REVIEW

Varidase: the science behind

the medicament
Varidase is used throughout the world for the topical treatment of purulent and suppurating wounds.
Its efficacy is centred on two enzymes, streptokinase and streptodornase. However, these represent only a small
proportion of the bulked solid. This article gives an overview of the preparation and mode of action
of Varidase, as well as showing some of the research and development that has gone into improving the
assessment of its quality and composition.

wide variety of agents and P.M. Rutter,1 BPharm, MRPharmS; plex emanating from dead cells or pus.

A approaches can be used to pro-

mote wound healing. Before treat-
ment can begin it is necessary to evaluate
B. Carpenter,1 BSc, PhD;
S.S. Hill,2 MSc, PhD, MRPharmS;
I.C. Locke,3 BSc, PhD

1. School of Pharmacy and Biomedical Science,

The evaluation of Varidase in a number
of aspects of wound therapy has been
reported. It has been described as an effec-
the wound and determine the wound tive agent for cleansing wounds contain-
University of Portsmouth, Portsmouth, Hants, UK
type. This characterisation will influence 2. Pharmaceutical Unit, St Mary’s Hospital, Penarth, ing necrous fibrin, pus and blood
treatment options, which include: Vale of Glamorgan, UK coagulates.8,9 Papers on the safety and the
■ Surgical techniques 3. Department of Molecular Pathology, The efficacy of Varidase compared to chemical
Windeyer Institute of Medical Sciences, University
■ Mechanical irrigation formulations of Betadine10 (a povidone
College, London, UK
■ Chemical formulations iodine compound), and hydrogen perox-
■ Antibiotics Varidase; Streptokinase; Streptodornase; ide,11 showed that in patients with
■ Hydrogels Topical wounds chronic venous leg ulcers, Varidase was a
■ Bioactive preparation safe wound cleanser with a rapid onset of
■ Biosurgery. action. It had advantages over Betadine
If necrotic tissue is present in the fore be left to appropriately qualified staff. when considering the area of granulation
wound, this must be removed before Mechanical debridement, using wet-to- tissue, and a similar efficacy to hydrogen
wound healing can commence.1 Necrotic dry dressings, is now uncommon in prac- peroxide. A similar study with dextra-
tissue is easily identified, as the wound tice as it results in pain on removal and nomer beads12 showed Varidase to be as
will show signs of yellow, green or grey may also remove healthy granulation tis- effective in cleansing necrotic ulcers.
slough. If this dead tissue becomes dehy- sue. Biosurgery, using sterile maggots of A comparative study undertaken to com-
drated the tissue will appear as brown or Lucilia sericata (the greenbottle), has again pare the enzyme preparation Trypure to
black devitalised tissue that hardens to become fashionable, and has achieved con- Varidase13 showed Varidase to be statisti-
form a thick, leathery eschar or scab. siderable success, especially on wounds that cally better as an adjuvant in removing pus
The debridement of necrotic tissue can are located in awkward areas or have failed and debris from ulcers. Trypure has a differ-
be achieved in five principal ways: to respond to conventional treatment.4-6 ent mode of action to Varidase in that its
■ Autolysis The use of enzymatic preparations in bioactive component is trypsin, which can
■ Surgical intervention the debridement process provides a novel cause widespread breakdown of protein.
■ Mechanical methods approach in facilitating the removal of A further study has compared Varidase
■ Biosurgery necrotic tissue. One such product, Vari- with a hydrogel in the debridement of
■ Enzymatic approaches. dase Topical, is licensed in the UK and is eschar.14 Both treatments were effective,
All have their place in therapy; but the useful for the removal of clotted blood, or however, the authors suggested that the
clinical need of the patient and the accept- in situations where there are fibrinous or hydrogel alone may be a more cost-effec-
ability of treatment must be the main purulent accumulations.7 tive approach to managing the removal of
deciding factors. Autolysis can be achieved Varidase contains two major bioactive eschar. This statement was based on the
by using several types of dressings includ- components: streptokinase and strepto- fact that the average number of days for
ing hydrogels, hydrocolloids, transparent dornase; these act on different con- eschar removal from patients using
films, alginates or foam.2 However, autoly- stituents of wound debris but have a hydrogel was less than for the Varidase
sis requires an extended time to remove mutually enhancing effect. group. However, the difference was not
necrotic tissue,3 in contrast to surgical Streptokinase, through activating the statistically significant.
debridement, where removal is instant. patient’s fibrinolytic system, brings about
Such surgical procedures require a high the dissolution of blood clots and fibrinosis Manufacture and preparation
degree of skill, and can remove viable as exudates, while streptodornase breaks Varidase is obtained from the extracel-
well as necrotic tissue; they should there- down nucleoprotein, a DNA-protein com- lular products of the bacterium Strepto-

JOURNAL OF WOUND CARE MAY, VOL 9, NO 5, 2000 223

ournal of Wound Care. Downloaded from magonlinelibrary.com by 130.194.020.173 on November 26, 2015. For personal use only. No other uses without permission. . All rights reserved
 REVIEW

coccus equisimilis. When the growing However, blood clots in patients who
bacterial culture reaches optimum con- had recently recovered from a streptococ-
ditions the cells are removed and a pre- cus infection produced antibodies that
cipitate is produced from the remaining lowered the activity of the filtrate. Ini-
cell-free growth medium by adding tially, the name streptokinase was given
reagents such as alcohol and trichloro- to the bioactive filtrate, but it subse-
acetic acid. quently became apparent that the activity
After further processing, the bulk prod- resided in a single protein molecule. This
uct, which is approximately 25% protein, protein was extensively characterised in
is transferred into individual vials. Each the 1960s–80s, when it was shown that it
vial gives the amount of streptokinase consisted of a single polypeptide chain
and streptodornase present in terms of with no cross linkages and no associated
their units of activity. lipid or carbohydrate moieties.
Until the late 1970s, the determination A complete analysis to determine the
of activity was based on the dissolution of a sequence of the amino acids in the strep-
standard fibrin clot for streptokinase, and tokinase molecule was achieved by Jack-
the reduction of viscosity of a DNA solution Fig 1. Polyacrylamide gel electrophoresis son and Tang in 1982.17 The results were
for streptodornase; these techniques have using SDS, of standard protein markers remarkable in that they showed one half
now been superseded. (M), secondary precipitation of Varidase of the protein had a very similar amino
Streptokinase activity is now measured using increasing quantities of ammonium acid sequence to bovine trypsin and to
by the rate of cleavage of a synthetic chro- sulphate (1-6), Streptokinase and albumin proteases from Streptomyces griseus.
mogenic substrate (S-2251, KabiVitrum (5) and Varidase alone (7). The amount of However, streptokinase is not a protease
Ltd) by the activated streptokinase-plas- material of each tracks is not the same. enzyme, as it cannot directly cleave pep-
minogen complex. The cleavage reaction tide bonds in proteins, nor is its action
results in the production of free p- Mechanism of action diminished by typical trypsin inhibitors.
nitoanaline, a yellow coloured com- Enzymes are proteins of precise composi- More recent observations have revealed a
pound, the intensity of which can be tion and structure, and usually achieve natural, but transient, pre-precursor for
measured using a spectrophotometer set maximum activity under a clearly defined streptokinase, and minor variations
to a wavelength of 405nm. set of solution conditions, which include between the amino acid sequences of
Current quality control of streptodor- pH, temperature and possibly the pres- streptokinases from different sources.
nase also relies on a spectrophotometric ence of metal ions or other small chemi- Research has indicated the possibility that
measurement. The native double helical cal entities that can act as co-factors to a number of streptokinase molecules of
structure of DNA is destroyed by strep- enhance activity. In contrast to the fac- similar but different structures can exist
todornase, and this can be monitored by tors that enhance enzyme activity others in a single bacterial species.18
observing the increase in absorbance at can reduce or inhibit it. Inhibition may be Streptokinase does not directly break
260nm of a DNA solution of suitable con- either reversible or irreversible, and as down fibrin clots; it acts as an effector
centration. The rapid linear increase in well as being brought about by small nat- molecule of a more complex lytic system.
absorbance on addition of Varidase to a urally-occurring entities the considered The molecule responsible for the break-
standard DNA solution is easily measured enzymes themselves may lose activity, down of fibrin is plasmin. This powerful
and can be used to directly define its becoming degraded by the presence of proteolytic enzyme is produced by chemi-
activity, in units of Δ(absorbance)min-1 other proteolytic enzymes. cal modification of the inactive pre-pre-
(Fig 1). The spectrophotometric determi- As streptokinase and streptodornase are cursor plasminogen.
nation of streptodornase activity has been both enzymes it is important to know The action of plasminogen stems from
further developed, and the results that their mechanism of action, the optimum the formation of an equimolecular com-
detail a fully automated assay procedure conditions for activity, possible inhibitors plex between plasminogen and streptoki-
published.15 and the likelihood of degradation when nase, which induces a conformational
The individual vials of Varidase for clini- Varidase is in the dry or reconstituted change to occur in the complex and an
cal use are reconstituted with either physio- form. The possibility must also be consid- active site to form in the plasminogen
logical saline or sterile water. ered that partially degraded streptokinase moiety of the complex. This complex has
The recommendation is for reconstitu- and streptodornase may have a modified a multiplicity of action. If left for five
tion with water if Varidase is to be applied action, and that other constituents of minutes at room temperature it will con-
in the form of a jelly. When mixed with Varidase may exercise a synergistic effect vert intramolecularly to become a strep-
inert gels such as K-Y or carboxymethyl with regard to the overall bioactivity of tokinase-plasmin complex.
cellulose, the product can be used without the medicament. It can also convert free plasminogen and
a dressing. This has an advantage in the plasminogen in other plasminogen-strep-
treatment of burns. Inorganic phosphate Streptokinase tokinase complexes to plasmin. Both free
salts are also present in each vial of Vari- In 1933, Tillet and Garner16 examined the plasmin and plasminogen-streptokinase
dase so that when reconstituted with metabolic products of a culture of β- complexes can disrupt fibrin clots, but the
20ml of saline or 5ml of water and 15ml haemolytic streptococci and found that activity of the latter is much greater.
of jelly, a physiologically acceptable pH of after removal of the bacteria the sterile fil- Plasmin is trypsin-like in that it cleaves
7.5 is achieved. trate rapidly dissolved fibrin clots. peptide bonds formed between the amino

224 JOURNAL OF WOUND CARE MAY VOL 9, NO 5, 2000

ournal of Wound Care. Downloaded from magonlinelibrary.com by 130.194.020.173 on November 26, 2015. For personal use only. No other uses without permission. . All rights reserved
 REVIEW

acids arginine and lysine. Hence it can Fig 2. Data output from a dye, in this case Coomassie blue, has to be
digest not only the fibrin network in the spectrophotometer accompanying applied to reveal their presence.
clot but fibrinogen and other clotting fac- the degradation of double helical The PAGE investigation of Varidase (Fig
tors which are protein in nature; therefore DNA. The increase in absorbance at 1) reveals not only the number of proteins
a large number of polypeptide degrada- 260nm with time is shown. present, but how preferential fractiona-
tion products are formed. 0.15 tion of these proteins can be achieved
Streptokinase-initiated plasminogen with different concentrations of the pre-
activation is an oxygen-dependent pro- cipitating agent ammonium sulphate.
cess requiring specific superoxide involve- The molecular size of the Varidase pro-
ment. Although human plasminogen is teins can be estimated from comparing
able to generate superoxide radicals, this the migration pattern of a mixture of
observation of oxygen involvement standard proteins (shown in track M).
endorses the concept that wound debride- 0.10 Track 7 contains a reference standard of
Absorbance (260 nm)
ment is enhanced in the presence of streptokinase together with the stabilising
active oxygen-producing reagents. agent albumin. Some of the tracks show
proteins from Varidase migrating just
Streptodornase below the streptokinase, which are proba-
Streptodornase is a term given to a group of bly the isokinases (streptokinase enzymes
extracellular product deoxyribonucleases 0.05
with similar activity, but also having very
produced by several haemolytic strepto- small changes in amino acid composi-
cocci. The precise classification of these tion) mentioned earlier.
enzymes is still a matter of scientific debate. Thus, changes from one molecular form
The central control of cellular activity to another or the loss or gain of protein
and morphology in both bacterial constituents will be accompanied by
(prokaryotic) and nucleated (eukaryotic) changes in the appearance of the bands
cells resides with the molecule of heredity within the polyacrylamide gel.
0.00
deoxyribonucleic acid (DNA). In both The effects of inorganic ions on
0.00 1.00 2.00
types of cell the DNA is intimately associ- streptodornase activity are readily followed
Time (min)
ated with protein, although in eukaryotic by observing modifications to the rate of
cells there is a much greater amount of absorbance change accompanying DNA
protein which produces specifically inhibited by them. It is worth noting that breakdown on the addition of Varidase (Fig
ordered structures (e.g. nucleosomes). nuclease enzymes occur in the skin and 2). The divalent cations Ca++ and Mg++
The basic protein-DNA complex is may make a natural contribution to have a profound effect on streptodornase
known as nucleoprotein, and when com- wound healing. activity. When the activity of this nuclease
pacted in the form of nucleosomes in the is monitored in the presence of only one of
nuclei of eukaryotic cells forms the chro- Research and development these cations maximum DNA degradation
mosome matrices. The nucleoprotein in Very small changes in enzyme structure, is achieved at concentrations of <1mM.
low-salt aqueous environments will take which may occur in storage, can dramati- When concentrations are increased to
up water to form a gel, and as such is cally reduce the enzyme’s activity. While 2–3mM the effect is extremely inhibitory.
found in the cellular debris residing in the spectroscopic techniques already men- However, when both cations are present
suppurative wounds. tioned would reveal overall changes in together at a concentration of 3mM the
The breakdown of DNA is a process that streptokinase and streptodornase activity, reactivity remains high. The activity pro-
continuously occurs both in normal and it is also important for product develop- duced by these conditions is greater than
pathological situations. The process is ment to try to monitor the accompanying the maximum activity achieved by any
usually mediated by deoxyribonuclease molecular events. single cation alone.
enzymes which attack the DNA Polyacrylamide gel electrophoresis Sodium ions are strongly inhibitory at
structure in a number of ways. (PAGE) is a technique which can be used all concentrations. At a concentration of
Those enzymes which cleave from the in this respect, as it can reveal the individ- 10mM, activity is almost completely cur-
ends of the DNA chains are referred to as ual proteins which make up the composi- tailed. However, addition of Mg++ and
exonucleases, while those cleaving from tion of Varidase.19 In PAGE the proteins Ca++ at appropriate concentrations will
within the chain are known as endonucle- which carry the electric charge migrate completely restore activity to streptodor-
ases. Some endonucleases recognise a spe- slowly through a precast slab of polyacry- nase inhibited by 10mM Na+.
cific pattern in the DNA base sequence and lamide gel under the influence of an There has recently been a considerable
will only cleave the DNA when these applied electric field. upsurge of interest in biosurgery, culmi-
sequences are present. Such enzymes are The inclusion of the ionic detergent, nating in it achieving ‘millennium prod-
referred to as restriction endonucleases and sodium dodecyl sulphate, into the gel and uct’ status.20 A comparison of the
are used extensively in genetic engineering. accompanying solvent buffers means that mechanisms of action of greenbottle lar-
The products of nuclease action are also the proteins will in general migrate in vae and Varidase in their ability to
very variable, and most nucleases, as pre- reverse order to their molecular size. The remove necrotic tissue and promote the
viously indicated, require metal ions as proteins present in Varidase, in common healing process may prove informative.
co-factors. However, sometimes they are with most other proteins, are colourless. A Although the larvae are living, Varidase is

JOURNAL OF WOUND CARE MAY, VOL 9, NO 5, 2000 225

ournal of Wound Care. Downloaded from magonlinelibrary.com by 130.194.020.173 on November 26, 2015. For personal use only. No other uses without permission. . All rights reserved
 REVIEW

also a product of a living system and both injected under dry scabs or applied to the 8. Forsling, E. Comparison of saline and streptokinase-
streptodornase in the treatment of leg ulcers. Eur J Clin
may involve similar bioactive molecules. surface of scabs that have been cross- Pharmacol 1988; 33: 6, 637-638.
The principal active components of Vari- hatched with a sterile scalpel. 9. Poulsen, J., Kristensen, V.N., Brygger, H.E., Delikaris, P.
dase are streptodornase and streptokinase, Varidase may also be reconstituted as a Treatment of infected surgical wounds with Varidase. Acta
Chir Scand 1983; 149: 3, 245-248.
but there are over 20 more proteins pre- jelly when mixed with a hydrogel. There- 10. Graham-Brown, R.A.C., Shuttleworth, D., Lister, D.M.,
sent, some of which may have as yet fore no standard or ideal method of appli- Millar, E. A comparative study of the safety and efficacy of
unidentified roles in its therapeutic cation exists, and the practitioner must topical enzymatic therapy vs standard antiseptic dressings in
the cleansing of leg ulcers In: Rue, Y. (ed.) A Biological
action. It could be asked whether some of decide on an individual patient basis as to Approach to the Wound Healing Process: A clinical update.
the proteins secreted from the larvae have the best approach to adopt when admin- Andover: Medifax International, 1987.
11. Manna, V., Bem, J., Marks, R. An animal model for
counterparts in Varidase. istering Varidase. Reconstituted solution chronic ulceration. Br J Dermatol 1982; 106: 2, 169-181.
has a shelf life of 24 hours and must be 12. Hulkko, A., Holopainen, Y.V., Orava, S., et al.
Conclusion stored in a refrigerator. ■ Comparison of dextranomer and streptokinase-
streptodornase in the treatment of venous leg ulcers and
Varidase occupies a niche role in wound other infected wounds. Ann Chir Gynaecol 1981; 70: 2, 65-70.
management. Its mechanism of action is The authors wish to acknowledge the contribu- 13. Suomalainen, O. Evaluation of two enzyme preparations
unique, relying on enzymatic activation of tion made to this article by Dr M D Ramsey. – Trypure and Varidase – in traumatic ulcers. Ann Chir
Gynaecol 1983; 72: 2, 62-65.
the fibrinolytic system and degradation of REFERENCES 14. Martin, S.J., Corrado, O.J., Kay, E.A. Enzymatic
1. Maklebust, J. Using wound care products to promote a debridement for necrotic wounds. J Wound Care 1996; 5: 7,
cell DNA. Fluctuation in storage and han- healing environment. Crit Care Nurs Clin North Am 1996; 8: 310-311.
dling of Varidase can alter enzyme activity 2, 141-158. 15. Locke, I.C., Cox, S.F., Hill, S.S., Carpenter, B.G. An
and therefore correct application of the 2. Sieggreen, M.Y., Maklebust, J. Debridement: choices and automated assay for the determination of
challenges. Adv Wound Care 1997; 10: 2, 32-37. deoxyribonuclease activity as exemplified by fractionation
product is essential. 3. Rodeheaver, G.T., Baharestani, M., Brabec, M.E. Wound
of the components of Varidase. J Aut Chem 1993; 15: 2,
The contents of each vial should be healing and wound management: focus on debridement.
65-70.
Adv Wound Care 1994; 7: 1, 22-36.
gently mixed (not shaken) with 20ml of 4. Stoddard, S.R., Sherman, R.M., Mason, B.E., Pelsang, D.J. 16. Tillet W. S., Garner R.L. J Exp Med 1933; 58: 485-502.
saline until the powder is completely dis- Maggot debridement therapy. J Am Pod Med Assoc 1995; 85: 17. Jackson, K.W., Tang, J. Complete amino acid sequence
4, 218-221. of streptokinase and its homology with serine proteases.
solved. The standard method of applica- 5. Thomas, S., Jones, M., Shutler, S., Andrews, A. All you Biochemistry 1982; 21: 26, 6620-6625.
tion involves pre-soaking a gauze swab need to know about… maggots. Nurs Times 1996; 92: 46, 18. Hill, S.S. Studies on Varidase. PhD thesis, University of
63-70. Portsmouth 1991.
with the reconstituted solution before 19. Locke, I.C., Cox, S.F, Carpenter, B.G. Purification of
6. Mumcuoglu, K.Y., Lipo, M., Ioffe-Uspensky, I. et al.
applying, after which the wound should Maggot therapy for gangrene and osteomyelitis. Harefuah Streptococcal deoxyribonuclease by affinity
be covered with a semi-occlusive dress- 1997; 132: 5, 323-325, 382. chromatography based on a DNA-cellulose matrix. J
7. Datapharm. Compendium of Data Sheets and Summaries Chromatography (A) 1997; 788: 75-80.
ing. However, alternative methods of of Product Characteristics. London: Datapharm Publications 20. Anon. Larval therapy given ‘millennium product’ status
application are advocated. It can be Limited, 1998. by Design Council. Pharm J 1998; 261: 732.

COMING EVENTS
at the York Racecourse subjects under discussion will be: exhibition will take place on
conference centre. The curriculum innovations; flexible 28-30 November at Harrogate
conference will offer an update learning; implementing International Conference Centre.
and information on current evidence-based healthcare; With a responsibility to promote
policy and research and examine philosophy and ethics; politics both clinical and cost
areas of good practice. Among and healthcare; and quality effectiveness through appraisal,
the speakers are Christina issues. guidance and clinical audit, the
The Editor welcomes Edwards, regional nurse director, Abstracts are now being National Institute for Clinical
information on relevant Northern and Yorkshire Region invited for submission, and those Excellence aims to provide an
NHS Executive, and Mary Ward, selected will be asked to present interactive, informative and
conferences, study days and
the Nursing Times Good Practice their ideas in a core paper, multidisciplinary forum.
courses. This issue features Network co-ordinator. theme paper or poster at the The conference is divided into
some forthcoming For more information, contact conference. two sections: Health Technology
healthcare events Mike Wilby, Healthcare Events and For more information contact the Assessment and Preparing a
Conferences, tel: 01904 700061. NET Conference Office on 01954 Submission to NICE on 28
252020, e-mail jra@easynet.co.uk November; and Setting
MAY 2000 or visit the website at www.derby. Standards and Spreading Good
SEPTEMBER 2000 ac.uk/hcs/net/nettext.html Practice, on 29-30 September.
Working In Partnership For a Call for Papers submission
The North Yorkshire Equipment Nurse Education Tomorrow form and guidelines, contact
Shared Practice Group is running The annual International Nurse NOVEMBER 2000 Charles Edwards, Sterling Events,
a conference on Working In Education Tomorrow Conference on 0151 709 8979 or e-mail
Partnership: A multi-agency (NET 2000) will be held at Setting Standards and charles@sterlingevents.co.uk
approach to equipment and the University of Durham on Spreading Good Practice For delegate information call Kelly
tissue viability across all care Tuesday 5 to Thursday 7 This year’s Clinical Excellence Owen and for exhibition information
settings on Friday 12 May 2000, September 2000. Among the 2000 annual conference and Cathy Phillips at the same number.

226 JOURNAL OF WOUND CARE MAY VOL 9, NO 5, 2000

ournal of Wound Care. Downloaded from magonlinelibrary.com by 130.194.020.173 on November 26, 2015. For personal use only. No other uses without permission. . All rights reserved