment", suggests that therapies which inhibit platelet aggregation may be useful for treating occlusive vascular disease. The study utilised aspirin, sulphinpyrazone, or aspirin and dipyridamole as the platelet aggregation inhibiting agents.

Unfortunately, long-term aspirin therapy may lead to severe gastrointestinal irritation and bleeding. Also, these and other known agents which inhibit platelet aggregation may have other undesirable side-effects that make them unsuitable for administration to patients who could benefit from such therapy. For pregnant women with pre-eclampsia or other platelet aggregation associated conditions, the administration of drugs may be undesirable in view of the potential effects of the same on the developing fetus.

According to this invention there is provided a method of treating blood which comprises contacting the blood with a blood platelet-aggregation inhibiting effective amount of ozone gas and ultraviolet radiation.

Preferably the ozone gas has a concentration of from about 0.5 to about 100 μg/ l.

Advantageously the ozone gas has a concentration of from about 5 to about 50 μg/ml.

Conveniently the ultraviolet radiation has a wavelength of from about 253.7 nm. Advantageously the blood is heated to a temperature of from about 0 to about 56°C while being contacted with the ozone gas and ultraviolet radiation. The preferred temperature ranges from 37 to 43°C and the preferred temperature is 42.5°C.

Preferably the method is employed on a 10 ml aliquot of blood. Preferably the blood is contacted with the ozone gas and ultraviolet radiation for a period of about 3 minutes.

The blood may be human blood.

The invention relates to blood treated by a method as described above and also relates to the use of blood treated by a method as described above and a method as described above in the preparation of a medicament.

The medicament may be for the treatment of peripheral vascular disease, a thrombotic disease, coronary thrombosis, pulmonary thrombosis, stroke, pre-eclampsia, and hypertension.

The invention will now be described in greater detail.

THIS PAGE IS FOLLOWED BY PAGE 7

As evidenced by the data set forth in Examples 1 and 2 below, Applicant has found that satisfactory inhibition of platelet aggregation can only be achieved when the blood is treated with a combination of ozone gas and ultraviolet radiation. Treatment of blood solely with ozone gas produces minimal inhibition of blood platelet aggregation. Moreover, treatment of blood solely with ultraviolet light produces no inhibition of platelet aggregation whatsoever. The combined treatment with ozone gas and ultraviolet light, however, has unexpectedly been found to produce significant inhibition of blood platelet aggregation, which is useful in treating a variety of disorders associated with blood platelet aggregation.

The term "aggregation of blood platelets" as used herein refers to the sticking together of platelets to other platelets and/or to the walls of a blood vessel. The ozone gas used in connection with the inventive method has a concentration of ozone of from about 0.5 to about 100 μg/ml. Preferably, the ozone gas has a concentration of from about 5 to about 50 μg/ml. Ultraviolet radiation having a wavelength of about 253.7 n has been found to provide the results of the invention, when utilized in conjunction with the ozone gas treatment. It is believed that ultraviolet radiation having emission wavelengths corresponding to standard UV-A and UV-B sources would also provide acceptable results.

The blood is preferably heated to a temperature of from about 0 to about 56 °C while being contacted with the ozone gas and ultraviolet radiation. The blood is preferably heated to about 37-43 °C, most preferably about 42.5 °C, while being contacted with the ozone gas and ultraviolet radiation.

The aliquot of blood treated by the inventive technique is withdrawn from the human patient in any conventional manner known in the art. The method preferably involves removing about 10 ml of blood, treating the same with ozone gas and ultraviolet radiation, then returning the treated blood to the patient by intramuscular injection. Other conventional techniques or readministering the blood may be employed, such as intravenous injection, subcutaneous injection, and intraperitoneal injection. The readministration of small volumes of host blood in this fashion is termed micro-auto-hemotherapy. The invention also contemplates an embodiment wherein the blood is continuously removed from the patient's body and circulated through an apparatus which treats the blood with ozone gas and ultraviolet light before returning the blood to the patient. This procedure would have particular utility, for example, during the performance of operative procedures, such as coronary bypass surgery.

The blood is contacted with the ozone gas and ultraviolet radiation for a period of time sufficient to effectively inhibit the aggregation of blood platelets. A treatment period of from about 1 minute to about 60 minutes, and preferably about 3 minutes, has been found to provide satisfactory inhibition of platelet aggregation.

The method should be carried out under sterile conditions known to those of ordinary skill in the art.

The method of the invention may be carried out using conventional apparatus for ozonating blood and irradiating blood with ultraviolet light known to those skilled in the medical art. Preferably, an apparatus as disclosed in U.S. Patent No. 4,968,483 is employed to carry out the method of the invention. The disclosure of U.S. Patent No. 4,968,483 is incorporated herein in its entirety by reference.

In a preferred aspect of the invention, a method of inhibiting the aggregation of blood platelets in a human is provided, which comprises:

(a) removing an aliquot of blood from a human;

(b) contacting the blood with a blood platelet- inhibiting effective amount of from about 5 to about 50 μg/ml of ozone gas and ultraviolet radiation having a wavelength of about 253.7 nm, while heating the blood to a temperature of from about 37 to about 43 °C; and

(c) read inistering the treated blood to the human. The invention also contemplates a method of treating a condition in a human associated with blood platelet aggregation, which comprises:

(a) removing an aliquot of blood from a human;

(b) contacting the blood with a blood platelet- inhibiting effective amount of ozone gas and ultraviolet radiation; and

(c) readministering the treated blood to the human. The useful and preferred ranges of ozone concentration, ultraviolet wavelength, temperature, and other parameters of the method of treatment are the same as described above with regard to the method of inhibiting blood platelet aggregation.

Those skilled in the art will appreciate that the method of inhibiting blood platelet aggregation provided by the invention will have therapeutic utility for treating a wide range of disease states associated with the aggregation of blood platelets in humans.

The term "treating" as used herein refers to the alleviation or prevention of a particular disorder. In the case of traumatic conditions such as stroke, preventative treatment is obviously preferred. Also, although the term

"human" is used to describe the preferred host, those skilled in the art will appreciate that the methods of the invention would have similar utility with other mammals.

The following diseases are illustrative of known conditions which may be associated with the aggregation of blood platelets, and which are treatable according to the inventive method: peripheral vascular disease; arterial and venus disorders including thrombotic diseases such as coronary thrombosis, pulmonary thrombosis, arterial thrombosis, and venus thrombosis; stroke; pre-eclampsia; and hypertension. This list is merely illustrative of conditions which are associated with platelet aggregation; those of ordinary skill in the art will appreciate that other disease states associated with an aggregation of blood platelets may be treated with the inventive technique.

With regard to peripheral vascular disease, the disease is thought to be associated with a reduction of endothelial- derived relaxing factor (EDGF) , low levels of which lead to a contraction of the smooth muscle of blood vessels, and hence a reduction in the diameter of the lumen of the vessel and a reduction in blood flow. The major naturally occurring EDGF is nitric oxide. In addition, nitric oxide stabilizes blood platelets, reducing their aggregation. An increase in EDGF (nitric oxide) levels, therefore, has a double beneficial effect on the circulatory system: it inhibits aggregation of platelets, making the blood more fluid, and it enlarges the diameter of the vessels, improving the flow. The reverse, a reduction in nitri oxide levels, is present in peripheral vascular disease.

As illustrated in Example 2 below, the method of th invention is believed to increase nitric oxide levels in the blood, which may explain the mode of action in the inventive treatment of peripheral vascular disease and other conditions associated with blood platelet aggregation. Pre-eclampsia may lead to eclampsia, an acute hypertensive crisis that may occur in the second or third trimester of pregnancy. Although the precise etiology is unknown, overactive platelet activity leading to the formation of thrombi in the placenta is believed to be a cause of the condition. The inventive method, which results in a stabilization of the patient's blood platelets and an inhibition of platelet aggregation, is therefore a potential treatment modality. In particular, the method of the invention may be preferred over conventional antiplatelet therapies, where the administration of drugs to the mother is counterindicated.

The following examples are given to illustrate the invention but are not deemed to be limiting thereof. All percentages given throughout the application are percents of platelet inhibition, unless otherwise indicated.

EXAMPLE 1 Inhibition of Blood Platelet Aggregation The following experiment was conducted to study the effects of ozone/ultraviolet light treatment on blood platelet activity.

Experimental Procedure Samples (20 ml) of peripheral blood were taken from 10 individuals for 13 separate experiments. Each sample was II divided into two aliquots. The first aliquot was treated according to the inventive technique, as follows:

The 10 ml aliquot was treated in vitro for three minutes with ozone gas (variable ozone concentration of 5-50 μg/ml) and ultraviolet light (253.7 nm) , at a temperature of 42.5°C. An apparatus as disclosed in U.S. Patent No. 4,968,483 was utilized to carry out the treatment of the blood sample.

The second 10 ml aliquot from each sample served as an untreated control.

Platelets were isolated from the control or treated samples by centrifugation, and their ability to aggregate in response to different concentrations of ADP (a natural platelet stimulator) was measured in an aggregometer. A sample of both ozone-treated and untreated blood was used for quantitation of platelet numbers, using a Coulter counter. In some of the experiments described below, aliquots of the blood were treated with different concentrations of ozone. In other experiments performed, the blood was treated in the presence and absence of UV- light irradiation.

Platelet aggregation in the ozone-treated blood was expressed as a percentage of aggregation in the same-person untreated control blood. Resul

As shown in Table l, the results of the experiments indicate that treatment of blood with ozone and ultraviolet light according to the invention inhibits the aggregation of \t blood platelets. Furthermore, there is an indication that this inhibition is dose related to the ozone concentration (See Table 2) .

The effect of high levels of ozone on

ADP-stimulated blood platelets

High levels of ozone (between 35 and 50 μg/ml) caused a measurable inhibition of ADP-induced platelet aggregation

(arbitrarily taken as 33.3% inhibition) in 11 of the 13 experiments (8 of the 10 individuals) . Taking all the data on all 10 individuals, the mean inhibition of platelet aggregation was 49.2 +/- 27.8% (mean +/- sd) . There was no significant difference between the inhibitory effects on blood taken from males and females (mean inhibition 48.1% and 50.7%, respectively).

This inhibition appears to relate to the concentration of ADP (aggregation stimulator) over the concentration range of 0.01-O.lmM ADP, with lower inhibition at higher concentration of platelet agonist. However, this relationship did not hold at higher ADP concentrations (Table 1) and could be spurious, although the level of inhibition at O.OlmM ADP is significantly greater than at O.lmM ADP (71% vs. 95%, p < 0.02). TABLE 1

The effect of high levels of ozone on the aggregation of human blood platelets in the presence of varying concentrations of ADP

Percent

Concentration Concentration Inhibition Date of ozone of ADP of Platelet Count

(IπdividuaO (u~/m\) Aggregation Before Ozone - A fter Ozone

21.11.91 50 10 100 (Fl)

27.11.91 50 (M l)

2.12.91 50 (F2)

3.12.91 50

(M2)

6.12.91 50 34 49

(M3)

11.12.91 50 46 93

(M4)

12.12.91 50 51 P !

(M5)

13.12.91 50 3₃ 87

(Fl)

9-01.92 50 34 40

(M6)

10.01.92 50 49 64

(F3)

ft

0.01 69.8

0.05 33.8

0.1 31.2

0.5 10.1

1.0 21.8

13.01.92 50 0.005 100 49 52 (F4) 0.01 100 0.05 95.2 0.1 92.9 0.5 95.8 1.0 91.6 5.0 95.8 10.0 80.0

15.01.92 40 0.01 90.0 66 (Fl) 0.05 71.4

0.1 40.7

0.5 87.0

1.0 81.8

5.0 95.5

10.0 85.2

50.0 84.0

100.0 79.1

21.01.92 35 0.01 67.1 68 79 (M2 )

The following is a summary of the data set forth in Table l:

ADP mM 0.01 0.05 0.10 0.50 1.00 5.00 10.0

% inhibition 70.8 53.5 34.7 37.6 50.3 60.7 60.7 of aggregation +/-20.9 +/-26.1 +Λ28.4 +/-3S.4 +Λ28.7 +/-3S.2 +/-30.4 N = 6 6 8 7 7 4 4

The effect of high levels of ozone on total whole blood platelet counts As any apparent reduction in platelet aggregation following ozone treatment of whole blood could be caused by a loss of platelets from the blood during treatment, total whole platelet counts were performed on the treated and untreated whole blood samples in 9 experiments on blood from 8 individuals. Overall, the platelet count was 115.5 +\- 59.8% of the untreated level following ozonization (range 82-264%) .

Thus, the total platelet counts before and after ozone/UV treatment do not indicate a major loss of platelets from the blood as a result of ozonization.

The effect of different concentrations of ozone on the inhibition of aggregation of human blood platelets stimulated with ADP Three different concentrations of ozone (5, 25, and 50 μg/ml) were used at a range of ADP concentrations in 4 experiments on 4 different individuals. Bulking the data for different ozone concentrations from each individual and calculating the mean for the data from the. 4 experiments indicated that there was some dose response relationship between the concentration of ozone used and the inhibition of platelet aggregation (See Table 2) . Although overall these differences were not significant, in two of the four individuals there was a significantly greater inhibitory effect of ozone at 50 μg/ml then at 5 μg/ml (See Table 3) .

TABLE 2

The effect of different concentrations of ozone on inhibition of platelet aggregation in the presence of ADP

5

25 50

5

25 50

10.01.92 5 49 73 (F3) 25 90 50 64

5

25 50

5

25 50

5

25 50

5

25 50

5

25 50

5

25 50

13.01.92 5 49 60 (F4) 25 85 50 52

5

25 50

5

25 50

5

25 50

5

25 50

5

25 50

The following is a summary of the data set forth in Table 2:

Concentration of ozone dig/ml) 5 25 50 Platelet aggregation (%) 38.5 +/-30.9 S6.5 +/-29.4 S5.9 +/-26.4

(mean +/- sd, n =4

TABLE 3

The effect of different concentrations of ozone on inhibition of platelet aggregation in two individuals

Concentration of ozone (ug/mll 5 25 50

Platelet aggregation M2 (%) Difference from 5 μg/ml

Platelet aggregation M6 (%) Difference from 5 μg/ml

ns=not significant

The effect of UV light on the response of platelets to ozone The effect of ozone on the aggregation of human blood platelets was investigated at different concentrations of ADP, in the presence or absence of UV light. The results, shown in Table 4, indicate that, although there may be some platelet aggregation-inhibitory response to ozone alone, this is nearly always greater in the presence of UV light and the effect of UV light was highly significant (p<0.001) in this single experiment. This result was also repeated in a second experiment, using a single concentration of ADP (0.01 mM) . The results of this second experiment are set forth in Table 5.

TABLE 4 The effect of UV light on the inhibition of ADP-induced platelet aggregation by ozone at a concentration of 40 μg/ml. (Experiment date 15.01.92, individual Fl)

Concentration ADP ( M) Inhibition of platelet aggregation (%)

+uv -uy

0.01

0.05

0.1

0.5

1.0

5.0

10.0

50.0

100.0

Mean +/- sd

M9.6(p<0.00n

TABLE 5

The effect of UV light on platelet aggregation induced by

ADP (0.01 M) in the presence or absence of ozone.

(Experiment date 21.01.92, individual M2)

Percent inhibition of platelet aggregation

Ozone 35 μg/ml + UV Ozone 35 μg/ml - UV No ozone, UV alone 83.4% 11.2% 0%

In summary, the results of Example 1 indicate that the in vitro treatment of an"aliquot of blood with ozone gas and ultraviolet light inhibits the aggregation of blood 2D platelets. This platelet inhibition has been found to be dose related to the ozone concentration. Further, platelet inhibition was found to critically depend on the combined treatment of ultraviolet light and ozone gas, as evidenced in Tables 4 and 5. Treatment with ozone gas alone resulted in minimal inhibition of platelet aggregation, while treatment with ultraviolet light alone produced no inhibition of platelet aggregation.

EXAMPLE 2

Measurement of Nitric Oxide In order to elucidate the mechanism whereby ozonization/ UV light affects the aggregation of platelets in treated blood, the concentration of certain oxidized forms of nitrogen were measured.

The direct measurement of nitric oxide is difficult to achieve. However, nitric oxide is an intermediate in a metabolic pathway in which arginine is converted to citrulline. Other stable end-products are nitrates and nitrites.

Accordingly, the nitric oxide content for several samples of blood treated with ultraviolet light and ozone gas according to Example 1 were indirectly determined by measuring the combined nitrate plus nitrite concentrations in the samples before and after treatment with ozone/UV light, after converting nitrite to nitrate.

The results show that there is a small increase in nitrate plus nitrite concentrations after treatment according to the invention. This increase was consistently found in samples treated with ozone gas/UV light. Thus, nitric oxide levels may be enhanced by the treatment with ozone gas/UV light, and this may be part of the mode of action by which an inhibition of blood platelet aggregation is achieved by the invention. This therapeutic effect would be consistent with the etiology of peripheral vascular disease described above.

Conclusions The data of Examples 1 and 2 suggest that the treatment of blood with ozone gas and ultraviolet light according to the invention is actually inducing an inhibition of platele aggregation for the following reasons:

1. The inhibitory effect is at least partiall dependent on the concentration of ADP, ozone being mor inhibitory at lower ADP concentrations. This may b interpreted as the higher agonist concentrations partiall overcoming the inhibitory effect of ozone b "hyperstimulating" the platelets. This suggests that th inhibition is at least partially reversible, and is probabl not acting by destroying the platelet's ability t aggregate.

2. The inhibitory effect appears to be dose related t ozone concentration, with higher concentrations of ozon resulting in a greater inhibition of platelet aggregation.

3. The inhibitory effect is UV-dependent, suggestin that this is not a non-specific toxic effect caused by th oxidative capacity of the ozone gas. The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications are intended to be included within the scope of the following claims.

Claims

-ZZ -CLAIMS :

1. A method of treating blood which comprises contacting the blood with a blood platelet-aggregation inhibiting effective amount of ozone gas and ultraviolet radiation.

2. The method of Claim 1 wherein the ozone gas has a concentration of from about 0.5 to about 100 μg/ml.

3. The method of Claim 2, wherein the ozone gas has a concentration of from about 5 to about 50 μg/ml.

4. The method of any one of the preceding Claim wherein the ultraviolet radiation has a wavelength of from about 253.7 nm.

5. The method of any one of the preceding Claims wherein the blood is heated to a temperature of from about 0 to about 56°C while being contacted with the ozone gas and ultraviolet radiation.

6. The method of Claim 5, wherein the blood is heated to a temperature of from about 37 to about 43°C while being contacted with the ozone gas and ultraviolet radiation.

7. The method of Claim 6 , wherein the blood is heated to a temperature of about 42.5°C while being contacted with the ozone gas and ultraviolet radiation.

8. The method of any one of the preceding Claims wherein the method is performed on an aliquot of blood of about 10 ml of blood.

9. The method of any one of the preceding Claims wherein the blood is contacted with the ozone gas and ultraviolet radiation for a period of about 3 minutes.

10. The method of any one of the preceding Claims wherein the blood is human blood.

11. Blood treated by a method of any one of Claims 1 to 10.

12. The use of blood according to Claim 11 in the preparation of a medicament.

13. The use of a method according to any one of Claims 1 to 10 in the preparation of a medicament.

14. Use according to Claim 12 or 13 wherein the medicament is for the treatment of peripheral vascular disease.

15. Use according to Claim 12 or 13 wherein the medicament is for the treatment of a thrombotic disease.

16. Use according to Claim 12 or 13 wherein the medicament is for the treatment of coronary thrombosis.

17. Use according to Claim 12 or 13 wherein the medicament is for the treatment of pulmonary thrombosis.

18. Use according to Claim 12 or 13 wherein the medicament is for the treatment of a stroke.

19. Use according to Claim 12 or 13 wherein the medicament is for the treatment of pre-eclampsia.

20. Use according to Claim 12 or 13 wherein the medicament is for the treatment of hypertension.