US3928587A - Method of conditioning vascular systems by administering 3,5 dichloroaspirin and method of evaluating pharmaceutical compounds - Google Patents

Abstract

3,5 DICHLOROASPIRIN IS ADMINISTERED ORALLY OR INTRAVENOUSLY TO RENDER MORE NEGATIVE THE SURFACE CHARGE OF THE INTERIOR SURFACE OF THE VASCULAR SYSTEM AND THEREBY TO DECREASE THROMBOTIC TENDENCY THEREIN. This also increases the streaming potential of the vascular interface and increases Lee White clotting times, thrombin times, and partial thromboplastin times and recalcification times. The overall effect is to decrease thrombotic tendencies and to increase blood flow in the extremities. A method is provided for evaluating 3,5 dichloroaspirin and other such compounds.

Description

United States Patent [1 1 Sawyer METHOD OF CONDITIONING VASCULAR SYSTEMS BY ADMINIS'IERING 3,5 DICHLOROASPIRIN AND METHOD OF EVALUATING PHARMACEUTICAL COMPOUNDS [76] Inventor: Philip Nicholas Sawyer, 606 Third St., Brooklyn, NY. 11215 221 Filed: Aug. f6, 1914 211 Appl. No; 497,851

[52] US. Cl. 424/230 [5|] Int. Cl. A61K 31/60 [58] Field of Search 424/230 [56] References Cited OTHER PUBLICATIONS Chem Abst. 64 126330 (I966).

Chem. Abst. 73 646040 1970).

[ Dec. 23, 1975 Primary Examiner-Stanley J. Friedman Attorney, Agent, or Firm-Roberts & Cohen [57] ABSTRACT 3,5 dichloroaspirin is administered orally or intravenously to render more negative the surface charge of the interior surface of the vascular system and thereby to decrease thrombotic tendency therein. This also increases the streaming potential of the vascular interface and increases Lee White clotting times, thrombin times, and partial thromboplastin times and recalcification times. The Overall effect is to decrease thrombotic tendencies and to increase blood flow in the extremities. A method is provided for evaluating 3,5 dichloroaspirin and other such compounds.

4 Claims, 6 Drawing Figures METHOD OF CONDITIONING VASCULAR SYSTEMS BY ADMINISTERING 3,5 DICHLOROASPIRIN AND METHOD OF EVALUATING PHARMACEUTICAL COMPOUNDS FIELD OF INVENTION This invention relates to pharmaceutical compounds for treating the vascular system and to methods of evaluating pharmaceutical compounds.

BACKG ROUN D Various cell systems have been investigated in an attempt to explain specificities in cell recognition, rejection and adhesion (Danielli, J. F. Cell to cell interaction. J. Physio. Vol. 98: 109, 1940). Evidence indicates that platelets and erythrocytes have common surface properties that may greatly influence cell-cell interactions (Seaman, G. V. et al. Cell interaction a surface charge phenomena. Biochem. Biophys, Vol 1 l7, l966; Mehrishi, J. N. Phosphate groups on the surface of human blood platelets, Nature, Vol. 226, 5244, i970; Davies, D. F., et al. The electrophoretic mobility of RBC a measure of the surface activity. Clin. Sci., Vol. l7, 1965). One such property is the negative surface charge of the membrane, which was first described by Abramson in 1928 (Abramson, H. A. Electrophoretic migration of inert particles and blook cells in sols. and gels. Amer. J. of Med. Sci. Vol. 167, 1928). Intact cells at a physiological pH tend to move toward an anode as can be shown by quantitative electrophoresis measurements. The effect is accounted for by an excess of anionic over cationic groups in the surface layer that determines electrophoretic mobility (Madoff, M. A. et at. Sialic acid of human blood platelets .I. of Clin. lnv. Vol. 43, No. 5, 1964). Platelets have a true iso-electric point at a pH of 3.5 to 4.5 which indicates that the net negative charge excess is considerable. Upon treatment with neurominidase, 60 percent of the charge can be removed. apparently due to the removal of sialic acid residues located on the cell surface. Platelets so treated tend to aggregate spontaneously. This then would appear to indicate that electrostatic repulsion is partly responsible for vascular homeostasis (Weiss, L. Biophysical aspects of initial cell interaction with solid surfaces. Fed. Proc. Vol. 30, No. 5, Sept-Oct, l97l', Salzman, E. Role of platelets in blood surface interaction Fed. Proc., Vol. 30: No. 5, Sept. Oct., l97l It is possible to cause platelets as well as other cells to aggregate by the addition of positively charged macromolecules such as polylysine and protamine sulfate (Hampton, J. R., and Mitchell, J. R. A. Modification of the electrokinetic response of blood platelets. Nature, l966; Massini, P. and Luscher, E. On the mechanism of which cell contact induces the release reaction of blood platelets, Thromb. et Diath., Vol. 27, I972). It has also been shown that positively charged metallic surfaces that exhibit positive potentials vs. NHE in blood/saline are thrombogenic (Sawyer, et al. Relation between Thrombosis on Metal Electrodes and the Position of Metal in the Electromotive Series, Nature, Vol. 2l5, No. 5109, p. 1494, Sept. 30, I967). Modifications of collagen prostheses to reduce surface negativity promotes thrombus formation in vivo (Sawyer, et at., Critical potential for Thrombus Deposition on Metal Surfaces in Vivo. Vascular surgical services and the Electrochemistry and Biophysical Labs. of the Dept. of

2 Surgery and Surgical Research, State Univ. of NY. Downstate Medical Center, Bklyn.).

The above supports the view that electrostatic repulsion per se is in part responsible for platelet noninteraction, at both cell-cell and blood vessel wall-blood cell levels under normal conditions. One explanation of platelet transformation into highly adhesive cells relates to surface charge. Metallic tube implants have demonstrated the relevance of surface potential and 0 thrombogenecity (Sawyer et al., Critical potential for Thrombus Deposition on Metal Surfaces in Vivo supra). Metallic tubes with varying spontaneous potentials in contact with blood were implanted in dogs. Those tubes which possess a homogeneous negative surface potential vs NHE in contact with blood remain patent far longer than did those implants with positive potentials (Sayer et al., Critical potential for Thrombus Deposition on Metal Surfaces in Vivo supra). Likewise, polymer materials with an excess of oriented negative charge residues, (i.e., SO, and COO*) in contact with blood also support the premise that a homogeneous negative surface potential is one criteria for antithrombogenicity (Sawyer et al., Critical potential for Thrombus Deposition on Metal Surfaces in Vivo).

Potential dependent cell adhesion characteristics were first observed in I963. The fact has been established, both in vitro and in vivo, that anodic potentials imposed upon a P1 wire produced cell precipitation. The effects of varying pH and heparin were also established. Results have demonstrated that an acidic pH potentiates cell adhesion, while a basic pH inhibits cell adhesion. A series of experiments produced a semi quantitative method of evaluating cell adhesion in a highly ordered and controlled environment.

Data is available which correlates potential dependent cell adhesion with the influence of various pharmacological agents on platelet adhesion and thrombus formation on vascular prostheses. Furthermore, various pharmacological agents have been shown to modify thrombus deposition in vivo on metallic prostheses, the injured blood vessel wall and collagen implants (Sawyer et at, Utility of Anticoagulant Drugs in Vascular Thrombosis: Electron Microscopic and Biophysical Study, Surgery, St. Louis, Vol. 74, No. 2, PP. 263-275, August, i973).

SUMMARY OF INVENTION It is an object of the invention to provide an improved technique for the treatment of vascular systems of warm blooded animals.

lt is another object of the invention to provide a new technique for rendering more negative the intimal surface charge of the vascular system.

Yet another object of the invention is to provide an improved technique for decreasing thrombotic tendency in vascular systems.

Still another object of the invention is to provide an improved technique for decreasing the thrombotic tendency in vascular systems as manifested by an increase in the streaming potential of the vascular interface, an increased clotting time, thrombin time, partial thromboplastin time, and recalcification time.

To achieve the above and other objects of the invention, there is provided a method which comprises administering 3,5 dichloroaspirin to a warm blooded animal to render more negative the surface charge on the interior surface of the vascular system and thereby decrease thrombotic tendencies therein. The 3,5 di- 3 chloroaspirin has the following structure COOH C OCH 3 c1 CI The 3,5 dichloroaspirin may in accordance with the invention be administered orally. It is preferably administered in an amount in the order of magnitude of about 15 mg./kg. of body weight daily.

The 3,5 dichloroaspirin may also be administered intravenously in accordance with the invention in an amount in the order of magnitude of about mg./kg. of body weight daily.

Other objects, features and advantages of the invention will be found in the detailed description which follows hereinafter.

BRIEF DESCRIPTION OF DRAWING In the drawing:

FIG. 1 diagrammatically illustrates apparatus for conducting tests in accordance with the invention:

FIGS. 2(a) and 2(1)) are charts indicating potential dependent platelet adhesion vs. surface potential in the apparatus of FIG. 1;

FIG. 3 illustrates ADP aggregation levels vs. surface potential aggregation levels in chart form; and

FIGS. 4(a) and (b) illustrate platelet dependent cell adhesion vs. surface potential.

DETAILED DESCIPTION As shown in FIG. I, a Bausch & Lomb movable stage microscope with phase contrast optics and an oil immersion lens was employed. The optics include a phase microscope using an oil immersion lens at 1000 magnification. The microscope is focused on one small area of the platinum electrode in order to observe platlet sticking. A lucite cell or chamber 12 with a volume of 2 ml was constructed so as to allow the easy insertion of a Pr wire electrode 14 and two salt bridges l6 and I8 (agar bridges used as electrolytic conductors).

A Pt electrode with surface properties that are well defined was chosen as the test surface. Of great importance in obtaining accurate results in cleanliness (demonstrably free of blood coagulation factors and other proteins or chemicals which might excite blood clotting), particularly of the lucite cell and the electrodes. To achieve the above, the cell is first rinsed with distilled water and washed with a mild soap solution. The cell is then treated with a solution of 50 percent water and 50 percent methanol in order to denature any proteins or accumulated thrombin. It is finally rinsed with triple distilled water and allowed to dry.

The electrode is cleaned first by immersion in nitric acid for at least 15 minutes and then flamed. It is then inserted into the lucite cell. The cell is filled with a protein-free plasma-like solution, namely Krebs l solution (Sawyer et al.. Electrochemical Precipitation of Human Blood Cells and its Possible Relation to Intravascular Thrombosis, Proceedings of the National Academy of Sciences. Vol. 51. No. 3. PP. 428-432, March. 1964). The electrode is further cleaned by passing a cathodic current and degassing the electrodev The modified Krebs 1 solution is then removed and fresh solution is added for actual experimentation.

Ten ml of human blood are drawn into a sterile syringe and placed into two citrated vacutainers. The blood is centrifuged at 1000 rpm for 20 minutes. This produces a supernatant plasma with a high platelet concentration. 0.5ml of platelet rich plasma (PR?) is then added to the cell already containing 1 ml of the Krebs 1 solution which is then filled (approximately two milliliters) for the purpose of obtaining a series of control values.

In determining the effects of pharmacological agents on the adhesive behavior of platelets, varying amounts of the test drug are added to the above solution. The experiment is run at a pH of 7.35. Tenth normal HCI and NaOH buffer solutions were used for buffering to alter the pH range.

The salt bridges connect with ionic solutions in vessels 20 and 22. The ionic solutions may be, for example, saturated potassium chloride solution or a solution of sodium chloride. A potentiostat 24 was used to control the surface potential of the Pt wire. Current was read by meter 26 and voltage was read by meter 28. First the potential was set at 6()O mv vs. NHE and allowed to remain there for 2 minutes. A count of platelets sticking to the electrode was then taken. The potential was then increased in steps of mv and another count taken 2 minutes later. This procedure was repeated until the potential was brought through 0 to +600 mv vs. NHE (normal hydrogen electrode). The initial potential was not set below 600 mv vs. NHE in an effort to avoid the formation of gas bubbles on the electrode.

After each run, the apparatus is cleaned as previously described and another run is made. Upon completion of a series of experiments, the number of adherent platelets are averaged at each potential and a deviation from the mean is tabulated. The experimental results can best be represented by graphs, where the number of platelets adhering to the electrode is plotted against potential of the electrode under varying experimental conditions.

Included among the agents evaluated were (a) three anticoagulants, namelyz. heparin, aspirin (ASA), and 3,5 dichloroaspirin, (b) coagulants, namely protamine sulfate and chlorpromazine, and (c) a proteolytic en'- zyme (brinase); code number herein C The drug concentrations used (except for 3.5 dichloroaspirin which was not previously used) were in most cases ten times less than normal, normal and ten times greater than normal with respect to a clinical therapeutic dosage. The term normal as used herein means the average concentration normally found in plasma when the drug is administered orally and implies the maximum normal blood level with non toxic effect by whatever route the material is administered.

On the basis of ten independent blood samples, a standard curve (see FIGS. 2(a) and 2(b)) was arrived at for the potential dependent adhesion characteristics of human platelets on P! wire. Analysis of the control graph shows a peak in the curve at 40() mv. NHE, far below the plateau region which starts at the anodic potential of +400 mv NHE. It is this point in the curve where one observes reversible migration of platelets toward the electode followed by subsequent primary stage platelet adhesion. This reversible aggregation phenomena also occurs in vivo with the blood vessel wall and has been reported in other test systems. evaluating platelet adhesion (Fulher, M. B., et al. Reversible alterations in platelet morphology produced by anticoagulants and cold blood. vol. 9, 602, 1954). One cannot, however, actually say whether the charge on the metal at this potential is comparable to the charge density of an equal area of the blood vessel wall.

At a potential near the point of zero charge (PZC of the Pt electrode what appeared to be first stage viscous metamorphosis (agglutination) was observed. This same observation was made in previously reported research, utilizing a mercury drop electode. In an attempt to establish whether the point of zero charge and any anodic potential thereafter was inducing the platelet release reaction, a series of experiments was conducted using ADP (adenosine diphosphate) results in aggregation curves utilizing known optical techniques. Stated otherwise, in order to determine if platelets or blood cell precipitation was related to point of zero charge, a series of experiments was carried out measuring the precipitation potential of blood cells on electrodes constructed of different metals. The available evidence indicates that the deposition of cells on the electrodes always occurred at approximately the same potential with respect to the normal hydrogen electrode rather than being related to the point of zero charge of the materials in question.

A series of experiments was carried out matching the effects of ADP and the anodic electrode on pertinent aggregation comparing increasingly anodic potentials to the two increasing concentrations of ADP. Thus platelet aggregation decreased turbidity at various concentrations of ADP in the cell was compared with anodic potential to produce the same degree of aggregation, that is, decreased turbidity.

Following this, samples of PRP (platelet rich plasma) which had been exposed to various anodic potentials for a period of 3 minutes were masured for optical density changes compared with no current control (FIG. 3). By inference, comparing the slope of the control curve, produced by varying concentrations of ADP with the curve produced by anodic potentials, the possible correlation between the anodic potentials, inducing platelet release reaction, through the release of endogeneous ADP, and ADP addition to the cell can be compared.

As one approaches the anodic region of the control curve there is a rapid increase in the number of cells adhering to the electrode. Visual observations indicate that second stage platelet adhesion is occurring at appoximately +400 mv NHE. Beyond this, the rate of increase in the number of cells adhering to the electrode levels off.

Experiments were conducted comparing heparin, a potent anticoagulant, as a standard and brinase(C a potent proteolytic agent (very capable of lysing proteins). The effect observed with these drugs is plotted in FIG. 2(a). It demonstrates a large reduction in the number of cells adhering to the electrode regardless of the surface potential. The results seem to suggest that heparin is reducing the adhesive behavior of the plate' lets through an absorption phenomenon. The chemical structure of heparin, which is a repeating polymer of mucopolysaccharide units with an abundance of SO, groups represents an ideal compound for increasing the mutual electrostatic repulsive forces between the cells and foreign surface, if absorption takes place. The

results with ASA and with 3,5 dichloroaspirin demonstrate a similar trend. The number of cells adhering to the surface of the electrode fall far below the number adhering in the control situation in the anodic region. At no time was any viscous metamorphosis (agglutination) observed with the addition of ASA. This might be explained by the data of other reseachers who suggest that ASA acetylates the platelet membrane and by blocking ADP release, suppresses the secondary wave of platelet aggregation in in vitro studies (Stemerman, M. B. Drugs, platelets and vascular injury, Proceedings of the 4th Inter. Hemostasis Conference No. 23, I972; Mustard, 1. F., et al. Modification of platelet function, Ann. of the N.Y. Acad. of Sci., Vol. ZOI, I972). The results with C the proteolytic enzyme, are conventional. Some cell lysis was observed. Almost no cell adhesion was seen with varying surface potentials.

The results with protamine sulfate (neutralizing agent for heparin) demonstrate (FIGS. 4(a) and 4(b) a marked increase in the rate and number of cells adhering to the electrode. What is observed, upon analysis of the curve, is that there is a shift in the potential at which adhesion occurs, i.e., from +400 mv NHE to 200 mv NHE, with an almost linear relationship be tween potential versus number of cells adhering to the Pt surface. The skeletal proteins of the platelet membrane provide a number of possible sites for bond formation with macromolecular (e.g., COO, NH and SH) groups. The absorption of protamine sulfate onto the platelet membrane through disulfide bridging or due to an acid-base level reaction between the COOH and amine groups can explain the observed behavior.

Chlorpromazine has been reported to block the glucosyltransferase reaction reported to be the basis for platelet adhesion to an organic substrate. The results obtained by the present experiments indicate a conflict. Chlorpromazine seems to enhance the adhesion characteristics of platelets. Here again one is not sure of the mechanism. Chlorpromazine may be involved metabolically, producing an external alteration thus enhancing platelet adhesion in the absence of collagen.

The data presented herein tends to support the promise that mutual electrostatic repulsion between blood platelets and the arterial wall is just one of the forces responsible for homestatis in the blood vascular system (Born, GVR. Current ideas on the mechanism of platelet aggregation, Ann of the NY. Acad. of Sci., Vol. 201, I972). The results indicate that anodic surface potentials enhance platelet adhesion to appropriately charged foreign surfaces in vitro. Visual observations link first stage viscous metamorphosis with a point near the point of zero charge of the metal. Maximum platelet adhesion occurs on a PI wire at a surface potential of +400 mv NHE.

Various anticoagulants such as ASA, 3,5 dichloroaspirin and heparin appear to alter the adhesion charac teristics of platelets by a simple adsorption process. Both protamine sulfate and chlorpromazine enhance the adhesive characteristics of platelets. In the latter case the results cannot be explained by a simple adsorption phenomenon, and metabolic involvement must be indicated. This test system seems to be a useful one in evaluating the effects of various pharmacological agents upon platelet adhesion characteristics in association with foreign surfaces.

The structure of 3,5 Dichloroaspirin is COOl-I C OCH3 Results using 3,5 dichloroaspirin are shown in the following table (reference being made to Applicants *3.5 Dichloroaspirin Streaming Potential (in vivo) Electrtmsmosis Electrophoresis in vivo dogs Zeta Potential Canine Vessels In Vivo Canine Platelets RBCs jugular artery rt artery lt Intime Adventitia Zeta Potential vein femoral Artery IS!) IT] (7) Control Control 24 hrs. l8.7 control U.l J.5 ().25 Control Vein 7.l 5.l (3) 76 hrs l6.9 crush +0.2 +0.5 Fogarty +0.5

3.5 l 3,5 Dichloro- Dichloro control ll.25 l .0 AH) 3.5 aspirin aspirin crush 4).l U.5 Dichloro- Artery HM I315 (2) 300 mg/ 31M) mg/ Fogarty ().5 aspirin day da 3(lllmg/ 2 day 24 hrs l7.l control tl.5 (l.h ().S orally Vein 9.0 13.5 (I) 76 hrs l8.2 crush l.2 (l.h Fogarty t).5

Coagulation Study Rat Mesentry TR APTT RCT Occlusion Current 65 Micro Amp sion 65 Micro Amp Femoral Femoral Total occlusion Time vein Artery Control 48.0 r min 3 Control control U.h (l.l lst day 5.9 l .0 20.5 crush ().25 ().3 4th day 6.6 ...(i 83.1 3,5 Fogarty +(l.l (l. l Dichloroaspirin 3.5 5mg/kg *Signs refer to the potential at Dichloroaspirin drug given the interface. 300 mg) day oral l.V. A: 93.0: 31.1) min.

lst day 5.3 lfi.5 74.4 hr. before 4th day 5.9 2L4 78.4 lst day 7.3 17.4 98.7 4th day 6.! 33.3 l()l.() Smg/kg lst day 7 h l2.2 3 l .5 IV. daily 70.0: 13 mins. 4th day 5 days prior US. Pat. No. 3,722,504).

Regarding the above electrochemical screen measurements of the canine vessel. tests on streaming potential under control conditions and in comparison with aspirin, electroosmosis of canine artery and veins while using this material. platelet electrophoresis determinations. the effect of 3.5 dichloroaspirin on the charge characteristics of platelets and red cells as indicated. coagulation studies and measurements of the time but increases the partial thromboplastin time (PTI) and the recalcification time (RCT) of blood from the four dogs in which the studies were carried out. Five milligrams per kilogram intraperitonealy onehalf hour before the study and 5 mgs/Kg intraperitonealy for each of 5 days before the test prolonged rat mesentery occlusion time from a control level of 48 minutes for a single intraperitonealy dose and minutes for a five day intraperitoneal dose.

Data indicates that 3.5 dichloroaspirin has a dramatic effect on the blood-vascular interface. Rather limited effects which are not entirely explained are seen. by electroosmosis of canine blood vessels, in animals given 300 mgs. per day orally and rather marked effects on platelets. The dramatically increased coagulation times and RC'T indicated that it has very fundamental effects on the coagulation enzymes.

A summary of its physiologic effect as shown by rat mesentery studies demonstrate prolonged rat mesentery electrical thrombosis times and therefore it has profound antithrombotic effects on the intact microcirculation.

2. A method as claimed in claim 1 comprising administering the 3,5 dichloroaspirin orally or intraperitonealy to increase the streaming potential of the vascular interface and to increase clotting time, thrombin time, partial thromboplastin time and recalcification time.

3. A method as claimed in claim 2 wherein the 3,5 dichloroaspirin is administered orally in an amount in the order of magnitude of about 15 mg./kg. of body weight daily.

4. A method as claimed in claim 2 wherein the 3,5 dichloroaspirin is administered intraperitonealy in an amount in the order of magnitude of about 5 mg./kg. of body weight daily.

Claims (4)

1. A METHOD FOR RENDERING MORE NEGATIVE THE INTERNAL SURFACE OF A VASCULAR SYSTEM IN ORDER TO DECRESE THROMBOTIC TENDENCY, SAID METHOD COMPRISING ADMINISTERING 3,5 DICHLOROASPIRIN IN THE ORDER OF MAGNITUDE OF ABOUT 5-15 MG./KG OF BODY WEIGHT DAILY TO A HOST REQUIRING SUCH TREATMENT, THE 3,5 DICHLOROASPIRIN HAVING THE STRUCTURE

2. A method as claimed in claim 1 comprising adMinistering the 3,5 dichloroaspirin orally or intraperitonealy to increase the streaming potential of the vascular interface and to increase clotting time, thrombin time, partial thromboplastin time and recalcification time.

3. A method as claimed in claim 2 wherein the 3,5 dichloroaspirin is administered orally in an amount in the order of magnitude of about 15 mg./kg. of body weight daily.

4. A method as claimed in claim 2 wherein the 3,5 dichloroaspirin is administered intraperitonealy in an amount in the order of magnitude of about 5 mg./kg. of body weight daily.

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