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Title:  Antivenom immunesera

United States Patent:  6,833,131

Issued:  December 21, 2004

Inventors:  Smith; Damon Charles (Essex, GB)

Assignee:  Protherics Inc. (Brentwood, TN)

Appl. No.:  137168

Filed:  August 5, 1994

PCT Filed:  April 24, 1992

PCT NO:  PCT/GB92/00761

371 Date:  August 5, 1994

102(e) Date:  August 5, 1994

PCT PUB.NO.:  WO92/19280

PCT PUB. Date:  November 12, 1992

Abstract

An antivenom comprising a mixture of monospecific antisera each raised against venoms of one species or sub-species is disclosed. Also disclosed is a pharmaceutical composition comprising the antivenom of the invention, and a method of treating envenomation in a mammal comprising administering the claimed antivenom.

Description of the Invention

FIELD OF THE INVENTION

1. Background of the Invention

The present invention relates to antivenoms and processes for their preparation. More particularly, the invention relates to snake antivenoms and processes for their preparation.

2. Description of Related Art

A number of animals including snakes, gila monsters, spiders and bees produce venoms which are hazardous to man. For example, approximately one million people throughout the world are bitten each year by poisonous snakes. It has been estimated that of these some 100,000 die and that another 300,000 will suffer some form of disability for the remainder of their lives. This is probably a gross underestimation due to lack of detailed records from some parts of the world.

Snake venoms, produced primarily for the procurement of prey or in a defensive role, are complex biological mixtures of upwards of 50 components. Death of prey from a snake bite is due to respiratory or circulatory failure caused by various neurotoxins, cardiotoxins (also called cytotoxins), coagulation factors, and other substances acting alone or synergistically. Snake venoms also contain a number of enzymes which when injected into the prey start tissue digestion. The venoms thus contain substances designed to affect the vital processes such as nerve and muscle function, the action of the heart, circulation of the blood and the permeability of membranes. Most constituents of snake venoms are proteins, but low molecular weight compounds such as peptides, nucleotides and metal ions are also present (1).

Poisonous snakes may be divided into 4 main families,

                            TABLE 1.1
                Classification of venomous snakes
              Class: Reptilla (Reptiles)
              Order: Squamata (Snakes and Lizards)
              Suborder: Serpentes (Snakes)
              Infra order: Alethinophidia (Spectacled Snakes)
              Superfamily: Colubroidea (Advanced Snakes)
    Family            Subfamily            Tribe
    Colubridae        Nactricinae (Nactricine Water Snakes)
    (Colubrid         Dispholidinae (African Rear-Fanged
    Snakes)           Snakes)
                      Atrctaspidinae (Burrowing False
                      Vipers)
    Elapidae          Bungarinae (Cobras)  Bungarini (Kraits)
    (Palatine                              Najini (Cobras)
    Erectors)         Elapinae             Elapini (American
                                           Coral)
                                           Maticorini (Asian
                                           Coral)
                                           Laticaudini (Sea
                                           Kraits)
    Hydrophhiidae     Oxyuraninae (Australasian Venomous Snakes)
    (Palatine         Hydrophiinae         Ephalophini
    Draggers)         (True Sea Snakes)    Hydrelapini
                                           Aipysurini
                                           Hydrophini
    Viperidae         Viperinae (Pitless   Viprini (True
    (Vipers)          Vipers)              Vipers)
                                           Azemiopini (Fea's
                                           Viper)
                                           Causini (Night
                                           Adders)
                      Crotaline (Pit Vipers) Lachesini (Bush-
                                           masters)
                                           Crotalini
                                           (Viviparous Pit
                                           Vipers)
                        TABLE 1.2
         Classification and geographical distribution of
                      subfamily Crotalinae.
        Tribe       Genus                  Habitat
        Lachesini   Lachesis (Bush-        Central and South
                    masters)               America
        Crotalini   Crotalus (Rattle-      North, Central and
                    snakes)                South America
                    Sistrurus (Mass-       North America
                    augas and pigmy
                    rattlesnakes)
                    Bothrops (New World    Central and North
                    pit vipers)            America
                    Trimeresurus           Asia and North
                    (Asiatic pit           America
                    vipers)
                    Hypnale                Asia
                    Agkistrodon            North America,
                    (Moccasin)             Southeast Europe,
                                           and Asia

the Colubridae, the Viperidae, the Hydrophidae and the Elapidae (2). The systematics of these snakes is described in Tables 1.1 and 1.2. Rattlesnakes which are particular to the American continent are members of a subfamily of venomous snakes from the Viperidae family known as Crotalinae, genera Crotalus or Sistrusus (rattlesnakes) Bothrops, Apkistrodon and Trimerisurus. The two rattlesnake genera may be broken down still further into species and sub species. These snakes are also called the `pit vipers` due to the presence of facial sensory heat pits, however their most prominent feature is the rattle which when present distinguishes them from all other snakes.

Each species or subspecies occupies a distinct geographical location in the North or South America. The venom of each species of rattlesnake contains components which may be common to all rattlesnakes, common to only some smaller groups or may be specific to a single species or subspecies (3).

Antivenom is the serum or partially purified antibody fraction of serum from animals that have been rendered immune to venom toxicity as a result of a regimen of injections of increasing doses of snake venom.

The scientific study of antivenom began with the work of Henry Sewell (5) in 1887 and has progressed throughout the present century. Currently, a large number and diversity of monospecific and polyspecific antivenoms are produced around the world.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein the term "monospecific antivenom" refers to an antivenom raised against the venom of a single species or sub-species of venomous animal. The term "polyspecific antivenom" refers to an antivenom raised against a mixture of two or more venoms from different species or sub-species of venomous animal.

The terms monospecific and polyspecific antisera are used herein in order to avoid the confusion caused by use of the common alternative expressions `monovalent` and `polyvalent` antisera. This terminology is used because the term "valency" is used by immunologists to express the number of binding sites possessed by an antibody or antibody digestion product, thus an IgG molecule is divalent whereas an F(ab) fragment, which has only one binding site, is monovalent. The use of the term "specificity" in describing an antiserum avoids any confusion.

In the pioneering work by Sewell, pigeons were inoculated with sublethal doses of rattlesnake venom followed by injections of increasing doses to levels above those which would, if injected initially, cause death. It was thus demonstrated that the birds had developed a resistance to the venom. In 1889, Kaufmann (6) using the European snake Vipera berus, obtained similar results and in 1892, Calmette (7) working in Saigon with Cobra venom reported that it was possible to confer resistance by a protocol of injections of venom. However, it was Kanthack (8) who first conferred resistance to another animal when, by mixing venom with blood from an immunised animal, he demonstrated resistance to lethal doses of the snake venom.

Calmette's basic schedule was to accustom the animal to frequent, repeated, gradually increasing doses of venom (usually cobra venom). He found that over a period of 16 months immunised horses become tolerant to 80 times the lethal dose of the venom. He also showed that the antivenom derived from blood taken from these horses had a neutralising effect of 20,000 units when applied to rabbits, that is 1 ml of serum could neutralise the minimum lethal dose of venom for 20,000 g of rabbits.

Most known antivenoms are refined concentrates of equine serum globulins prepared in a liquid or dried form. The antivenoms are obtained from horses that have been immunised against a single venom, to produce a monospecific antivenom, or a mixture of a number of venoms, to produce a polyspecific antivenom. Antivenoms have been prepared for the treatment of most types of snake venom poisoning. Methods of production have changed little since the pioneering times of the last century. Immune horse serum may undergo a crude purification step usually employing ammonium sulphate to precipitate the globulin fraction and in some cases this is the form of the final product. Since antivenoms in this form can give rise to severe serum reactions, it is known to employ pepsin digestion to remove the Fc part of the immunoglobulin which is primarily responsible for such immunogenic reactions.

The effectiveness of the known antivenoms in neutralising both the deleterious and seemingly non-deleterious effects of a specific venom may vary considerably and depends upon a number of factors. The most important of these factors are the specificity of the antivenom, the titre of the antibodies produced and the degree concentration or purification of the final product.

In general, the more specific an antivenom the greater the likelihood that it will neutralise the challenging venom. Monospecific antivenom, raised against single venoms, are therefore most effective against their homologous venom. However, such antivenoms are only of use in the treatment of a snake bite when the species or subspecies of the offending snake has been identified. When the offending snake has not been identified, as is usually the case in a "field" situation, a polyspecific antivenom, raised against a spectrum of different venoms, is preferred in order to improve the likelihood of the antivenom being effective against the venom of the unidentified snake. Conventional polyspecific antivenoms, however, lack the specifity of monospecific antivenoms and are, therefore, less effective in neutralising the pharmacological activity of a venom.

The Applicant has made the unexpected and surprising discovery that an antivenom (herein referred to as a "mixed monospecific antivenom") comprising a mixture of different antisera raised separately to different venoms is more effective in neutralising the pharmacological activity of a venom than a conventional polyspecific antivenom prepared by raising a single antiserum to a spectrum of venoms, but retains the broad specificity of polyspecific antivenoms.

According to a first aspect of the present invention there is provided an antivenom comprising a mixture of at least two different antisera raised to different venoms.

It is postulated that antivenoms comprising a mixture of different antisera are more effective than conventional polyspecific antivenoms because the former may contain a higher proportion of antibodies directed against the low molecular weight and/or poorly immunogenic components of venoms.

Snake venoms are complex multicomponent mixtures of protein, nucleotides and metal ions. These components differ in molecular weight, in their degree of antigenicity and in their concentration in the venom. When venom is injected into an animal to raise an antiserum a number of antibody populations may be produced. The concentration and affinities of the antibodies raised will vary according to various criteria, for example, the number of epitopes on the surface of a component, the immunogenicity of each epitope, the concentration of each component. The lethal, neurotoxic components of venoms (including, for example, rattlesnake venoms) often comprise low molecular weight, poorly immunogenic components present only in low concentrations. Such components are unlikely to elicit high titre antibodies.

It is postulated that this problem is exacerbated in the production of a polyspecific antivenom by using an immunising mixture comprising a mixture of venoms in which the low concentration, low molecular weight and poorly immunogenic components are further diluted by highly immunogenic components. Production of a polyspecific antivenom therefore results in an antivenom in which antibodies to some components do not exist or are in such low concentration that their effectiveness is negligible.

In contrast, the mixed monospecific antivenom of the present invention comprises a mixture of antisera raised to different venoms in separate groups of animals. By raising the antisera separately, the number of possible antibody populations that is available for each antiserum is the same but the number of epitopes in the immunogen is significantly less. Thus, it is postulated that the component antisera contain a higher proportion of protective antibodies against low molecular weight, poorly immunogenic components than polyspecific antivenoms. Combination of the monospecific antisera to produce a mixed monospecific antiserum results in an antivenom which has all the populations of the monospecific serum, and therefore conveys better protection, but also has the advantages of a polyspecific antivenom in that the cross reactivity of the antivenom has been maximised.

It will be appreciated that each component antivenom of the mixed monospecific antivenom of the present invention may itself be a monospecific antivenom or a polyspecific antivenom. For example, the mixed monospecific antivenom may comprise a mixture of a polyspecific antivenom raised to venoms A+B and a monospecific antivenom raised to venom C.

Preferably, each component antivenom is a monospecific antivenom. For example, the mixed monospecific antivenom may comprise a mixture of monospecific antivenoms raised to venoms A, B and C.

The antisera which comprise the mixed monospecific antivenom may be mixed in any suitable proportion. Preferably the mixed monospecific antivenom contains antisera mixed in a proportion appropriate to the geographical area in which the mixed monospecific antivenom is intended for use. Factors that may be taken into consideration when producing such a "bespoke" mixed monospecific antivenom are the population, distribution, behaviour and toxicity of a particular venomous animal within a particular area.

The composition of the mixed monospecific antivenom may be determined by a statistical analysis of bites on humans in a particular geographical area by particular species or sub-species of venomous animal. Preferably, each component antisera of the mixed monospecific antivenom is present in direct proportion to the relative frequency of bites on humans in a particular geographical area by the particular species or sub-species of venomous animal against whose venom the antiserum is raised.

For example, the Diamond-back rattlesnake is separated into two geographical types known as the Eastern (C.adamanteus) and the Western (C.atrox) Diamond-back. A mixed monospecific antivenom can therefore be produced which caters for the snakes of a particular geographical area. The inclusion of antisera against snakes not found in that area, which might dilute the effectiveness of any product, is therefore not necessary. This ability to produce bespoke antivenoms allows the mixed monospecific antivenoms of the present invention to approach or even to better the effectiveness of an homologous monospecific antivenom without knowledge of the offending snake by statistically compensating for the type of snake bite in a geographical region.

The antisera which comprise the antivenom may be raised in any suitable animal, for example, a mouse, rat, sheep, goat, donkey or horse. Preferably, the antisera are raised in sheep. The raising of antisera in sheep is particularly advantageous over the traditional method of raising antisera in horses as antisera raised in sheep contains none of the particularly immunogenic IgG and IgG(T) components of horse antisera which cause undesirable immunogenic serum reactions in humans or animals to whom the antivenom is administered.

The antisera which comprise the antivenom may be whole antisera. Preferably, the antisera may be partially digested to the F(ab')2 or F(ab) fragments. Removal of the Fc fragment is advantageous in reducing the immunogenic reaction of the patient to the antivenom. Preparation of antibody fragments may be accomplished by conventional techniques, for example by pepsin or papain digestion (15).

The antisera which comprise the antivenom may be raised against the venom of any venomous animal, including snakes, gila monsters, spiders and bees. The antivenom may comprise antisera raised to the venom of a single type of animal, for example, antisera raised to the venom of different species or sub-species of snake. Alternatively, the antivenom may comprise antisera raised to the venom of more than type of animal. Preferably, the venom is snake venom. More preferably, the venom is rattlesnake venom.

The venom against which each antiserum is raised may comprise whole venom, partially purified venom, or one or more selected components of a venom. Preferably, the venom comprises whole venom.

According to a second aspect of the present invention there is provided a process for the preparation of an antivenom according to the first aspect of the invention comprising mixing at least two different antisera.

According to a third aspect of the present invention there is provided a pharmaceutical composition comprising an effective amount of antivenom according to the first aspect of the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

Preferably, the pharmaceutical composition is suitable for parenteral administration to a patient. More preferably, the pharmaceutical composition is suitable for intravenous injection.

According to a fourth aspect of the present invention there is provided a method of counteracting a venom comprising administration to a subject suffering from the effects of the venom an antivenom according to the first aspect of the present invention in an effective amount.

According to a fifth aspect of the present invention there is provided a kit for administering antivenom to a human or animal body comprising:

(a) an antivenom according to the first aspect of the present invention, and

(b) means for injecting the antivenom into the body.

Claim 1 of 3 Claims

I claim:

1. An isolated snake antivenom comprising a mixture of four different monospecific IgG, F(ab')2 fragment, or Fab fragment populations each of which is obtained from different ovine antisera, wherein each of said antisera is separately raised to a different snake venom, and wherein said snake venom is selected from a group consisting of A. piscivorus, C. adamanteus, C. atrox, and C. scutulatus.


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