WO1995012403A1

WO1995012403A1 - Use of low anticoagulant heparinderivatives

Info

Publication number: WO1995012403A1
Application number: PCT/SE1994/001030
Authority: WO
Inventors: Lars Å. HANSON; Andrej Tarkowski; Bruno Lomonte
Original assignee: Pharmacia Ab
Priority date: 1993-11-02
Filing date: 1994-11-02
Publication date: 1995-05-11
Also published as: SE9303612D0; AU8119594A

Abstract

The present invention relates to the use of low anticoagulant heparin (LA-heparin), low molecular weight-low anticoagulant heparin (LMW-LA-Heparin) or low anticoagulant heparan sulphate for the manufacture of a medicament regulating phospholipases A2 (PLA2). The medicament can be used against myotoxic phospholipases A2 (PLA2) and against bites from snakes or insects. The medicament also inhibits cellular PLA2 in human leukocytes and can be used for treating tissue damages caused by PLA2 and function as an anti-inflammatory medicament. The medicament can be adapted for injectable, topical or oral administration.

Description

USE OF LOW ANTICOAGULANT HEPARINDERIVATIVES

The invention relates to the use of low anticoagulant heparin (LA- heparin), low molecular weight- low anticoagulant heparin (LMW-LA- Heparin) or low anticoagulant heparan sulphate for the manufacture of a medicament regulating phospholipases A2 (PIA2). The medicament can be used against myotoxic phospholipases A2 (PLA2) and against bites from snakes or insects. The medicament also inhibits cellular PLA2 in human leukocytes and can be used for treating tissue damages caused by PIA2 and function as an anti-inflammatory medicament.

The medicament could be adapted for injectable, topical or oral administration.

INTRODUCTION

Heparin is a sulphate containing polysaccaride which on a large scale is isolated from intestinal mucosa from swine or lung from cow. It has for several decades been used clinically as an effective drug for the prevention and treatment of thromboembolic disorders but it sometimes causes bleeding complications.

Low anticoagulant heparin (LA-heparin) can be obtained by fractionation of standard heparin by affinity chromatography on antit-hrombin -Sepharose (SE 760340-2) and collecting the low affinity fraction or alternatively by selective chemical treatment of standard heparin (SE 9191155-1). Low anticoagulant heparins generally have an anti-FXa less than 40 IU/mg compared to standard heparin which gererally has an anti Xa activity of 120-200 IU/mg. Low molecular weight heparins (LMW heparin) can be obtained either by fractionation of standard heparins or more commonly by depolymerization of standard heparin by chemical or enzymatic methods. LMW heparins are characterized by average molecular weigts in the range 2 000 - 10 000 Da. Their anticoagulant activity can vary considerably but generally is above 70 IU/mg anti FXa. LMW- LA-heparin as well as heparan sulphates of high and/or low molecular weights ( which always are of low anticoagulant activity) can be obtained in different ways for example from side fractions in the manufacturing of heparins and/or LMW-heparins.They can also be obtained by chemical or enzymatic cleavage of purified heparins or heparan sulphate followed by suitable purification which may or may not involve fractionation on antithrombin-Sepharose. LMW-LA heparins and low molecular weight heparan sulphates typically have an average molecular weigt in the range 1000 - 10 000 Da and an anti-FXa activity below 70 IU/mg. Heparan sulphates have very similar structure to heparin but are less sulphated. They can have average molecular weights of 10 000 - 40 000 Da. The described heparins with reduced anticoagulant activity all show a reduced haemorrhagic effect compared to heparin. (Palm M, Mattson C, Svahn CM and Weber M. Thrombosis and Haemostasis 64 (1990), 127-132)

In recent years, hepaiin has been shown to interact in vitro with several types of phospholipases A₂ (PLA₂'s) (Horigome et al., 1987; Diccianni et al., 1990; 1991) and in some cases, to inhibit their enzymatic activity. PLA2's are notoriously abundant and widely distributed in snake venoms, showing a variety of toxic activities, such as neurotoxicity, myotoxicity, anticoagulant effect, and edema-forming activity (Kini and Evans, 1989). Non immunologic inhibitors of myotoxic PLA2's could be of interest therapeutically, especially considering that antivenom immunoglobulins do not completely prevent the muscle damage that follows envenomation (Gutierrez et al., 1981; Ownby et al., 1983). In addition, inhibitors may constitute useful tools for understanding the mechanism of action of myotoxic Pl-A^'s.

The neutralization of myotoxic activity of the venom of Bothrops jararacussu, a crotalid species from Brazil, by heparin has been reported (Melo and Suarez-Kurtz, 1988; Melo et al., 1993). This interesting finding has been further investigated, using myotoxin π, a basic PLA₂ purified from the venom of B asper (Lomonte and Gutierrez, 1989), and with a special interest on heparin derivatives with little or no effect on the coagulation system, i.e. heparin with low affinity for antithrombin (LA-heparin). Myotoxin II is a natural PLA2 isoform devoid of enzymatic activity, mainly due to the critical amino acid substitution at position 49 (Asp→Lys) (Francis et al., 1991), but still exerts myotoxic activity. In some experiments, another isoform that has PLA2 activity, myotoxin III (Kaiser et al., 1990), was utilized to investigate the effect of heparins on the enzymatic activity.

Basic myotoxic phospholipases A₂ from the venom of Bothrops asper are cytolytic to a variety of cell types in culture, including L6 myoblasts and tEnd capillary endothelial cells.

In Immunology Today, Vol 12, No 5, 1991, 143-46. W Pruzanski et al have given evidence supporting PLA2 as a mediator of inflammation, specifically in linking local and systemic effects. Different pathophysiological effects of PLA2 are shown in Fig 2 in the article.

Glycosaminoglycans of the heparin/heparan sulfate family have now been found to be potent blockers of this cytolytic action, and, as well, to be able to neutralize the muscle damaging activity of purified myotoxins and crude venom in vivo. Using the cell cytotoxicity system as a model for myotoxin action, the cytolytic activity of myotoxin π (a lysine-49 phospholipase A₂ devoid of enzymatic activity) and its inhibition by glycosaminoglycans was characterized. The blocking activity of heparin did not depend on its anticoagulant activity, since both standard heparin, heparin with low affinity for antithrombin (LA- heparin) and low molecular weight LA-heparin (LMW-LA- heparin) had a similar activity. Heparan sulfate and low molecular weight heparin also neutralized the toxic activity of myotoxin II. In contrast, different heparin-derived disaccharides were unable to block cytolysis, implying a requirement for carbohydrate chains comsisting of more than two saccarides for the interaction with the protein. By affinity chromatography and gel diffusion, it was demonstrated that heparins form a complex with all isoforms of venom myotoxins, held at least in part by electrostatic interactions. The results of this study demonstrate the use of low anticoagulant or non-anticoagulant heparins and heparan sulphate as an aid in the treatment of bites from snake or insects from certain species, and also as useful tools for understanding of the mechanism of action of myotoxic phospholipases A2.

It has now, for the first time, been found and shown that the ability of heparin to form complexes with myotoxins and to block their cytolytic action did not depend on its anticoagulant activity, since the same results i.e. complete neutralization, were obtained when comparing conventional heparin with heparan sulphate, LA-heparin, and LMW-LA heparin, although not with a series of heparin-derived disaccharides. Heparan sulphate has a similar structure to standard heparin but has no or little anti-coagulant effect. LA-heparin and LMW-LA-heparins have less hemorrhage effect than standard heparin and thus less anticoagulant effect.

This is of great clinical value since e.g. Bothrops venoms contains potent hemorrhagic toxins and also severely disturb coagulation.

We have also shown that LMW-LA- heparin could reduce the PLA2 activity measured as a decrease of the production of the leucotrienes B4 and C4 in human leukocytes, which suggests a considerable potential for therapeutic use.

The present invention thus relates to the use of low anticoagulant heparan sulphate, low anticoagulant heparin (LA-heparin) or low molecular weight- low anticoagulant heparin (LMW-LA-Heparin) for the manufacture of a medicament regulating phospholipases A2 (PLA2), and especially against myotoxic phospholipases A2 (PLA2).

It also relates to the use of heparan sulphate, low anticoagulant heparin (LA-heparin) or low molecular weight- low anticoagulant heparin (LMW-LA-Heparin) for the manufacture of a medicament against bites from snakes or insects. Preferably the medicament is against snake venom containing myotoxic phospholipases A2 (PLA2) and more particular for the treatment of bites from Crotalids family, especially Bothrops species. The invention also relates to the use of low anticoagulant heparan sulphate, low anticoagulant heparin (LA-heparin) or low molecular weight- low anticoagulant heparin (LMW-LA-Heparin) for the manufacture of a medicament inhibiting PLA2 in human leukocytes. The regulation, especially of extra cellular PLA2 in humans, can be used for preventing and treating tissue damages and functioning as an anti- inflammatory medicament. Anoxie, such as in stroke and neonatal asphyxia can be mentioned as examples of diseases with tissue damages where PLA2 seems to play an important role.

The medicament could be adopted for injectable, topical or oral administration.

The injection could be be subcutanous or intravenous. The topical form can e.g. be as powder or ointments.

The oral form of the medicament can e.g. be pills, lozenges, tablets, capsules, liquids or other suitable forms. If absorption of the medicament is to take place in the intestine, the composition may be given an enteric coating such as cellulose acetate-phthalate or styrene- maleic anhydride copolymers. Enteric coatings are well known in the art and are discussed for example in Remington' s Practice of Pharmacy.

The medicament can also be in a composition as disclosed in WO 9319737.

The medicament comprises preferably at least a tetrasaccarid of heparan sulphate and more preferably a LMW-LA heparin/heparan sulphate with a molecular weight of at least 2000.

LMW heparins and heparan sulphate normally have a molecular weight less than 10 000 kDa.

The invention also relates to a method for regulating phospholipases A2 (PLA2) and for treatement of bites from snakes or insects by administration of low anticoagulant heparan sulphate, low anticoagulant heparin (LA-heparin) or low molecular weight- low anticoagulant heparin (LMW-LA-Heparin). It also relates to a method for treatement of tissue damages and/or inflammatory diseases.

FIGURE LEGENDS

Fig. 1: Myotoxic action of whole B. asper venom in mice. (Example 1) Fig. 2: Lactic dehydrogenase (LDH) release from damaged tEnd (m) and L6 (1) cells. (Example 2) Fig. 3A: Cytotoxic activity of myotoxin II.(Example 3) Fig. 3B Cytotoxic activity of myotoxin II. (Example 3) Fig. 4: SDS-PAGE (Example 4) Fig. 5: Myotoxic activity. (Example 5)

MATERIALS AND METHODS

Glycosaminoglycans Standard heparin (5000 IE/ml), heparin with low anticoagulant effect, i.e. heparin with low affinity for antithrombin (LA-heparin; 7 IU anti- factor Xa/mg, M_r 15 kDa), low molecular weight heparin (Fragmin®, 25000 IE/ml, M_r 5 kDa) and low molecular weight- low anticoagulant heparin (LMW-LA -heparin. Mr 2,5 kDa) were provided by Kabi Pharmacia (Sweden). Heparitinase, heparan sulfate and a series of heparin-derived disaccharides, were purchased from Sigma Chemical Co. (St Louis, MO, USA).

Myotoxin purification Crude B. asper venom from Costa Rica was fractionated by CM-Sephadex chromatography as previously described (Lomonte and Gutierrez, 1989) to obtain pure myotoxin π, as evaluated by SDS-PAGE (Laemmli, 1970) and cathodic native PAGE at pH 4.3 (Reisfeld et al., 1962). B. asper myotoxin III (Kaiser et al., 1990). Myotoxic activity

Myotoxicity was evaluated in mice (Swiss, 10-24 g) by injecting either crude venom or pure myotoxins, alone or after incubation with heparins ( 15 min at room temperature), by i.m. route (gastrocnemius), and then measuring the serum creatine kinase (CK; EC 2.7.3.2) activity after 3 hr ( Gutierrez et al., 1981). In some experiments, samples of injected tissue were evaluated histologically on hematoxylin-eosin stained sections to confirm data from the enzymatic assay. In addition, neutralization of myotoxicity by heparins was evaluated qualitatively using an intravital microscopic technique to study the effect of local application on the mouse cremaster muscle.

In vitro cytotoxicity

The activity of myotoxin II was studied in vitro by a cytotoxicity assay using two cell lines. L6 rat myoblasts (ATCC CRL 1458) and tEnd cells, a polyoma virus-transformed mouse endothelial cell line of thymic capillary origin (Bussolino et al., 1991) were used. Cells were routinely grown in Iscove's medium (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (FCS; Biological Ind., Haemek, Israel), 2 mM L- glutamine, 5xl0^-5 M 2-mercaptoethanol, and 0.05 mg/ml gentamycin. In order to quantify cytotoxicity, cells were seeded at l-4xl0⁴/well in 96-well plates and grown for 2-4 days. At the moment of the assay, culture medium was removed and replaced with 150 μl/well of medium with 1% FCS containing the tested agents. The FCS concentratio was lowered to 1% in order to minimize the basal lactate dehydrogenase (LDH) activity of the medium. After 3 hr of incubation at 37°C, 100 μl of supernatant were assayed for LDH released from damaged cells. As controls for 100% and 0% cytotoxicity, cells were incubated with 0.1% Triton X-100-containing medium, or plain medium, respectively. All samples were assayed in triplicate wells. For neutralization experiments, myotoxin was preincubated with the tested agents for 15 min at room temperature, and then applied to cultures. In order to determine if the inhibitory activity of heparins on myotoxin-induced cytolysis was due to an effect on the target cells, cultures were preincubated with heparin (170 μg/ml) for 2 hr at 37°C, washed, and then exposed to myotoxin as described. Phospholipase A2 assay

An indirect hemolytic assay in fluid phase, based on the gel diffusion technique described by Gutierrez et al. ( 1988) was utilized. Washed sheep red blood cells were suspended at 1.5% (v/v) in 0.12 M NaCl, 0.04 M sodium phosphate buffer, pH 8.1, containing 1% egg yolk as a source of phospholipids and 0.09 mM CaCl2. To 500 μl of this suspension, 5 μl (containing 5 μg) of myotoxin III alone, or after preincubation with heparins (at a ratio of 5 μg heparin/μg toxin), were added and incubated for 30 min at 37°C. Then, 3 ml of buffer were added to each tube, and hemolysis was read at 540 nm after centrifugation. To assure the PLA2 dependency of the lysis and the lack of direct hemolysis, parallel myotoxin samples were run using red blood cells in the absence of egg yolk phospholipids.

Affinity chromatography

Crude B. asper venom. (25 mg) was applied to a column of heparin- agarose (Sigma) equilibrated with phosphate-buffered saline (PBS), pH 7.2. After the absorbance at 280 nm of the eluent returned to baseline, elution of the heparin-binding fraction was performed by either a stepwise or a linear gradient to 1 M NaCl. The eluted fraction was analyzed by SDS-PAGE and native cathodic PAGE as described above.

Gel diffusion The interaction between heparins and either crude venom or myotoxin II was tested by gel diffusion in 1% agarose-PBS plates

(Ouchterlony and Nilsson, 1978). After 24 hr of incubation at room temperature, gels were washed with PBS and stained with Coomassie blue R-250.

EXAMPLES

Example 1. Heparins neutralize the myotoxic action of whole B. asper venom in mice.

Venom and different amounts of heparins were mixed, incubated 15 min at room temperature, and then injected i.m. into groups of four mice (50 μg venom/ mouse). After 3 hr, creatine kinase (CK) levels in serum were determined as an indicator of skeletal muscle damage (% myonecrosis). CK values obtained with venom alone were taken as 100%. St heparin: standard heparin (empty bars); LA heparin: low affinity heparin (filled bars); PBS: phosphate-buffered saline. Bars represent mean ±SD of four determinations. All values are significantly (p<0.05) lower than the venom control. See figure 1

When whole B. asper venom, was preincubated with either standard or LA-heparin, and subsequently injected into mice, its myotoxic action was significantly reduced in a dose-dependent manner (Fig. 1). This result was confirmed by histological evaluation and by the use of intravital microscopy. In the latter system, widespread muscle fiber damage regularly developed 4-6 min after application of venom alone, while, when it was mixed with standard or LA-heparin before application, muscle fibers were protected throughout the observation period of 30 min. In contrast to LA-heparin, standard heparin markedly increased the amount of hemorrhage induced by the venom, evident both histologically and intravitally.

This clearly shows that LA-heparin, which does not give the hemorrhage side effect of standard heparin, has advantageous therapeutic effect.

Examρle2. Mvotoxin II is cvtotoxic to cultured rat mvoblasts (L6) and mouse capillary endothelial cells (tEnd). To analyze in more detail the neutralizing ability of heparins towards myotoxins of B. asper venom, an in vitro cytotoxicity assay was developed.

Figure 2:shows (A) Time-course of lactic dehydrogenase (LDH) release from damaged tEnd (m) and L6 (1) cells after incubation with myotoxin 11 (10 μg/well). LDH release is expressed as a percentage, considering the enzyme activity of 0.1% Triton X-100 treated cells as

100%.

(B) Dose-response curves of cytotoxic activity of myotoxin π on tEnd (1) and L6 (m) cells, measured by release of LDH at 3 hr. Each point represents the mean ±SD of triplicate wells. Myotoxin II was cytotoxic not only to the L6 myoblasts, but also to tEnd cells, in the concentration range of 25-150 μg/ml. The endothelial cells were significantly more susceptible than L6 cells to the cytotoxic action of myotoxin (Figs. 2 A and 2B). An incubation time of 3 hr (Fig. 2A) and a toxin challenge of 20 and 10 μg/well, for L6 and tEnd cells, respectively (Fig. 2B), were selected for all subsequent experiments, to assure 100% cytotoxicity in the absence of a neutralization effect.

Example 3. Standard heparin and LA-heparin block the cytotoxic activity of mvotoxin II.

Either standard (St-H; empty symbols) or low affinity (LA-H; filled symbols) and low molecular weigt-low anticagulant (LMW-LA-Hep) heparins were mixed with myotoxin π at the indicated proportions, incubated for 15 min at room temperature, and then assayed for cytotoxic activity on tEnd (circles) and L6 (triangles) cells. The toxin challenge for tEnd and L6 cells was 10 and 20 μg/well, respectively. Cytotoxicity was measured by the release of lactic dehydrogenase (LDH) from cells at 3 hr. Each point represents the mean ±SD of triplicate wells. See Figures 3A and 3B.

When preincubated with myotoxin II, standard as well as LA-heparin and LMW-LA-heparin blocked its cytolytic activity on both cell types. Cells were completely protected at approximate ratios of 0.02 and 0.3 μg heparin/μg myotoxin, for L6 and tEnd cells, respectively, with no observable differences in the inhibitory efficiency of the three heparin types (Fig. 3 A and 3B). Complete neutralization was also achieved with heparan sulfate and the low molecular weight heparin, although not with a series of heparin-derived disaccharides (Table 1). On the other hand, pretreatment of the cells with heparins, followed by washing, had no protective effect on myotoxin II-induced cytolysis.

Table 1 shows that heparan sulfate and low molecular weight heparin (Fragmin®), but not heparin disaccharides of different types as explained in the table, block the cytotoxic activity of myotoxin II on L6 myoblasts and tEnd endothelial cells. Fragmin® has a higher hemorrhagic effect than heparan sulfate and therefore heparan sulfate is here the preferred heparin derivative.

labl≤J Tested Agent, ratio % LDH* Release, mean ±SD L6 cells tEnd cells

Myotoxin II control** 96 ± 3 99 ± 2

Heparan sulfate, 0.0075 μg/μg myotoxin n.t. 101 ± 1

, 0.075 μg/μg myotoxin 95 ± 1 82 ± 2 " " , 0.75 μg/μg myotoxin 4 ± 1 2 ± 1

Fragmin®, 0.75 μg/μg myotoxin 3 ± 3 5 ± 1

Heparin disaccharides, 0.75 μg/μg myotoxin I-A: α-ΔUA-2S-[l→ 4]-GlcNAc-6S 97 ±3 n.t. II-A: α-ΔUA-[l→4]-GlcNAc-6S 99 ±2 n.t. III-A: α-ΔUA-2S-[l→ 4]-GlcNAc 101 ±2 n.t.

I-H: α-ΔUA-2S-[l→ 4]-GlcN-6S 102 ± 1 n.t. II-H: α-ΔUA-[l→4]-GlcN-6S 102 ± 1 n.t. I-S: α-ΔUA-2S-[l→ 4]-GlcNS-6S 101 ± 1 n.t. II-S: α-ΔUA-[l→4]-GlcNS-6S 99 ± 1 n.t. III-S: α-ΔUA-2S-[l→ 4]-GlcNS 100±2 n.t.

IV-S: α-ΔUA-[l→4]-GlcNS 100±2 n.t. n.t.: not tested.

*LDH: lactic dehydrogenase.

**the toxin challenge was 10 and 20 μg for tEnd and L6 cells, respectively.

The finding in table 1 are interesting as they show that heparin-derived disaccarides are not capable to block the cytolytic effect of myotoxic PLA2. It has been shown that heparins with low anticoagulant effect are as good as heparin in blocking the cytolytic effect of myotoxic PLA2. When giving the medicament to a patient in need of an agent against the poison from an animal, the risk of bleeding is thus -minimized and side-effects are reduced.

The low molecular heparin with low anticoagulat activity for antithrombin is preferred, as the low molecular heparins have higher bioavaibility.

Example 4. B. asper venom myotoxins bind to heparin. Whole venom was fractionated on a column of heparin-agarose (Sigma) as described in Materials and Methods, and a heparin-binding fraction was obtained by salt elution. (A) analysis of the heparin-binding fraction by SDS-PAGE, 15%. Lane 1: molecular weight standards (106, 80, 49.5, 32.5, 27.5 and 18.5 kDa); lane 2: crude B. aspervenom, 10 μg; lane 3: myotoxin II, 5 μg; lane 4: heparin-binding fraction, 20 μg. All samples were reduced with 2-mercaptoethanol at 95°C. Coomassie blue R-250 stain, anode is at the bottom. (B) Analysis of the heparin-binding fraction by cathodic PAGE under native conditions.

Lane 1: crude B. aspervenom, 10 μg; lane 2: heparin-binding fraction, 20 μg; lane 3: myotoxin 11, 10 μg; lane 4: mixture of myotoxins I and III, 10 μg. Coomassie R-250 stain, cathode is at the bottom. (C) gel diffusion in 1% agarose-PBS. Wells were filled with 20 μl of the following solutions: 1 and 3: crude B. aspervenom, 20 mg/ml; 2: myotoxin II, 0.5 mg/ml; 4: standard heparin, 0.17 mg/ml; 5: LA- affinity heparin, 0.17 mg/ml. Coomassie blue R-250 stain. See Figure 4.

The heparin-binding components in the venom were isolated by affinity chromatography, eluting at approximately 0.5 M NaCl concentration. SDS-PAGE of this fraction showed a main band of 14-15 kDa under reducing conditions, which corresponds to the subunit molecular weight of myotoxins, and very small amounts of few other components (Fig. 4A). Native PAGE for basic proteins showed that all described myotoxin isoforms bound to heparin (Fig. 4B). Gel diffusion demonstrated that heparins form a precipitable complex with venom myotoxins (Fig. 4C). Example 5. Heparins block the myotoxic activity of mvotoxin III, but no its phosphQ-Upase Az activity.

Either standard (St-Hep) or low affinity (LA-Hep) heparins were mixed with myotoxin III at a ratio of 5 μg heparin/μg myotoxin, and incubated for 15 min at room temperature. Then, mixtures were assayed for in vivo myotoxic activity (50 μg myotoxin/mouse) or in vitro phospholipase A₂ activity (5 μg myotoxin/ tube). Activities are expressed as a percentage, considering the activity of the toxin alone as 100%. Bars represent mean ±SD of triplicate deteπninations. See Figure 5.

Since affinity chromatography and electrophoresis showed that heparin interacts not only with myotoxin II, but also with other isoforms present in this venom, the effect of heparins on the enzymatic activity of myotoxin HI was investigated. Both types of heparins significantly reduced the myotoxic effect of this isoform in vivo, without inhibiting its enzymatic activity (Fig. 5).

Example 6. Measurement of inhibition of PLA2 in human granulocytes. The production of the of leucotrienes B4 and C4 (LTB4 and LTC4) was measured according to the method described by Odlander B et al, "A rapid and sensitive method for measurement of leucotrienes based on HPLC" in Biomed. Chromatogr. 1987:2:145-7. LA-LMW-heparin was added in an amount of 10 to 5000 μg/per 15xl0⁶ leukocytes.

The leukocytes were prepared according to the method described by Shimizu t et al, "Effects of Pancreatic and Snake venom Phospholipase A2 on the Generation of Leukotriene B4 and C4 by Human Leukocytes in Vitro" in Pancreas, Vol 9, No 1, 37-41, 1994. The results are given in Table 2

(The mean value of three experiments). Table 2

LA-LMW-Heparin LTB4 LTC4 μg/5xl06leukocvtes % of control

10 100 100

100 79 83

500 84 74

1000 85 78

5000 50 57

This experiment shows that human PLA2 can be inhibited and that a high concentration of LA-LMW-heparin is needed to inhibit the intracellular PLA2.

DISCUSSION

The present results demonstrate that glycosaminoglycans of the heparin/ heparan sulfate family block the cytolytic action of basic myotoxic PLA₂'s from the venom of B. asper, both in cell culture and in vivo. This inhibition is clearly due to the formation of a complex, which is held, at least in part, by electrostatic interactions between the negatively-charged groups of heparins or heparan sulfate and the numerous positively-charged amino acid residues of myotoxins. These conclusions are supported by the direct observation of a precipitate in gel diffusion, and by the dissociation of myotoxins from the heparin affinity column with an increasing ionic strength. The structural nature of this interaction is currently being investigated. Melo et al. (1993), using a gel filtration technique, also obtained evidence for the formation of a complex between standard heparin and a myotoxic PLA₂ from B. jararacussu venom that has similar antigenic (Lomonte et al., 1990a) and physicochemical (Cintra et al., 1993) characteristics to the B. asper myotoxins utilized in this study. Previous work has shown that venoms from many species of Bothrops, distributed in Latin America, as well as other crotalids such as Trimeresurus Ωavoviήdis from Japan, contain components that cross-react antigenically with the group of B. asper myotoxin isoforms (Lomonte et al., 1990a), and therefore are presumably myotoxic PLA2's with similar properties, as confirmed in several cases (Lomonte et al., 1990b; Kihara et al., 1992; Diaz et al., 1992). Thus, the present results will apply not only to B. asper myotoxins but also to other myotoxic PLA2's.

Interestingly from the medical point of view, the ability of heparin to form complexes with myotoxins and to block their cytolytic action, did not depend on its anticoagulant activity or its molecular weight, since the same results were obtained when comparing conventional heparin, LA-heparin, LMW-heparin and LMW-LA-heparin. This is of clinical relevance since Bothrops venoms contain potent hemorrhagic toxins an also severely disturb coagulation (Gutierrez and Lomonte, 1989; Tan and Ponnudurai, 1991). Indeed, the potentiating effect of standard heparin on the microvessel damaging action of the venom was evident in the intravital microscopy experiments, emphasizing the potential risks of its use.This makes thus LA-heparin and LMW-LA-heparin more interesting for clinical use than standard heparin.

As shown by the electrophoretic analyses, all myotoxin isoforms described in B. aspervenom (Lomonte and Carmona, 1992) could be recovered from a heparin affinity column. Of these, myotoxin II, a Lys- 49 PLA₂ isoform, was studied in more detail in respect to its interaction with the heparins. Since the muscle damaging effect of myotoxic PLA₂'s is currently considered to be related to their ability to penetrate phospholipid bilayers and alter cell membrane permeability (Gutierrez et al., 1984a; Diaz et al., 1991; Rufini et al., 1992; Bultrόn et al., 1993; Bruses et al., 1993), an in vitro cytotoxicity system was utilized. Myotoxin II was clearly cytolytic to skeletal muscle myoblasts and capillary endothelial cells, and this effect was efficiently prevented by preincubation of the toxin with heparins or heparan sulfate. The ratios at which heparins completely blocked cytotoxicity suggest that several toxin molecules may simultaneously be neutralized by one heparin molecule. This could be explained by the polysaccharide nature of heparin, with its linear repeating unit structure (Kjellen and Lindahl, 1991). However, the neutralizing efficiency of heparins in the in vivo myotoxicity tests was lower than in the in vitro cytotoxicity assay. This could be due to several reasons. On one hand, although the group of basic PLA2 myotoxins has been shown to be the main mediator of muscle damage in this venom (Lomonte et al., 1987; 1990c), it is possible that other factors not affected by heparins, for example acidic hemorrhagic toxins causing ischemia, could contribute to some degree to myonecrosis (Ownby, 1990). Nevertheless, this possibility would only partially explain the observed difference, since even when using purified myotoxins, the neutralizing efficiency of heparin was lower in vivo than in vitro. An alternative, explanation is that myotoxins would have a significantly higher affinity for mature muscle fibers than for myoblasts (or other cell types) utilized in culture. Although it is clear that several types of cells can be killed in vitro by myotoxic PLA2's (Bruses et al., 1993; present study), the only cell type described to undergo necrosis in vivo is the mature muscle fiber (Gutierrez et al., 1984a). Immature muscle cell precursors are likely to be less susceptible to myotoxin action, as suggested by the regeneration of affected muscles (Gutierrez et al., 1984b; 1991; Harris and Cullen, 1990). The determination of the binding affinity of myotoxins to heparin, and to diverse cell types in culture, particularly to more differentiated muscle cell stages such as myotubes, would shed light on this hypothesis. Still another possibility to explain the lower in vivo efficiency of heparins would be a partial dissociation of myotoxin/heparin complexes, due to the competition caused by high affinity heparin-binding factors, presumably absent in the in vitro cytotoxicity system.

The interaction of heparin with PLA2 of different origins has been reported to affect enzymatic activity in some cases (Diccianni et al., 1990; 1991; Franson et al., 1974; Avila and Convit, 1976), but not in others (Condrea and de Vries, 1964; Ho et al., 1986; Horigome et al., 1987). In the case of myotoxin III, its PLA₂ activity was unaffected by heparins, despite that its myotoxic effect was significantly reduced. Thus, the two toxin actions were clearly dissociated, in agreement with earlier results obtained with neutralizing monoclonal antibodies (Lomonte et al., 1992).

The present results demonstrates the possibiUty of utilizing heparan sulphate, LA-heparin, and also smaller heparin fragments devoid of anticoagulant effects, as an aid in the treatment of snakebites from certain species. Bergonzini et al. (1992) demonstrated that bioavailability and distribution of heparins in biological compartments depend on their molecuar weight, favouring the use of LMW-LA- heparins over LA-heaprin. The screening of different heparin-derived disaccharides showed that the neutralizing effect cannot be achieved with such small fragments, and therefore requires slightly larger oligosaccharides, as has been shown for the interaction of heparin with other proteins (Maccarana and Iindahl, 1993).

The present result also shows the possibility of using heparin derivatives devoid of anticoagulant effect in the treatment of diseases where PLA2 plays an important role in induction of tissue damage and inflammation. This effect is of great clinical value, because there is need for better drugs against anoxie, such as stroke and neonatal asphyxia and varios forms of inflammation, having less side effects.

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Claims

1. Use of low anticoagulant heparin (LA-heparin), low molecular weight- low anticoagulant heparin (LMW-LA-Heparin) or low anticoagulant heparan sulphate for the manufacture of a medicament regulating phospholipases A2 (PLA2).

2. Use according to claim 1 for the manufacture of a medicament against myotoxic phospholipases A2 (PLA2)

3. Use according to claim 1 or 2 for the manufacture of a medicament against bites from snakes or insects.

4. Use according to any of claims 1 - 3 in which the medicament is against snake venom containing myotoxic phospholipases A2 (PLA2).

5. Use according to any of claims 1 - 4 for the treatment of bites from Crotalids family, especially Bothrops species.

6. Use according to any of claims 1-3 for the treatment of bites from an insect.

7. Use according to claim 1 for the manufacture of a medicament inhibiting PLA2 in human leukocytes.

8. Use according to claim 7 for the manufacture of a medicament for preventing and treating tissue damages.

9. Use according to claim 8 for the manufacture of a medicament, for treating anoxie, such as in stroke and neonatal asphyxia.

10. Use according to claim 7 for the manufacture of an anti- inflammatory medicament.

11. Use according to any of claims 1 to 10 in which the medicament is adapted for injectable ad-ministration.

12. Use according to any of claims 1 to 10 in which the medicament is adapted for topical administration.

13. Use according to any of claims 1 to 10 in which the medicament is adapted for oral administration.

14. Use according to any of claims 1 to 13 in which the medicament comprises at least a tetrasaccarid of low anticoagulant heparan sulphate or low molecular weight- low anticoagulant heparin (LMW-LA- Heparin).

15. Use according to any of claims 1 to 14 in which the medicament comprises LA-heparin or low anticoagulant heparan sulphate with a moelcular weight of at least 2 000.

16. Method for regulating phospholipases A2 (PLA2) by administration of low anticoagulant heparan sulphate, low anticoagulant heparin (LA- heparin) or low molecular weight- low anticoagulant heparin (LMW- LA-Heparin).

17. Method according to claim 16 for treatement of bites from snakes or insects.

18. Method according to claim 16 for prevention and treatement of tissue damages and/or i-nflammatory diseases.