Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

• 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.
• 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 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 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)
• PLA2 's phospholipases A 2
• Horigome et al., 1987; Diccianni et al., 1990; 1991 phospholipases A 2
• 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).
• inhibitors may constitute useful tools for understanding the mechanism of action of myotoxic Pl-A ⁇ 's.
• 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.
• Basic myotoxic phospholipases A 2 from the venom of Bothrops asper are cytolytic to a variety of cell types in culture, including L6 myoblasts and tEnd capillary endothelial cells.
• 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.
• myotoxin ⁇ a lysine-49 phospholipase A 2 devoid of enzymatic activity
• 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.
• 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).
• LA-heparin low anticoagulant heparin
• LMW-LA-Heparin low molecular weight- low anticoagulant heparin
• heparan sulphate low anticoagulant heparin
• LMW-LA-Heparin low molecular weight- low anticoagulant heparin
• 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.
• PLA2 myotoxic phospholipases A2
• 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.
• LA-heparin low anticoagulant heparin
• LMW-LA-Heparin low molecular weight- low anticoagulant heparin
• the medicament could be adopted for injectable, topical or oral administration.
• the injection could 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.
• PDA2 phospholipases A2
• LA-heparin low anticoagulant heparin
• LMW-LA-Heparin low molecular weight- low anticoagulant heparin
• Fig. 1 Myotoxic action of whole B. asper venom in mice.
• Fig. 2 Lactic dehydrogenase (LDH) release from damaged tEnd (m) and L6 (1) cells.
• Fig. 3A Cytotoxic activity of myotoxin II.
• Fig. 3B Cytotoxic activity of myotoxin II.
• Fig. 4 SDS-PAGE (Example 4)
• Fig. 5 Myotoxic activity. (Example 5)
• 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).
• mice 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).
• samples of injected tissue were evaluated histologically on hematoxylin-eosin stained sections to confirm data from the enzymatic assay.
• 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.
• L6 rat myoblasts ATCC CRL 14578
• 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.
• myotoxin was preincubated with the tested agents for 15 min at room temperature, and then applied to cultures.
• cultures were preincubated with heparin (170 ⁇ g/ml) for 2 hr at 37°C, washed, and then exposed to myotoxin as described.
• 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.
• PBS phosphate-buffered saline
• Example 1 Heparins neutralize the myotoxic action of whole B. asper venom in mice.
• heparin 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 ⁇ g 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
• LA-heparin which does not give the hemorrhage side effect of standard heparin, has advantageous therapeutic effect.
• 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.
• FIG. 2 shows (A) Time-course of lactic dehydrogenase (LDH) release from damaged tEnd (m) and L6 (1) cells after incubation with myotoxin 11 (10 ⁇ g/well). LDH release is expressed as a percentage, considering the enzyme activity of 0.1% Triton X-100 treated cells as
• Example 3 Standard heparin and LA-heparin block the cytotoxic activity of mvotoxin II.
• 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.
• I-H ⁇ - ⁇ UA-2S-[l ⁇ 4]-GlcN-6S 102 ⁇ 1 n.t.
• II-H ⁇ - ⁇ UA-[l ⁇ 4]-GlcN-6S 102 ⁇ 1 n.t.
• I-S ⁇ - ⁇ UA-2S-[l ⁇ 4]-GlcNS-6S 101 ⁇ 1 n.t.
• II-S ⁇ - ⁇ UA-[l ⁇ 4]-GlcNS-6S 99 ⁇ 1 n.t.
• III-S ⁇ - ⁇ UA-2S-[l ⁇ 4]-GlcNS 100 ⁇ 2 n.t.
• IV-S ⁇ - ⁇ UA-[l ⁇ 4]-GlcNS 100 ⁇ 2 n.t. n.t.: not tested.
• **the toxin challenge was 10 and 20 ⁇ g for tEnd and L6 cells, respectively.
• 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.
• Lane 1 crude B. aspervenom, 10 ⁇ g; lane 2: heparin-binding fraction, 20 ⁇ g; lane 3: myotoxin 11, 10 ⁇ g; lane 4: mixture of myotoxins I and III, 10 ⁇ g.
• Coomassie R-250 stain, cathode is at the bottom.
• C gel diffusion in 1% agarose-PBS. Wells were filled with 20 ⁇ l 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.
• 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 ⁇ g heparin/ ⁇ g myotoxin, and incubated for 15 min at room temperature. Then, mixtures were assayed for in vivo myotoxic activity (50 ⁇ g myotoxin/mouse) or in vitro phospholipase A 2 activity (5 ⁇ g myotoxin/ tube). Activities are expressed as a percentage, considering the activity of the toxin alone as 100%. Bars represent mean ⁇ SD of triplicate dete ⁇ ninations. See Figure 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 ⁇ g/per 15xl0 6 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
• glycosaminoglycans of the heparin/ heparan sulfate family block the cytolytic action of basic myotoxic PLA 2 '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.
• myotoxin isoforms described in B. aspervenom (Lomonte and Carmona, 1992) could be recovered from a heparin affinity column.
• myotoxin II a Lys- 49 PLA 2 isoform, was studied in more detail in respect to its interaction with the heparins.
• myotoxic PLA 2 Since the muscle damaging effect of myotoxic PLA 2 '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; Bultr ⁇ n 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 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 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.
• a 2 to phospholipid micelles importance of the amino-te-rminus.

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