GB2453251A

GB2453251A - Viricidal formulations

Info

Publication number: GB2453251A
Application number: GB0817673A
Authority: GB
Inventors: David Okhono Ulaeto; Lyn Margaret O'brien; Joanne Elizabeth Thwaite
Original assignee: UK Secretary of State for Defence
Current assignee: UK Secretary of State for Defence
Priority date: 2007-09-26
Filing date: 2008-09-26
Publication date: 2009-04-01
Also published as: WO2009040543A3; GB0718814D0; WO2009040543A2; GB0817673D0

Abstract

A viricidal formulation comprising a bioactive peptide and a detergent or surfactant. Also claimed are kits comprising a bioactive peptide and a detergent or surfactant and a cleaning or hygeine product comprising the viricidal formulation. The bioactive peptide may be LL37, magainin II, uperin 3.1 or indolicidin.

Description

BIOACTIvE PEPTIDE This invention relates to a viricidal formulation comprising a hioacrive peptide and a detergent or surfactant wherein the detergent or surfactant enhances any viricidal activity of the hioactive peptide.

Bioactive peptides are short peptides that have anti-microbial properties. They were originally k)Und IC) he present in many plants, animals (thus the rag "animalcules") and microbes as part of that organism's natural defence mechanism. In the case of a microbe, having bioactive peptides that killed or inhibited other microbes in its immediate vicinity would increase its own survival rate. In the case of animals, it is now accepted that these peptides contribute to their innate immunity against various microbial pathogeiis.

Two major classes of bioactive peptides are produced by mammalian skin: 3-defensins and cathelicidins. Both limilies of compounds have antimicrobial activities against bacterial and fungal pathogens.

Howell et al, (Howell et al, The Journal of Immunology, 2004, 172: 1763-1767) have suggested that some bioactive peptides also possess anti-viral activity. In particular, Howell ci al, reported that one type of antimicrobial peptide, the catheljcjdin LL-37 and its murine homologue CRAMP (cathelicidin-related antimicrobial peptide), has an anti-viral effect in vitro against the vaccinia virus. Howell et al also predicted that this anti-viral effect was restricted to cathlicidi,is since no anti-viral effect was found when the dcfcnsins (HBD-I and f-IBD-2) were used. Howell et at., (Howell et al.. J ::5 Allergy Clin Immunol, 2007, 1022-1025) have subsequently found that human 13-* . defensin 3. another bioactive peptide known to have effect against a variety of bacteria, has anti-viral activity against vaccinia virus.

The present inventors have found that the addition of a detergent or surfactain to a formulation comprising a bioactive peptide can significantly enhance the viricidal activity of the hioactive peptide. In the first aspect of the invention, therefore, there is provided a viricidal foimulacioi comprising a bioactive peptide and a detergent or surfaciant wherein the detergent or surfactant enhances aiiy viricidal activity of the bioactive peptide.

As used herein. "bioactive peptide" means a short peptide, preferably no more than 40, 45, 50, 55. 60, 65. 70, 80, 90, 100 amino acids in length that possesses anti-bacterial, anti-fungal or anti-viral activity. Preferably, the bioactive peptide is a peptide that was originally identified in nature, produced by an animal, plant, fungus IS or bacterium as part of their natural defence or competition mechanism.

By "Anti-bacterial activity" is meant any activity that is capable of killing a bacterium or preventing its growth (i.e. includes both bactericidal and bacteriostatjc activities).

Methods of measuring such activities are well known to a skilled person in the art. In one example, bactericidal activity can be measured by seeing whether a lawn of bacteria growing on an agar plate are lysed, leaving a clear agar patch on the agar plate where the bacteria had previously been, when a compound being tested for anti- *::::* bacterial activity is added. In a further example bactericidal activity can be measured by seeing whether treatment can result in a reduction in the number of bacterial :.:s colony-forming units (CFU) able to initiate growth on an agar plate.

By "Aiui-fungal activity is meant any activity that is capable of killing or inhibiting the growth (i.e. includes both fungicklal or fungistatic activities) of a fungal cell.

Methods of measuring such activities are well known to a skilled person in the art.

By Thuti-viral activity" is meant any activity that inactivales a virus, that is, prevents a virus from replicating some or all of its essential genetic material and/or disseminating it.

Enhancement of viricidal activity can he measured using methods well known to a person skilled in the art. For example, viricidal activity can be measured by incubating preparations of virus (e.g. vaccinia) with different candidate bioactive peptide concentrations for a certain period of time, e.g. one hour, washing the virus and ascertaining the remaining viable virus on cell monolayers. To test whether a detergent or surfactant enhances viricidal activity of a bioactive peptide, the above assay can he carried out in the presence and absence of the detergent or.surfactant and the results compared. If there is a statistically significant lowering of the TClD,/nil-in the presence of detergent or surfactant compared to the absence of detergent or surfactant, then there is considered to be an enhancement of viricidal activity by the detergent or surfactant. TCID50 stands for 50% tissue-culture infectious dose and is a * .* measure of the virus dose that will result in virus replication in 50% of cultures to S...

which the dose of virus is applied. The lower the TCID59 reading, the lower the amount of viable virus being recovered. This assay is just one example of how S.....

* enhancement of viricidal activity can be measured. Other tests will be well-known to a skilled person in the art. In summary, any effect by a detergent or surfactant on a * .. S I * S hioactive peptide will be considered as an enhancement of viricidal activity if there are statistically significant more viruses inactivated in the presence of the detergent or surfactant and hioactive peptide compared to the absence of the detergent or stirfactant using the same concentration of bioactive peptide.

As used herein, Thurfacrant" means surface-active agents which must be soluble.

contain groups of opposite polarity and solubilising tendencies, form oriented monolayers at phase interfaces, form micelles, have detergency, foam, wetting, emulsifying and dispersing properties. As used herein, "detergent" means a water-soluble, surface-active agent capable of wetting a variety of surfaces, removing greasy and oily deposits and retaining the dirt in suspension for ease of rinsing.

Preferably, the detergent of the first aspect of the invention is a non-ionic detergent.

Non-ionic detergents are less harsh than ionic detergents and are thus more suited to I 5 products that are intended for use near, in or on humans or animals, including some of the products of the present invention.

More preferably, the non-ionic detergent is selected from a group consisting of tween- (also known as polyoxyethylene sorbitan monolaurate; poly(oxyethylene) sorbitan monolaurate; pol yox yethylene (20) sorbitan monolaurate: IC! Americas, Inc), octoxynol-9 (TRITON X-l0O, Rohm & I-Iaas) and polysorhate 80 (TWEEN 80. IC! **S.

Americas, Inc., Wilmington Del.). * . **.

* S *s** * The present inventors tested various bioactive peptides that had been previously shown to exhibit anti-bacterial, anti-fungal and/or anti-viral activities and found that *.**S. * *

the viricic.lal effects of hioactive peptides LL37. magainin II and uperin 3. I were found to be enhanced in the presence of detergent or surfactant. In one embodiment of the first aspect of the invention, the bioactive peptide of the first aspect of the invention is selected from a group comprising LL37, magainin 11 and uperin 3. I or a functional variant thereof.

By LL37 is meant a polypeptide having the sequence set out in SEQ ID NO: I. By magainin 11 is meant a polypeptide having the sequence set out in SEQ ID NO:2. By uperin 3. I is meant a polypeptide having the sequence set out in SEQ ID NO:3. By indolicidin is meant a polypeptide having the sequence set out in SEQ ID NO:4.

Preferably. the bioacrive peptide of the first aspect of the invention consists of a sequence selected from the group of SEQ ID NO: 1. SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

IS

By "functional variant" is meant any polypeptide having a sequence that is homologous or analogous to the SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 and that also has an anti-viral effect that is at least 90, preferably 95, 96, 97, 98. 99 or 100% identical to that anti-viral effect observed with the polypeptide having the original sequence (i.e. SEQ ID NO:!, SEQ ID NO:2. SEQ ID NO:3 or SEQ ID NO:4). The percentage identity of activity can be measured by substituting the original bioactive peptide with the candidate biouctive peptide in a formulation of the S...

first aspect of the invention and comparing their relative anti-viral activities.

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S..... * . * S. * S * S...

* SSSS. * .

The two terms homologous" 1Lnd "analogous' as used herein, are used interchangeably. Two polypeptides are said to he "homologous" or "analogous". if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide, that is, they share more than 70, 75. 80. 85. 90 or 95%. More preferably. the two sequences share more than 80, 81. 82.

83. 84. 85. 86, 87. 88, 89, 90. 91. 92. 93, 94. 95, 96, 97. 98, 99% identity or similarity.

"Identity", when referring to a polypeptide, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.

"Similarity", when referring to a polypeptide, indicates that, at any particular position in the aligned sequences. the amino acid residue is of a similar type between the sequences. For example, amino acid residues can he grouped by their side chains.

Glycine. alanine, valine, leucine and isoleucine all have aliphatic side-chains and amino acids in this group may be regarded as similar. Proline, although a cyclic amino acid, shares many properties with the aliphatic amino acids and may also be I 5 regarded a being grouped with the other aliphatic amino acids. Another group is the hydroxyl or sulphur containing side chain amino acids. These are serine, cysteine, threonine and rnerhionine. Phenylalanine, tyrosine and tryptophan are grouped together as the aromatic amino acids. Histidine, lysine and arginine are the basic amino acids. Aspartic acid and glutamic acid are the acidic amino acids and asparagine and glutainine are their respective amides. Also included in these groups *:*::* are modified amino acids (i.e. non-naturally occurring amino acids) that have side-S...

chains that share similar properties with the naturally occurring amino acids.

Members of a particular group can be regarded as being "similar". Swapping one

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* S....

* * amino acid from a group with another amino acid from the same group is often termed :::: 25 a conservative substitution.

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*SS..S * S The definition of a "homologous" or "analogous" polypeptide may also include a polypeptide that has had one or more amino acids deleted or inserted into the sequence. or combinations thereof, as long as the overall identity or similarity is 70.

75. 80. 85. 90 or 95%. The amino acids that are iiiserted or substituted may be non-conservative amino acid changes as long as the overall identity or similarity falls within the given percentages. Homologous or analogous polypeptides may include natural biological variants. In the case of LL37, this includes for example, CRAMP, the niurine homologue of human LL37. Homologous or analogous polypepticles may also include fragments of a polypeptide.

Degrees of identity and similarity can be readily calculated using known computer programs (see Computational Molecular Biology. Lesk, A. M., ed., Oxford University Press. New York. 1988; Bioconiputing. Informatics and Genome Projects, Smith, D. W.,ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Parti, Griffin, A. M. . and Griffin, H. G. , eds., l-lumana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). For example, simple sequence comparisons can he done on web-sites such as the NCBI website: http://www.ncbi.nlm.nih.gov/BLAST, (version * S. 2.2.11). As used herein, percentages identity or similarities between sequences are S...

S..' measured according to the default BLAST parameters, version 2.2.11. For **.S polypeptides, blastp is used with the following settings: advanced blasting, low S. S*** * complexity, expect 10. word size 3, hlosuii 62 matrix, existence: II, extension: I gap costs, inclusion threshold 0.005 and alignment view: hit table. * S

Such deletions, additions, substitutions and insertions may he naturally occurring or deliberately engineered.

Methods of making synthetic structurally equivalem polypeptides aie well known ifl the art and include techniques such as site-directed mutagenesis (see Deng, W.P. and Nickoloff, TA.. Anal. Biochem. 200. 81-88 (1982)), polymerase chain reaction, chemical gene synthesis and chemical polypeptide synthesis.

The present inventors found that the enhancing effect of detergents or surfactants is not observed with all hioactive peptides and is not limited to a particular group of bioactive peptides. Indolicidin for example is a cathelicidin, like LL-37, and has been found to have an inhibitory effect on HIV-I integrase in vitro (Marchand et al., Nucleic Acids Research, 2006. 34(18). 5157-5165). However, unlike LL-37, inclolicidin had no discernible viricidal effect on vaccinia virus, and addition of detergent or surfactant did not cause it to have an effect. This is why it is important to test the different bioactive peptides for an enhanced effect.

Further, it has been found that having more than one species of hioactive peptide in the formulation does not have ally synergistic or inibitory effect Ofl the viricidal effect *:*::* of the formulation (see Figure 8 where there is no statistical difference between the S..

TCID5Os for LL-37 alone, Magainin II alone and LL37 and Magainin I! in *: combination). In one embodiment of the first aspect of the invention, therefore, there S. .*SS * is provided a formulation of the first aspect of the invention wherein the formulation :.::::25 comprises a single species of bioactive peptide.

*S...* * . Some of the bioactive peptides used in the formulation of the present invention are known haciericides. In contrast to the enhancement effect of the detergent or stirfactain on viriciclal activity. ii was lound that addition of detergent or surfactant to some hioactive peptides actually inhibits the bactericidal activity (e.g. the bactericidal activity of LL37 is completely abrogated by addition of a non-ionic detergent (see Figure 9). Given that possible uses for the formulation is in cleaning products, it would he useful to have a single product that could kill both bacteria and viruses, that is. a cleaiiing product that is multi-cidal rather than having to use multiple products to get the same effect. The present inventors have found that although a detergent or surfactant will inhibit the bactericidal activity of magainin II to some extent, it does not inhibit the activity completely. Thus, using magainin II and a detergent or surfactant in a formulation would be suitable for multi-cidal cleaners. In a preferred embodiment of the invention, the bioactive peptide retains some bactericidal or fungicidal activity in the presence of the detergent or surfactant. Preferably, the bioactive peptide of the first aspect mentioned in the first aspect of the invention is magainin 11 or a functional variant thereof. More preferably, when the bioactive peptide is magainin II or a functional variant thereof, the detergent is non-ionic, even more preferably. Tween-20. * ..

Bioacrive peptides, detergents and surfactants are often toxic to animals and in ***.

particular, human hosts when ingested or administered parenterally. However, *..

hioactive peptides, detergents and surfactants can be tolerated without toxicity if *.*** * applied topically to an animal in sufficiently small doses or if present for a short :.:..5 period of time e.g. once the formulation has killed the viruses, the formulation could **.*.* * * * 9 be removed by washing. In one embodiment of the first aspect of the invention, therefore, the formulation additionally comprises a component suitable for topical adniinistratjcm. Components suitable for topical administratioi may include. hut are nor limited to known moisrtirjsers and/or scent.

Although toxic to mammals when ingested or administered parenterally, bioactive peptides can he delivered to the respiratory airways without killing the host. Indeed.

some bioactive peprides such as -clefensins and cathelicidins are known to he naturally expressed in the epithelium of the human lung and thought to contribute to the innate immunity of the host. Bioactive pepticles have even bccn suggested as potential treatment for cystic fibrosis patients owing to the large number of deaths in these patients caused by bacterial infections of Pseiidomona.r aeruginosa.

As well as bacterial infections, animal hosts are also susceptible tO viral infections in IS their respiratory systems such as those caused by rhinovirus, influenza virus, adenovjrus, paraintluenza virus and respiratory syncytial virus. Applying the formulation oF the first aspect of the invention to the respiratory system of animals suffenng from these infections would help rid the infection from the animal, thus alleviating them of the disease.

In the second aspect of the invention, therefore, there is provided a method of treating * 1* *.a.' an animal inlCctcd or at risk of being infected with a virus, comprising the step of *::::* administering the formulation of the first aspect of the invention to the respiratory *:: passage of the animal. Prcfci-ably, the animal is inlCcicd or is at risk of being inlCcted with a rhinovirus, influenza virus. adcnovirus, paraiiilluenza virus or respiratory

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S..... * .

syncytial virus. In this aspect of the ivnention, the formulation of the first aspect of the invention should have a detergent or surfactant that is hiocompatihle SO that administration of the derergeiit or surfactant does not harm the animal hut given at a sufficient dosage diat that the viricidal activity of the hioactive peptide is enhanced.

Preferably. the surfactant is DL-a-phosphatidylcholjne dipalmiroyl. By "hiocompatihle, as used herein, is meant any substance that has been accepted by a national licensing authority as being suitable for human or animal use.

In the third aspect of the invention, there is provided the formulation of the first aspect of the invention for use as a niedicament. The rnedicament can be prophylactic or therapeutic. The formulation may further comprise a pharmaceutically acceptable carrier. Such carriers are known to a person skilled in the art and may he solid, liquid or gas. They may sLiitably be formulated for administration via a IxirticLilar route of delivery. Cornpositioiis are suitably prepared in unit dosage forms, as conventional in the art. They are administered at dosages which are determined using clinical practice, and depend upon factors such as the nature of the patient and the severity of the condition. In particular, the carrier may help deliver the formulation to the respiratory system of an animal infected or at risk of being infected with a respiratory system associated virus.

In one embodiment of the third aspect of the invention, there is provided the S...

s.. formulation of the first aspect of the invention for use 10 treat a viral infection.

Preferably. the viral iiifectioii is selected from a group consisting rhinovirus, influenza * virus, adenovirus, parainfluenza virus or respiratory syncytial virus. * *. * ,,

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S..... *

II

Also, there is provided the use of the formulation of the first aspect of the invention in the inaniifactui*e of a medicament for treating a viral infection. Preferably. the infection is selected from a group consisting o rhinovirus, influenza virtis.

adenovirus. paraiiifluenza virus or respiratory syncytial virus.

In the fourth aspect of the invention, there is provided a kit comprising a hioacrive pepride and a detergent or surfactant. In one embodiment of the fourth aspect, the bioactive peptide and detergent or surfactant are present in the kit as separate items.

The bioactive peptide and detergent or surfactant are then mixed together before being used. Preferably, the kit additionally comprises a suitable vessel in which the bioactive peptide and detergent or surfactant can be mixed. The advantage of this embodiment is that the hioactive peptide can be stored in a state which is optimum for its stability (e.g. dry powder) and mixed with the detergent or surfactant when necessary, thus ensuring maximum viricidal efficacy is obtained. Preferably, the kit IS of the fourth aspect of the invention comprises a non-ionic detergent, more preferably, Tween-20.

In another embodiment of the fourth aspect of the invention, the bioactive peptide and detergent or surfactant are present in the kit in a single item. The advantage here is that no pre-mixing is required before use making it easy and convenient to use. * ** S* a * S. 555.

* The kit may additionally contain a cloth or other absorbent material to which the S...

bioactive peptide and detergent or surfactant can he applied. * . * *5 *, S S... a

b5**SS * In the fifth aspect of the invention, there a provided a domestic or industrial cleaning product comprising the formulation of the first aspect of the invention.

By domestic cleaning product is meant any product that is suitable for cleaning surfaces found in a domestic environment (e.g kitchen surfaces, clothes and other laundry. bottles, other materials such as contact lenses) or suitable for cleaning liquids found in a domestic environment (e.g. liquids found in cisterns), cleaning or improving the air-quality of a localized area (e.g. the cleaning product could be in the form of an air freshener). By domestic environment is meant that environment that to can be expected to he found in a home and also includes objects that can he found in such an environment such as telephones, floors, carpets, furniture etc. By industrial environment is meant that environment that can be expected to be found in a place of work, including, but not limited to, an office, hospital, kitchen, factory, and includes furniture that may be found in places of work such as computer screens, telephones, desks, floors, etc found therewith.

The doniestic or industrial cleaning product may be provided in the form of, hut nor limited to. cloths (including disposable cloths such as a tissue or napkins) impregnated with the formulation of the first aspect of the invention (e.g. wet-wipes), shampoos (e g. carpet shampoo), cleaning liquids, cleaning sprays/aerosols, cleaning mousses * S. and cleaning creams. * *.. * S * S..

*: In the sixth aspect of the invention, there is provided a hygiene product comprising * 25 the formulation of the first aspect of the invention. By hygiene product is meant any * S. * S S S..

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*S.,.S * S prolticr that is intended for improving the hygiene of an animal. particularly a human.

by coniaci of said product with said animal. Such hygiene products, include, hut are not limited to. cloths (including. hut not limited to disposable cloths such as a tissue or napkin) impregnated with the formulation of the first aspect of the invention (e.g. wet-wipes). shampoos, dental products such as toothpaste and mouth wash, face products. hair products, hand products. foot products, body products, such as face.

hair. hand, foot or body soaps, liquid soaps, creams or sprays/aerosols or mousses.

Preferably, the cleaning product of the fifth aspect of the invention or the hygiene product of the sixth aspect of the invention comprises the formulation wherein the hioaciive peptide is magainin II since this has been found to exhibit anti-bacterial activity even in the presence of a non-ionic detergent (see Figure 10) thus giving the product a niulti-cidal activity, which is a useful quality to have in such products as it saves using multiple products to obtain a clean environment. More preferably, when the bioactive peptide is magainin 11, the formulation comprises a non-ionic detergent, even more preferably, Tween-20.

As described above, the formulation of the first aspect of the invention is suitable for many uses where an environment devoid of viruses is required. In the seventh aspect of the invention, therefore, there is provided the use of the formulation of the first aspect of the invention to inactivate a virus. Examples of a virus being inactivated by * *.

the formulation of the first aspect of the invention includes vaccinia, rhinovirus, *..S * * *S..

influenza virus, adenovirus, parainfluenza virus, respiratory syncytial virus, l-IIV **.S * . (including HIV-l), herpes simplex virus (including HSV-2), vesicular stomatitis virus, *SS*..

: *** :25 cytomegalovirus, papillovirus, echovirus, reovirus, parainfluenzavirus (including Ply-

S * S

3) and i)orovirus The method by which the onmilaiion can be used to Inactivate a virus is to administer the formulation to an area suspected of Containing the virus. In the eighth aspect of the invention, therefore, there is provided a method of inactivating a virus comprising adiiiinisteriiig a formulation of the first aspect of the invention to an area suspected or containing the virus. Owing to the problems with the toxicity of hioactive peptides and detergents when ingested or administered parenterally, preferably, the method of the eighth aspect of the invention is a method wherein the torrniilarion is not ingested or administered parenterally.

In a ninth aspect of the invention, there is provided a method of making the formulation of the first aspect of the invention comprising the steps of mixing together a bioactive peptide and a detergent or surfactant. Care should be taken that the vessel in which the components of the first aspect of the invention are mixed should not react with the componetits that are being mixed. Preferably the method comprises mixing together the hioactive peptide and detergent or surfactant in a plastic or glass vessel.

More preferably, when the vessel is plastic, the plastic is polypropylene.

DESCRIPTION OF TilE DRAWINGS

Figure 1 Treatment of EEV-and IMV-enriched virus preparations with LL-37 in the absence of tween-20. Virus preparations were enriched for EEV (A, two representative experiments shown) or IMV (B, two representative experiments *:*::* shown) as described in Example 4. Six equal aliquots were taken from each virus * . * *.* preparation and adjusted to 25 () or 0 (*) tM LL-37, as two sets of triplicate *S.S samples as described in ExampleS. Alter incubation for 1.5 hours, the virus was ***..S * . * ** * S S 5*55

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S..... * S

washed. resuspeittleci an(l titrated as described in Example 2. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8.

Figure 2 Treatment of EEV-and IMV-enriched virus preparations with LL-37 in the presence of tweeii-20. Virus preparations were enriched for EEV (A, one representative of three experiments) or IMV (B, one representative of Iwo experiments) as described in Example 4. Six equal aliquots were taken from each virus preparation and adjusted to 25 () or 0 (I) tM LL-37 in 0.5% tween-20, as two sets of triplica te samples as described in Example 5. After incubation for I.5 hours.

I () the "irus was washed, resuspended aixi titrated as described in Example 2. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8.

Figure 3 Treatment ol EEV-and IMV-enriched virus preparations with indolicidin in the absence of twcen-20. Virus preparations were enriched for EEV (A. two representative experiments shown) or IMV (B. two representative experiments shown) as described in Example 4. Six eqLial aliquots were taken from each virus preparation and adjusted to 0. I () or 0 (U) mg/mi indolicidin, as two sets of triplicate samples as described in Example 5. After incubation for I.5 hours, the viius was washed, resuspended and titrated as clcscribccl in Example 2. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8. * S. * S S * *S * SS* * S * S5*

Figure 4 Treatment of EEV-and IMV-cnriched virus preparations with indoliciclin in **.I the presence of tween-20. Virus preparations were enriched for EEV (A, one S.....

: .*s:25 representative of Iwo experiments) or IMV (B, one representative of two experiments) *S55 *S*... * S

as described in Example 4. Six equal aliquots were taken from each virus prearat ion and adjusted to 0. I ( or 0 (U) mg/mi indolicidin in O.5% tween-20, as two sets of triplicate samples as described in Example 5. After incubation for 1.5 hours, the virus was washed, resuspended and titrated as described in Example 2. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8.

Figure 5 Treatment of EEV-and [MV-enriched virus preparations with uperin 3.1 in the absence of tween-20. Virus preparations were enriched for EEV (A, two representative experiments shown) or [MV (B, two reprcscntativc experiments JO shown) as described in Examplc 4. Six equal aliquots were taken From each virus preparat loll and adjusted to 0.1 () or 0 (U) mg/mi uperin 3.1, as two sets of triplicate samples as described in Example 5. After incubation for I.5 hours, the virus was washed, resuspended and titrated as described in Example 2. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8.

Figure 6 Treatment of EEV-and IMV-cnriched virus preparations with uperin 3. I in the presence of twcen-20. Virus preparations were enriched for EEV (A, one representative of two experiments) or IMV (B, one representative of two experiments) as described in Example 4. Six equal aliquots were taken From each virus preparation and adjusted to 0. I () or 0 (U) mg/mi Lipcrin 3. I in 0.5% twccn-20. as two wts oF triplicate samples as described in Example 5. After incubation for 1.5 hours, the virus :.:: was washed, resuspended amid titrated as described in Example 2. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8. * * * *. * * S

**S. -

S

**SSSS * * Figure 7 Treatment of EEV-and IMV-enriclied virus preparations with magaillin II in the presence and absence of tween-20. Virus preparations were enriched for EEV (A) or IMV (B) as described in Example 4. Six equal aliquots were taken from each virus preparation and adjusted to 0. I ( or 0 () nig/ini niagainin II in the presence and absence of 0.5% rween-20, as two sets of triplicate samples as described in Example 5. After incubation tbr I.5 hours, the virus was washed, resuspended and titrated as described in Example 2. Data is presented as means of triplicate samples.

Statistical analysis is presented in Example 8.

Figure 8 Trcmmciu of EEV-and IMV-cnrichcd virus preparations with magainin II and LL37 in the prcscncc of iween-20. Virus preparations were enriched for EEV (A) or IMV (B) as described in Example 4. Twelve equal aliquots were taken from each virus preparation and adjusted to 0.1 mg/mI for both LL-37 and magainin II (p). 0.1 mg/mi LL-37 ( 0. I mg/mi magainin 11(0), or no peptide () in the presence of 0.5% twccn-20. as four sets of triplicate samples as described in ExampleS. Alter incubation for 1.5 hours, the virus was washed, resuspended and titrated as described in Example 2. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8.

Figure 9 Treatment of F. Co/i preparations with LL37 in the presence and absence of Lwccn-20. E. co/i XL I-Blue cells were prepared as described in Example 6. Twelve * .. *, * * S.

equal aliquots were taken and adjusted to 0.1 () or 0 (*) mg/mi LL-37 in the S. * presence and absence of 0.5% tween-20, as four sets of triplicate samples as described S...

*:! in Example 6. After incubation for 10 minutes, the bacterial cells were washed, * .. * * a...

I

S..... * S

resuspended and titrated as described in Example 3. Data is presented as means of triplicate samples. Statistical analysis is presented in Example 8.

Figure 10 Treatment of E. colt preparations with magainin ii in the presence and absence of tween-20. � co/i XL1-Blue cells were prepare(l as described in Example 6. Twelve equal aliquots were taken and adjusted to 0. 1 () or 0 (U) mg/mi magainin II in the presence and absence of 0.5% tween-20, as four sets of triplicate samples as described in Example 6. After incubation for 10 minutes, the bacterial cells were washed, resuspended and itratecl as dcscrihcd in Example 3. Data is presented as tO means of triplicate samples. Statistical analysis is presented in Example 8.

Figure 11 Treatment of E co/i preparations with LL37 and magainin 11 in the presence and absence of tween-20. E. co/i XL I-Blue cells were prepared as described in Example 6. Twelve equal aliquots were taken and adjusted to 0. I mg/mI for both IS LL-37 and magainin II (, or no peptide (*)in the presence and absence of 0.5% tween-20. as fotii sets of triplicate samples as described in Example 6. Afler incubation for 10 minutes, the bacterial cells were washed, resuspended and titrated as described in Example 3. Data is presented as means of triplicate samples.

Figure 12 Treatment of E. co/i preparations with LL37 and magainin II in the presence and absence of twcen-20. E. coli XL I-Blue cells were prepared as described * ** in Example 7. Twenty-four equal aliquots were taken and adjusted to 0.1 mg/nil for **-,.

both LL-37 and magainin II (), 0.1 mg/mI LL-37 () 0.1 mg/mI magainin 11(Q). or *SS.

*: no peptide (U) in the presence of 0.5% tween-20, as eight sets of triplicate samples as * *. 25 described in Example 7. After incubation for 10 minutes, the bacterial cells were S. *

S s.... * S

washed, resuspended, and the antibacterial effect assessed as described in Example 7.

Data is presented as means of triplicate samples.

SEQUENCES

SEQ ID NO: 1 (LL37)

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

SEQ ID NO: 2 (nagainin 11)

GIGKFLHSAKKFGKAFVGEIMNS

SEQ ID NO:3 (uperin 3.1)

G VLDAFRKIAT V VKN V V

SEQ ID NO:4 (indolicidin)

ILPWKWPWWPWRR

EXAMPLES

In the following examples that involve viruses, vaccinja virus has been used to exemplify the present invention. Vaccinia virus exists in IWO morphologically distinct forms: the extracellular enveloped virus (EEV) form arid the intracellular mature virus (IMV) form. The present inventors have chosen to further characterise the viricidal *:*::* enhancing effect of detergents by determining which of the two (or both) ***.

morphological forms of vaccinia is being affected. *S.. * **..

The present inventors have found that in all instances, the enhanced effect of ::::.25 detergent on hioactive peptides is observed on both the IMV and EEV forms of

S

*S. .55 a * vacciiiia. The net effect is that the examples demonstrate that detergents enhance the viricidal activity of bioactive peptides.

Example I Cell lines and viruses Monolayers of RK 13 rabbit epithelial kidney cells were maintained in Dulbecco's Modified Eagle's Medium (Sigma) supplemented with 10% foetal bovine serum (Sigma), 2 mM L-gltitarnine, 100 units/mI penicillin and streptomycin at 37°C in a 5% C0, humidified atmosphei-e. The IHD-J strain of Vaccinia virus was grown in RK 13 cells in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 3 mM glutamine and 100 units/mI penicillin and streptomycin. Stock virus was harvested by Dotince homogenisation of infected cells and purified by ultra-centrifugarjoii at 25.000xg through a 36% w/v sucrose cushion for 90 minutes. Virus pellets were resuspended in H20, titred and stored in aliquots at -80°C. Titrat ions were undertaken as described below. E. coli strain XL-1 blue IS (Stratagcne), was cultured in L-broth and on L-agar plates.

Example 2 Quantification of virus The virus suspension was dispensed in 100 tl volumes iii each well of the first column of a sterile tissue-culture 96-well microtitre plate, into which 100 tI of tissue-culture medium had previously been dispensed. The remaining columns were filled **..* with 200 tl/well of culture medium. The virus aliquots were serially diluted across S.. * S S..

the remaining columns of the plate in StepS of 1:3, transferring 100 L1 volumes. A *SS* 100.tl volume of each serial dilution was transferred to a replica plate containing * . freshly coniluent monolayers of RK 13 cells. Cultures were maintained at 37°C in a * .. * S S *S..

5% C02, humidified atmosphere for six to seven days before fixing with I % *SI*.S * . formaldehyde and staining with crystal violet. TCID50 was calculated by Reed-Muerich analysis of virus positive wells using a Microsoft excel spreadsheet designed for this PurPose.

Example 3 Quantification of viable bacteria Bacterial colony forming units were quantitated by titration on L-agar plates.

Bacterial suspensions were serially diluted in 10-fold steps, serially transferring 30 p1 volumes unto predispensed aliqtiots of 270 p1 of L-hroth. For each dilution, 10 p1 aliquots were placed in the centre of eight wells of a 24-well tissue-culture plate, where each vell contained 1 ml of L-agar. The plates were incubated at 37°C in air in a non- humidified incubator for 24 hours, after which growth-positive wells were scored. For ease of scoring, the eight wells for each dilution were on two separate plates. to form two single columns of four wells at equivalent positions on both plates.

The 50% colony torniiiig dose was calculated according to the method of Reed & Muench, using a Microsoft excel spreadsheet designed for this purpose.

Example 4 Preparation of EEV-and IMV-enriched virus EEV-enriched virus was prepared by synchronously infecting a freshly-confluent monolayer of RK 13 cells in a T-25 tissue-culture flask (Falcon), a multiplicity of 10. After incubation at room temperature for 30-60 minutes with continuous rocking, the inoculurn was removed and replaced with 10 nil of fresh culture medium. The *::::* cultures were incubated for 16-24 hours at 3TC in a 5% CO,, humidified atmosphere.

After the incubation, the cultures were observed Visually under a microscope to *SSs confirm 100% cytopathic effect (CPE), after which the culture supernate was : .25 carefully removed by pipetting, taking care not to disturb the cell monolayer. The *S.. s * *

supernte was subjected to room temperature centrifugation at 2500 rpm in a bench-top centrifuge for 5 minutes. after which the superuate was transferred to a clean tube and the Ieller discarded. The supernate was taken as being EEV-enriched, and was uced fresh on the (lay of preparation, being maintained at 37°C until use. After removal of the EEV-enriched supernate from the cell monolayer, 5 ml of fresh culture medium was added to the 1-25 flask, and the flask then frozen at -20°C in a horizontal position. U) I upture cell membranes and release IMV particles. After thawing, the medium was removed and then treated as for preparation of EEV, to give a corresponding IMV-enriched preparation which was used immediately.

Example 5 Peptide treatment of virus preparations Peptides (made to order by Altabiosciences, Birmingham, UK) were diluted to a working stock of lOX-final desired concentration in PBS, and 5 j.tl aliquots dispensed mU) triplicate screw-cap microcencrifuge tubes. Negative control tubes received 5tl IS of PBS. For treatment in the presence of tween-20, an additional aliquoc of 5 t1 of 5% (w/v) tween-20 in H0. or 5 uI of H20 was dispensed into appropriate triplicate tubes.

Finally. the prepared EEV-or IMV-enriched VIILIS was dispensed into appropriate triplicate tubes in 40 or 45 tl volumes, to give a final volume of 50 jil per rube. The tubes were capped, vortexed briefly, and incubated for I -1.5 hours at 37°C. After incubation, 1.5 ml of PBS was added to each tube, mixed by vortexing, and the tubes sul,jected to ceinrifugation in a bench-top microcentrifuge for 10 minutes at 213,000 xg. After centrifugation, the supernate was carefully removed with a I ml finnpipecce and discarded. A further 1.5 ml of PBS was added to each tube, the tubes vortexed, S.....

* and the centrifugat ion repeated. After the second ceritrifugation, the supernate was :.::::25 again discarded, and the invisible pellet resuspended in 1.0 ml of tissue-culture S..... * S

medium. The viral suspensions were separately transferred to a safe-break potter-elvejhem homogeniser (wheatoii) and subjected to 5 manual strokes, after which they were transferred to a polystyrene hijou tube. The quantity of recovered virus was determined by limiting dilution titration as described above.

Example 6 Peptide treatment of bacterial preparations E. co/i strain XL-I blue was obtained from Stratagene as standard aliquots ot frozen competent cells. For each experiment, a single aliquot was thawed at room temperature and immediately inoculated into 5 ml of L-broth at a lxi dilution factor in an upright T-25 tissue-culture flask. The flask was shaken in an orbital incubator at 37°C overnight, after which a lx102 dilution in L-broth was made, from which aliquots were taken immediately and treated as described above for virus preparations, with the exception that incubation with peptide was for 10 minutes only, the final resuspension was in 150 p1 of L-broth, and the homogenisation step after final resuspension was omitted. The quantity of recovered bacterial cells was determined by limiting dilution titration as described above.

Example 7 Peptide treatment of bacterial preparations In a further example. E.coli strain XL-I blue was obtained from Stratagene as standard aliquots of frozen competent cells. For each experiment, a single aliquot was * ** thawed at room temperature and immediately inoculated into 5 ml of L-broth at a *S..

1x106 dilution factor in an upright T-25 tissue-culture flask. The flask was shaken in **Se an orbital incubator at 37°C overnight, after which a lx l02 dilution in L-broth was * S * made, from which aliquots were taken immediately and treated as described above for s:: 25 virus preparations. with the exception that incubation with peptide was for 10 minutes * S. S S S * S only. the final resuspension was in 5 ml of L-hroth, and the homogenisation step after fiuial resuspension was omitted. The bactericidal effect of the treatnient was assessed by placing the 5 ml samples, in their entirety. in separate upright 1-25 tissue-culture flasks and shaking in an orbital incubator at 37°C for 7.5 hours, after which the optical density of the cultures was determined as absorbance at 600 nm wavelength (OD600) iii a spectrophorometei Example 8 Statistical Analysis Statistical analysis was by ANOVA, using Microsoft excel. The experimental design facilitates the combination of repeat experiments into a single analysis using a 2 way ANOVA where one of the parameters is variation between experiments. It is to be expected that in some cases there will he significant variation between repeat experiments because the starting titre of virus preparations is not known in advance.

However, in all cases where there was a significant variation between experiments, IS the analysis showed that there was no interaction between the inter-experiment variation and the effects, if any, of peptide treatment. For individual experiments, the group (11) is always 3. Where multiple identical experiments have been pertbrmed, the data is combined in order to increase the overall group size and thus increase the power of the analysis. P values are presented. A P value of less than 0.01 indicates that a highly statistically significant difference between the experimental groups being compared. If 0.01< P <0.05, this indicates statistical significance with a lesser degree *s..

of confidence, and in this example is indicative of a small but significant difference between the experimental groups being compared. If P >0.05, this indicates that there S....

* is no difference between the experimental groups being compared. * S.

* . .,, 5*a --*S. a*. * .

Agent Comparison Tween-20 No of Combined I' - ________________________ iflCILIde(I expis GroUp Size Vaccjnii (EEV) LL- 37 v PBS YES 3 9 IxI(T Vaccirita (EEV) LL-37 v PBS NO 6 18 5.97x I0 Vaccinia (EEV) Magainiii liv PBS YES 2 6 l.02x104 Vaccinia (EEV) Magainin II v PBS NO I 3 0.2 Vaccinia (EEV) Indolicidin v PBS YES 3 9 0.494 Vaccinia (EEV) indolicidin v PBS NO 3 9 0.487 Vaccinia (EEV) Uperin 3.1 v PBS YES 2 6 8.8IxI0 Vaccinia (EEV) Uperin 3.1 v PBS NO 2 6 0.044 Vacciiiia (EEV) LL- 37 � Magainin YES I 3 3.4lxl0 ______________ 11 v PBS _________ ______ Vaccinia (EEV) LL-37 � Magainin YES I 3 0.490 _________________ Ii v LL- 37 alone ___________ _______ Vacciiija (EEV) LL-37 + Magainin YES 1 3 0.300 Ii v Magiiinin ii _________________ alone Vaccjnia (IMV) LL-37 v PBS YES 2 6 0.009 Vaccinja (iMV) LL-37 v PBS NO 4 12 0.544 Vaccinia(IMV) Magainm liv PBS YES 3 9 0.019 Vaccinia (1MV) Magainin 11 v PBS NO 2 6 0.343 Vaccjnia (IMV) Indolicjdjn v PBS YES 2 -6 0.7 16 Vaccjnja (IMV) Indoiicjdjn v PBS NO 2 6 0.665 Vaccinia(IMV) Uperin3.i v PBS YES 2 6 9.55x105 Vaccinia (IMV) perin 3.1 v PBS NO 2 6 0.0 18 E. coil LL-37 v PBS YES 2 6 0.765 E. co/i LL-37 v PBS NO 2 6 0.0043 E. coil Magainiii liv PBS YES 2 6 l.22x10" E. co/i J Magainin ii v PBS NO 2 6 5.73xl0 * ** *. * * a. *.S. * S *._. S... S *

*S SI 55 -. * *5 S. * S...

*SSISS * S

Claims

CLAI MS

I. A viricidal forimilation comprising a hioactive pepti(Ie and a detergent or surfactant wherein the detergent or surfactant enhances any viricidal activity of the bioactive peptide.
2. The viricidal lormulation of claim I wherein the detergent is a non-ionic detergent.
3. The viricidal formulation of any one of the previous claims wherein the bioaccive peptide is selected from a group comprising LL37, magainin II and uperin 3. I or a functional variant thereof.
4. The viricidal formulation of any one of the previous claims wherein the hioactive peptide consists of a sequence selected from the group of SEQ ID NO: 1, SEQ ID NO:2. SEQ ID NO:3 or SEQ ID NO:4.
5. The viricidal formulation of any one of the previous claims wherein the formulation comprises a single species of bioactive peptide.
6. The viricidal formulation of any one of the previous claims wherein the *:::* bioactive peptide retains some bactericidal or fungicidal activity in the presence of the *.S.

detergent or surf actant. * I *p..

*:
7. The viriciclal formulation of claim 6 wherein the bioactive peptide is magainin :i**.75 II or a functional variant thereof.
S

**.*** * I 8. The viricidal formulation of any one of the previous claims wherein the surfacrant or detergent is a biocompatible surfactant or hiocompatible detergent.
9. The viricidal tormulatioii of claim 8 wherein rhe hiocomparible surfactani is DL-a-phosphai idyichol me, dipalmitoyl.
10. The viricidal formulation of any one of the previous claims additionally comprising a component suitable for topical administration. l0
I I. A method of treating an animal infected or at risk of being infcted with a virus, comprising the step of administering the formulation of claim 8 or 9 to the respiratory passage of the animal.

IS
12. The method of claim I I wherein the aninial is inlcted or is at risk of being infected with a rhinovjrus, influenza virus. adcnovirus, paraintluenza virus or respiratory syncytial virus.
13. The viricjdal formulation of claim 8 or 9 for use as a medicarnent.
14. The viricidal formulation of any one of claim 8 or 9 for use to treat a viral intcction. S... * * *.

S S.... * S * .. * S S *.SS

S

*S.S.S * S
I 5. The viricidal formulation of claim 14 for use to treat a viral infection selected from a group Consisting of rhinovirus, influenza virus, adenovirus, parainfluenza virus or respiratory syncytial virus.
16. Use of the viricidal formulation of any one of claim 8 or 9 in the nianufacritre of a rnedicament for treating a viral infection.
17. The use of claim 16 wherein the viral infection is selected from a group consisting of rhiiiovirus, influenza virus, adenovirus, parainfluenza virus or respiratory syncytial virus.
18. A kit comprising a bioactive peptide and a detergent or surfactant.
19. The kit of claim 18 wherein the hioacrive peptide and the detergent or surfactant are present as separate items.
20. The kit of claim 19 wherein the bioactive peptide and detergent or surfactant are present in the kit in a single item.
2 1. The kit any one of claims 18 to 20 additionally containing a cloth or other absorbent material to which the bioactive peptide and detergent or surfactant can be * .* applied. **.* * * S... S...
22. A domestic or industrial cleaning product comprising the viricidal formulation S.....

of any one of claims I to 9. S...

S

S..... * .
23. A hygiene product comprising the viricidal formulation of any one of claims I tO 9.
24. The cleaning product of claim 22 or the hygiene product of claim 23 wherein the bloactive peptide is magainin II.
25. Use of the viriciclal formulation of any one of claims I to 9, the kit of any one of claims 18 to 2 I. the cleaning product of claim 22 or 24, or the hygiene product of claim 23 or 24 to inactivate a virus.
26. A method of inactivating a virus comprising administering a viricidal torniulation of any one of claims 1 to 9. the cleaning product of claim 22 or 24, or the hygiene product of claim 23 or 24 to an area suspected or containing the virus.
27. A method of making the formulation of any one of claims I to 9 comprising the steps of mixing together a bioactive peptide and a detergent or surfactant. * ** * * * * ** * S ***. S... * . S...

S. S*** * . * S. * . * S...

S

**.... * .