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Home , Botulinum toxin, Clostridium botulinum, Neurotoxin

Eye (1988) 2, 16--23

Clostridium Botulinum : Nature and Preparation for Clinical Use

1. MELLING, P. HAMBLETON and C. C. SHONE Salisbury

Summary C. botulinum are acutely toxic materials and act by inhibiting release of the . The specific nature of this inhibition is discussed and the preparation and purification of Type A specifically for clinical use is described.

There have been few whose mode and Thus when it comes to pharmaceutical pro­ site of action and detailed have duction C. can be viewed as been so well understood prior to their clinical one of the first of a group of high activity application as Type A botulinum toxin. products prepared from cultures of Indeed, the nature of this family of neu­ micro-organisms and the problems and solu­ roparalytic agents as the most acutely potent tions in handling such materials have general microbial toxins and the cause of the food­ applications. borne illness has made regulatory The purpose of this article is to review the authorities particularly cautious in approving nature of C. botulinum toxins and to describe their clinical application. Nevertheless the use the procedures involved in the production, of the toxin particularly to treat a variety of purification and presentation of the type A dystonic conditions represents a most elegant, toxin for clinical use. precise and safe treatment which reflectscon­ siderable credit on the pioneering work of The Nature of the Toxins Scotti who first instituted the use of the toxin Structure for therapy. Complex toxins. There are eight serologically ( ) The therapeutic, as well as the prophylactic distinct toxins A, B, Cb C2, D, E, F and G () use of microbial products is, of produced by C. botulinum that are among the course, well established and as well as native most powerful neuroparalytic agents known. products, especially antibiotics, also includes The toxins so far studied all appear to occur bacterial asparaginases used to treat acute both naturally and in in vitro culture com­ lymphoid and lymphoblastic leukaemia in plexed with non-toxic which may children. 2 Increasingly we are also seeing co-purify with the toxin moiety (Fig. 1). Of human gene products such as insulin, growth these complexes the smallest, the M or hormone and interferons produced in micro­ medium complexes, comprise the organisms and used for human therapy. It is moiety (ca. 150,000 daltons) associated with a virtually certain that many other potent similar sized non-toxic protein of undeter­ human regulatory proteins will also become mined biological activity and are the only available by this route. forms found for the types C3, E4.5 and P and

From Vaccine Research and Production Laboratory, PHLS, Centre for Applied Microbiology and Research, , Salisbury, Wiltshire. Correspondence to: J. Melling, CAMR, Porton Down, Salisbury, Wilts SP4 OJG, UK. CLINICAL USE OF BOTULINUM TOXIN 17

Sedimentation coefficent 19S 16S 12S 7S

Molecular weight (kdaltons) 900 500 300 150

haemagglutinin

non-toxic protein

neurotoxin � iii II

LL L M S

Toxin type A B C1 D E F G

Compl exes LL,L,M,S. L,M,S. M,S. L,M,S. M,S. M.S. ? Fig. 1. haemagglutinin-negative strains of type D7. The composition of several Types AS, B9 and haemagglutinin-positive toxin types (A, B, C, D, E and F) have been D7 also occur as trimolecular or L (large) com­ evaluatedI6.18.20-22 and similarities are appar­ plexes comprising the M complex in associ­ ent. In particular all have high contents of ation with a non-toxic protein having hydrophobic amino acids residues which may haemagglutinin activity. The L complexes be relevant to the membrane binding/inter­ (Ca, 500,000 daltons) are the largest complex nalisation of the toxin as discussed later. The states of these toxins with the exception of similarities in amino acid compOSItion type A toxin which also occurs as a complex of together with their shared pharmacological about 900,000 daltons. This latter complex, action imply that, despite their lack of anti­ which is thought to consist of dimers of the L genic cross-reactivity, there may well be complex, is the molecular size of the type A regions of structural homology within the toxin in the crystalline state. neurotoxins. Neurotoxins. The various C. botulinum neu­ Neurotoxin subunits. Reduction of the disul­ rotoxins, although serologically distinct, phide link(s) between H and L subunits appear to be structurally very similar to each generally to a loss of and a other; the purified neurotoxins having similar reduction in the solubility of the toxin. The molecular sizes and sharing a common subunit subunits of reduced neurotoxin remain associ­ structure (Table II). These single chain pro­ ated by covalent forces but these can be dis­ genitor toxins may be cleaved by proteolytic sociated and solubilised by urea and to give biologically active dichain subsequently separated chromatographi­ toxins (Fig. 2). cally. 16-18.23-25 The dichain toxins consist of one large Purified H and L subunits of the neurotox­ (heavy, H) and one small (light, L) subunit ins are of greatly reduced specific toxicity linked by at least one disulphide bridge; the H «0.5 per cent) compared to the intact toxin; subunits are about double the molecular size the residual toxicity probably being due to of the L subunits4.6.10-19 (Table I). trace contamination with intact toxin.16 Sep­ arated neurotoxin subunits appear to main­ PROGENITOR TOXIN

s-s s-s s-s s-s tain their structural integrity and can be P/ZZZZ)?IIZZZZZ?l EZZZ2'J p zz 77ZZZ))! recombined into intact neurotoxin by mixing equimolar portions of each subunit, in reduc­ Fig. 2. ing conditions, and slowly removing the 18 J. MELLING ET AL.

Table I Molecular size and subunit structure of C. botulinum neurotoxins

Molecular size (k daltons)

Toxin type Holotoxin H subunit L subunit Reference

A 145 95- 97 55 10, 11 B 155-170 101-104 51-59 12-14 C1 141-144 92- 98 52-53 15-17 D 140 85 55 18 E 147-150 102 50 4 F 155 105 56 6, 19 G Ca.150? Not yet chracterised reducing agent. Some 30 per cent of the digestive tract prior to its uptake into the potential of reconstituted types A23 and lymphatic systemsY·28 and B24 toxins were regained and up to 70 per cent for type C, toxin.16 Mode of Action Toxicity. When administered by the parent­ The botulinum neurotoxins act primarily on eral route the non-toxic proteins of the com­ the peripheral where, by plexes appear not to play a role in toxicity, inhibiting the release of the neurotransmitter indeed, the highest specific toxicities are acetylcholine, they cause the widespread mus­ observed with the purified neurotoxin compo­ cular characteristic of the fatal syn­ nent (Table II). For example, the purified drome botulism. Studies with isolated 145,000 type A neurotoxin (S-form) preparations show has a specifictoxicity about 3 fold higher than that the action of the toxin is not mediated that of its L complex. In contrast, the complex merely by its binding to the nerve membrane; toxins are more toxic than purified neurotox­ since binding is rapid, whereas paralysis has a ins when administered orally (Table II). This slower onset. 30 Furthermore, in contrast to difference is even more pronounced with the the rapid binding, stimulation of the nerve is type B toxin where oral toxicities of the S, M required to induce paralysis which is tempera­ and L forms are in the ratio of 112/1,600 ture sensitive and requires the presence of respectively.26 Differences in the oral tox­ in the fluid bathing the neu­ icities of toxins from two different type B romuscular junction.3H3 strains of C. botulinum were attributed It is now believed that at least three steps entirely to differences in the properties of the are involved in the inhibitory action of bot­ non-toxic proteins of the respective complex ulinum toxins. Firstly, there is a binding step, toxins. The enhanced oral toxicity of the com­ whereby the toxin attaches rapidly and avidly plex toxins probably reflects the ability of the to the presynaptic nerve membrane. non-toxic proteins to protect the neurotoxin Secondly, an internalisation step in which moiety from the hostile environment of the toxin crosses the presynaptic membrane and a finalstep, or steps, whereby toxin inhibits the release of the neurotransmitter substance, Table II Enteral and parenteral toxicities of different acetylcholine. molecular forms of C. botulinum Type A toxin Toxin binding. Selective, saturable binding of botulinum neurotoxins to presynaptic nerve Intraperitoneal Oral Route membranes of both peripheral and central route mouse mouse LD\olmg Molecular form LD50lmg protein protein nerves has been shown using labelled toxin molecules.34-36 Neurotoxin visualised with fer­ LL 3.8x107 3.2x105 ritin-labelled was observed to bind L 4.8x107 2.2x104 in discrete patches of varying size rather than 8.0x107 2.2x104 M uniformly over the nerve surface.36 S 1.6x108 3.7X103 Studies using synaptosomes from rat or Data calculated from Sakaguchi et aP" mouse have shown that the binding of CLINICAL USE OF BOTULINUM TOXIN 19 radio-labelled toxin is inhibited by an excess The single chain forms of Band E toxins bind of unlabelled toxin, indicating that a limited to synaptosomes with similar affinities to number of binding sites are available.37 Two nicked, active toxinl4.42 implying that the rela­ types of acceptor component appear to be tive non-toxicity of the single chain molecule involved; a small proportion of sites bind neu­ does not reflect an inability to bind to the rotoxin with high affinity (Kd20 nM) (Table III). If one Direct evidence of toxin internalisation was assumes that each neurotoxin contains but provided by experiments using hem i­ one binding (active) site then it seems most diaphragms incubated with radio-labelled likely that the toxin forms a 'good fit' with the Type A neurotoxin. 34 Electron micro-auto­ high affinity sites but a 'poor fit' with the low radiographs showed that following binding to affinity ones. the nerve terminal membrane transfer of labelled toxin across the plasma membrane Table III Binding constants for high and low affinity was evident after about 20 minutes and was synaptosomal acceptor sites for botulinum toxins maximal after 90 minutes. The translocation process which did not require the presence of Kd (nM) Ca2+ ions nor indirect stimulation of the nerve was abolished at low temperatures or by Toxin type High Low Reference inhibitors of energy production. The latter did A 0.6 25 37 not, however, prevent binding to the mem­ B 0.3-0.5 20 14 brane surface indicating that energy is C 0.08 35 38,39 required only for internalisation. From elec­ 0.023 35 18 D tron micrographs of nerve preparations in which toxin internalisation was prevented it Although the similar structural and phar­ was estimated that there were between 150- macological properties of the various bot­ 500 acceptor sites per �m2 on the nerve sur­ ulinum neurotoxin types suggest they may face.34 Detailed quantitation of EM auto­ have similar modes of action, it would appear radiographs revealed that not all bound that the synaptosomal acceptor sites may dif­ material was internalised and that some 60 per fer from some toxin types. There may, cent remained associated with the membrane. indeed, be at least two groups of synaptoso­ Neurotransm itter release. Since botulinum mal acceptors, one recognised by the A and E toxins appear to inhibit neither neu­ toxins and one by the Type B toxin, since rotransmitter synthesis nor storage disruption there is little or no competition between Types of the Ca2+ -mediated acetylcholine release A and B or B and E for binding to synapto­ mechanism seems probable. Three pos­ somes whereas Types A and E appear to com­ sibilities exist; toxin could inhibit Ca2+ influx pete for the same acceptor sites.14.37.40 during nerve stimulation or stimulate Ca2+ Similarly, binding of Type D toxin is com­ efflux, or it could act directly on some stage in pletely inhibited by CI toxin, suggesting com­ the release mechanism itself. No hypothesis mon acceptor components.18 Whether these adequately accounts for all the available data are similar to the acceptors for either A and E although most evidence argues against bot­ or B toxins is not yet known. ulinum toxin acting by inhibiting Ca2+ influx. The role of subunits in binding to acceptors. In In the early stages of botulinum toxin poi­ those botulinum neurotoxins so far studied soning of the neuromuscular junction the the H subunit appears to contain the active effects can be reversed by increasing the flow site which binds to synaptosomal acceptor. of Ca2+ into the nerve terminal.43 However, as For both A and B toxins homologous the progresses so the ability of Ca2+ unlabelled H-subunit reduces binding of to reverse the toxic effects is diminished. It is radio-labelled toxin, whereas the L chain is conceivable then, that the botulinum toxin without effect. Similarly Type D toxin binding may act by reducing the sensitivity of the neu­ is inhibited by Types C, and D H subunits. rotransmitter release mechanism to Ca2+ such 20 J. MELLING ET AL.

that the normal intracellular Ca2+ levels are into a 30 1 fermenter operated under ana­ insufficient to promote release. erobic conditions and toxin production and Similar conclusions are indicated by studies other culture parameters are monitored with substances such as tetraethylam­ (Fig. 3). When the maximum yield of toxin monium43 and 4-aminopyridine44-46 which, in has been attained (2x106 mouse LDsolml) prolonging the depolarisation/repolarisation usually after 72 hours the toxin is harvested by cycle of the nerve, allow higher con­ centrifugation after acidifying the culture. In centrations of Ca2+ to build up in the nerve this form the toxin can be stored prior to terminal. In these conditions the end plate purification. Cultures are extensively checked potentials of mildly botulinum-poisoned neu­ for toxin activitiy, identity and absence of romuscular junction preparations can be contamination. restored to normal. Many key aspects of the action of the bot­ 10 ulinum neurotoxins still await clarification, . including the appearance of minature end . pH,., plate potentials shortly after the onset of "; E poisoning and particularly the long duration o E U"l c 0105 o of the toxic effects. One possibility is that the -' 1.0 � :li W toxin evokes a permanent change in an :J o , or other vital system, having a long :;; . half life. 47,79 Indeed it has been proposed that, 5 in common with some other microbial toxins, the toxin itself may be an enzyme; the pro­ L------� ------L--� 01 � tracted action and high specificity of the toxin 50 ,OO appears to support this view. Time (h) Fig. 3. Preparation of Type A Toxin for Clinical Use For a product to be acceptable and licensable Purification. The precipitated crude toxin is for clinical use appropriate standards of re-dissolved and purified by a series of pro­ quality, safety and must be achieved. cedures involving ammonium sulphate pre­ Thus material must be reproducible in its cipitation and -exchange chromatography composition and potency from batch to batch (Table IV). During these procedures the pro­ and stable during a particular period under cess is monitored for the absence of con­ defined and achievable storage conditions. tamination with extraneous micro-organisms Safety and efficacy need to be assessed both as well as the toxicity and protein content of by animal experimentation and in human the extracts. trials and to be verifiable on subsequent Formulation and Freeze Drying. The potency batches by the application of relevant quality of the purifiedtoxin is assessed and an appro­ control tests. All of the above requires the priate quantity of the purifiedtoxin solution is application of Good Manufacturing Practice added to a diluent containing and (GMP) techniques. GMP builds on labora­ human serum albumin. The diluent is tory biochemistry and microbiology and designed to provide protection for the toxin translates a Research and Development pro­ during freeze-drying and to act as a bulking cess into an effective pharmaceutical oper­ agent for the freeze dried product. Prior to ation. The importance of standardising freeze drying, the diluted toxin is dispensed conditions for production and purification is into vials and at the completion of the drying clearly apparent given the effect of proteolytic process vials are checked for integrity, ster­ 'nicking' on toxic activity and the further sus­ ility, moisture content and potency. ceptibility of the protein toxin to inactivation by proteolytic enzymes. Clinical Use and Dosage Culture. Cultures of C. botulinum from a veri­ C. botulinum toxin has now been used to treat fied seed stock are grown up and inoculated both experimentally and routinely a range of CLINICAL USE OF BOTULINUM TOXIN 21

Table IV Purification ote. botulinum Type A Hemagglutinin-neurotoxin Complex48

Toxicity

Total Recovery % Protein mouse MLD50fmg Stage procedure (mg) LD50 protein Stage Ov erall

1 Whole culture (20.1) 1010 100 100 2 Precipitation at pH3 .5, and adjusted with 3N H 2 S04 4,600 7.5x109 1.6x 106 7 5 7 5 3 Extraction with 0.2M phosphate buffer at pH 6.0 1,300 6.8x109 5.2xl06 91 68 4 Ribonuclease treatment (100 ug/ml, 34°, 3 h) 1,300 6.8x 109 5.2x106 100 68 5 Precipitation at 60 per cent saturation (at 25°) of ammonium sulphate 6.5x109 9 6 65 6 DEAE-Sephadex A50 batch preabsorption 860 5.0x109 5.8x106 77 50 7 DEAE-Sephacel ion-exchange chromato- graphy at pH 5.5 19 5 4.8x109 2.5x107 9 6 48 8 Precipitation at 60 per cent saturation (at 25°) if ammonium sulphate 195 4.8x109 2.5x107 100 48 conditions where deliberate paralysis of a par­ As the data presented above show, not only ticular muscle may be of benefit. The condi­ is the mode and site of action of the toxin tions include ,I ,49 highly specific but it also possesses a further lateral rectus paresis,50 hemifacialspasm,51 important characteristic which is its avidity for Meige syndrome,52 after fac­ the site of action. Taken together these ial nerve palsy and . 53 characteristics allow local injection into a Clearly the muscle sizes, and most critically muscle with little or no general dissemination. the number of cholinergic synapses, vary con­ It will eventually be interesting to investigate siderably among these conditions as it is clear the clinical use of the pure 150,000 MW neu­ does the dose of toxin required for effective rotoxin as this molecule which does not have action. It is likely that for the effective treat­ the haem agglutinin moiety of the complex ment of patients and the convenience of clini­ toxin may give even better precision and cians toxin will need to be made available in a reproducibility of action. range of doses appropriate to the particular Further development may be required if condition and even for one condition individ­ there is evidence of production of antibody by ual patient responses may dictate varying patients receiving toxin. Should this become a doses. Accordingly we decided to present the problem it would be necessary to make avail­ toxin which has been used for blepharospasm able other of the serologically distinct toxin and strabismus treatment as a weight of active Types B through G and the information protein determined by mouse potency; the currently being pursued on the structure and use of units of activity based on human phys­ activity of these other toxins should prove iological activity may follow as more clinical invaluable in assessing their suitability for data become available. clinical use. The stability of the toxin preparation over time is clearly critical to building up an effec­ References tive clinical data base and we are able to con­ 1 Scott AB, Rosenbaum AL, Collins CC: Phar­ firm that our freeze-dried toxin retains macological weakening of extra ocular muscles. 4° Inv est Ophthalmol1973 , 12: 924-7. stability even when kept at C for not less 2 3 Report of Working Party on Leukaemia in ­ than years. Thus the potency of the prep­ hood: Improvement in treatment for children aration is a constant factor even when used in with acute lymphoblastic leukaemia. Lancet different clinics, countries and over a long 1986, 1: 408. time period and should enable a consistent 3 Syuto B and Kubo S: Purification and chrystallisa­ tion of botulinum type C toxin. Jpn J dose regime to be established for a range of Vet Res1972. 20: 19-30. conditions. 4 Kitamura M, Sakaguchi S, Sakaguchi G: Purification 22 J. MELLING E r At.

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