GB2595633A

GB2595633A - Anti-infective agents

Info

Publication number: GB2595633A
Application number: GB2005492.0A
Authority: GB
Inventors: Thierry Sireau Nicolas; B Roberts Norman; Bou-Gharios George; a gallagher James; Ranganath Lakshminarayan
Original assignee: Liverpool Univ Hospitals Nhs Foundation Trust; University of Liverpool
Current assignee: Liverpool Univ Hospitals Nhs Foundation Trust; University of Liverpool
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2021-12-08
Also published as: GB202005492D0

Abstract

The benefit of inhibiting the activity of the enzyme homogentisate 1, 2 dioxygenase when homogentisic acid is used as an antibacterial agent is disclosed. Inhibitors of homogentisate 1, 2 dioygenase (HGD) such as homogentisic acid derivatives of formula 1 (wherein: R1 is H, OH, or a halogen; R2 is H, OH, C1 to C4 linear or branched alkyl, or a halogen; R3 is H, OH, or a halogen; R3 is H, OH, or a halogen; and X is -CH2- or -CH2CH2-) and inhibitory antisense nucleic acids such as siRNA that can reduce mRNA translation and preferably has at least 80% sequence identity to the full antisense cDNA or mRNA sequences of HGD are disclosed. The combined therapeutic use of homogentisic acid (HGA) with an inhibitor of homogentisate 1, 2 dioxygenase is disclosed alongside methods for the screening for suitable homogentisate 1, 2 dioxygenase inhibitors.

Description

ANTI-INFECTIVE AGENTS

Technical field

The technical field of this invention generally relates to anti-infective agents and methods of treating infectious diseases with the same.

Background

Infectious diseases present a global health challenge. Despite many advances in the treatment of infectious diseases, there are many pathogens that are now resistant to previously efficacious antibiotic agents. Accordingly, antibiotic resistance is a growing global health concern.

Antibiotics have proven to be one of medicine's most effective tools at fighting disease. Pathogens, such as bacteria, can infect a host and cause a variety of complications, ranging from a simple cough or a fever to sepsis or encephalitis, and can ultimately cause the death of the host. Prior to the 20th century infectious diseases such as diphtheria, pneumonia and syphilis were widespread, and the average life expectancy was no more than 50 years. The discovery of penicillin by Sir Alexander Fleming and the subsequent developments by Ernst Chain and Howard Florey ushered in a new era of healthcare where people were no longer at risk of dying from simple infectious diseases.

In the years following this, several classes of antibiotics were discovered, and life expectancy continued to rise. However, the continued and overuse use of antibiotics has forced bacteria to evolve ways of surviving exposure to antibiotics. These include mutations that prevent antibiotics from engaging their original target proteins, the evolution of effective efflux pumps that remove the antibiotic from the cell or enzymes that break down the antibiotic, and even the evolution of new pathways that bypass the pathway once inhibited by the antibiotic. Resistance can spread quickly, and even between diverse species of bacteria. This has resulted in many species of bacteria that are no longer susceptible to currently known classes of antibiotics, and once readily treatable infections are now proving harder, if not impossible, to treat. As a result of this, there are approximately more than 700,000 deaths annually due to an infection caused by antibiotic resistant bacteria, with millions more suffering serious complications. The 'post-antibiotic era' is a real possibility, in which any infection could lead to death, with some predicting that annual deaths attributable to infectious diseases from 1.

antibiotic resistant bacteria will reach 10 million by 2050 This will outpace cancer, diabetes, and dian-heal disease as the leading cause of death Despite the growing threat of antibiotic resistance, there have only been a few new classes of antibiotics developed since the 1970s, and most developments in this area have typically focused on structural modifications of existing antibiotics. Faced with the continuing emergence and spread of antibiotic resistance, there is an urgent and ongoing need to identify new agents and strategies for treating infectious disease.

Homogentisic acid (2, 5-dihydroxyphenylacetic acid, HGA) is an intermediate in the metabolism of phenylalanine and tyrosine. Studies as far back as 1948 identified that HGA has antibacterial activity (Abbott LD Jr el al, Proc. Coc. Exp. Biol Med. 1948, 69 (2), 201). Moreover, HGA conforms to Lipinski's 'rule of five', indicating it has physical characteristics suggesting it is a good candidate for an orally active drug, as it has a molecular weight 168 Da, 1 H-bond acceptor, 3 H-bond donors and a LogP value of 0.86. Crucially, however, HGA rapidly metabolised in viva by the enzyme homogentisic 1, 2 dioxygenase (HOD), and thus its use as an antibiotic to date has been ruled out.

Summary of the Invention

A general aim of the present invention is to cause a transient increase in HGA in a patient in need thereof to treat an infection in a patient. The invention achieves this by inhibiting the activity of HOD, or temporarily inactivating the HOD gene by inhibiting the transcription of HOD or decreasing the level of mRNA encoding HOD in a subject in need thereof In a first aspect the invention provides a polynucleotide as defined in claim 1. In a second aspect the invention provides a pharmaceutical composition as defined in claim 13. In a third aspect the invention provides a conjugate as defined in claim 14. In a fourth aspect the invention provides a polynucleotide for use as a medicament, as defined in claim 15. In a fifth aspect the invention provides a polynucleotide for use in the treatment of an infection, as defined in claim 16. In a sixth aspect the invention provides a inhibitor of FIGD for use as a medicament, as defined in claim 17. In a seventh aspect the invention provides an antibacterial topical composition, as defined in claim 18. In a eighth aspect the invention provides homogentisic acid for use in the treatment of an infection in a patient, as defined in claim 19. In a ninth aspect the invention provides a method of screening for an inhibitor of homogentisate 1, 2 dioxygenase, as defined in claim 20. In a tenth aspect the invention provides nitisinone for use in reversing homogentisate 1, 2 dioxygenase inhibition therapy, as defined in claim 21. In a eleventh aspect the invention provides a polynucleotide a composition for use in the treatment of an infection in a patient, as defined in claim 22. In a twelfth aspect the invention provides HGA for use in the treatment of an infection in a patient, as defined in claim 23. In a thirteenth aspect the invention provides a kit as defined in claims 24. In a fourteenth aspect, the invention provides an antibiotic coating as defined in claim 25. Further features of the invention are defined in the dependent claims.

Detailed Description of the Drawings

Figure 1 A schematic of part of the phenylalanine/tyrosine metabolic pathway. The metabolite (I) is metabolised by the enzyme (2) indicated on the left in italics. The enzyme inhibited by nitisinone (3) is indicated, as is the reaction involved in diseases (4) associated with this pathway, on the right in bold Figure 2 shows how HGA inhibits E. coil on an agar plate.

Figure 3 shows HGA levels in AKU mice urine "HGA' = homogentisic acid. Error bars represent standard error of the mean (SEN/1) N=48 Figure 4 Top Panel -shows relative expression of HGD gene compared to the expression of a control gene (beta-actin) in HUH7 cells following treatment with (i) polynucleotide targeting HGD mRNA (knockdown'); (ii) polynucleotide with a random sequence (scrambled') and (iii) vehicle only ('control'). Bottom Panel -shows HGD knockdown in cells treated with a polynucleotide as a percentage of HGD expression in cells treated with vehicle control. "Knockdown-= cells treated with polynucleotide, "scrambled" = cells treated with scrambled polynucleotide, "control" = cells treated with vehicle.

Figure 5 shows expression of liver enzymes in the metabolism of phenylalanine can be knocked down with a polynucl eoti de. Results are expressed as % of average control (Phosphate buffered saline, (PBS)). "PBS" = treatment with PBS, "Ctrl ASO" = treatment with a scrambled antisense oligonucleotide, -41-1PD ASO" = antisense oligonucleotide targeting 4HPD mRNA. N= 12 per group.

Figure 6 shows the effect of nitisinone treatment on HGA levels in AKU patients. "uHGA74" (urine HGA) is expressed as the total number of moles of HGA present in urine collected over a 24-hour period and "sHGA" (serum HGA) is expressed in number of moles per unit volume. The comparison is between no-treatment and nitisinone treatment groups over 48 months in SONIA 2. *p<0.05; **p<0.01; ***p<0.001.

Figure 7 shows the number and type of infections in AKU patients over the course of the nitisinone treatment. "[E]" represents the number of events. "PT": Preferred term; "PYRs": Patient years; "SOC": System Organ Class. -n": Number of patients observed Percentage calculated on N (patients in treatment group).

Figure 8 shows the breakdown in the infections and infestations seen in AKU patients being treated with nitisinone. "[E]" represents the number of events. "PT": Preferred term; "PYRs": Patient years; "SOU' System Organ Class -n"-Number of patients observed Percentage calculated on N (patients in treatment group).

Figure 9 shows the sequences of SEQ ID NOs 1-9.

Figure 10 shows the sequences of SEQ ID NOs 10-39.

Figure!! shows the sequences of SEQ ID NOs 40-46.

Detailed description of the Invention

Homogentisic acid (HGA) is rapidly systemically metabolised, preventing its usefulness as an oral antibiotic. HGA is an intermediate metabolite in the phenylalanine/tyrosine metabolic pathway. In the process of phenylalanine metabolism in mammals, such as humans, phenylalanine is first hydroxylated into tyrosine, which is then deamidated and oxidised to produce 4-hydroxyphenylpyruvic acid. This is then reduced and hydroxylated to produce homogentisic acid. Through the action of homogentisic 1, 2 dioxygenase (HGD), homogentisic acid is then converted into maleylacetoacetic acid, which is then further metabolised to be fully reutilised by the body, fumarate being glucogenic and acetoacetate being lipogenic. By temporarily reducing or inhibiting the action of HGD, the phenylalanine/tyrosine metabolic pathway can be specifically disrupted at the point of conversion of homogentisic acid into maleylacetoacetic acid, resulting in the accumulation of homogentisic acid in the body.

Homogentisic acid is toxic to the body. Long term accumulation of HGA causes damage throughout the body, particularly in bone, cartilage tissue and heart valves. The effects of long term HGA accumulation can be seen with patients suffering from alkaptonuria, which is a genetic autosomal recessive disorder caused by a mutation in the HUD gene. Studies have shown nitisinone could be effective in lowering HGA. Generally speaking, such patients are asymptomatic for the first few decades of life until around their 30s, after which symptoms of alkaptonuria appear, with symptoms gradually becoming more severe with an. This suggests that the mammalian body tolerates increased levels of HGA reasonably well, provided the increase is temporary. HGA is rapidly and efficiently excreted by the kidney and minimised circulating concentrations. Consuming more tyrosine could lead to more HGA.

In an aspect, the invention provides a polynucleotide which binds to mRNA encoding mammalian homogentisate 1,2 dioxygenase. The polynucleotide may at least partially inhibit translation thereof The polynucleotide may cause at least a 50% inhibition of translation compared to a control, preferably at least a 60% inhibition compared to a control, preferably at least a 70% inhibition compared to a control, preferably at least an 80% inhibition compared to a control and even more preferably at least a 90% inhibition of translation compared to a control. The polynucleotide may cause a 100% or a substantially 100% inhibition of translation compared to a control. The control may comprise a polynucleotide with the same number and/or constituent nucleotides as the polynucleotide but with the sequence scrambled, or is random, or wherein the sequence does not bind HGD mRNA. The polynucleotide may cause a decrease in the amount of mRNA encoding for mammalian homogentisate 1, 2 dioxygenase. The decrease may be at least a 60% decrease compared to a control, preferably at least a 70% decrease compared to a control, at least an 80% decrease compared to a control, and even more preferably at least an 85% decrease compared to a control. The decrease may be a 100% or a substantially 100% decrease compared to a control. The control may comprise a control polynucleotide with the same number and/or constituent nucleotides as the polynucleotide but where the sequence is scrambled, or is random, or wherein the sequence does not bind HGD mRNA. Preferably, the polynucleotide binds to mRNA encoding mammalian homogentisate 1,2 dioxygenase and at least partially inhibits translation thereof Preferably, the polynucleotide binds to mRNA encoding mammalian homogentisate 1,2 dioxygenase and at least partially decreases the amount thereof The mRNA may encode for human homogentisate 1,2 dioxygenase. The polynucleotide may comprise DNA, RNA, or a combination of DNA and RNA. Preferably, the polynucleotide can be an isolated polynucleotide.

The polynucleotide may comprise a first sequence. The first sequence may have near-perfect contiguous complementarity to mRNA encoding mammalian homogenti sate 1,2 dioxygenase. The polynucleotide may comprise a first sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, i.e. the complementary RNA sequence to human homogenti sate 1,2 dioxygenase mRNA, or SEQ ID NO: 2, i.e. the DNA equivalent of SEQ ID NO: 1. The first sequence may have 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1 is the complementary RNA sequence to the mRNA of human HGD (SEQ ID NO: 46). SEQ ID NO: 46 was determined from NCBI Reference Sequence NM 000187.4 (SEQ ID NO: 45). The first sequence may comprise from 5 to 60 nucleotides, preferably from 10 to 40 nucleotides, preferably from 11 to 39 nucleotides, preferably from 12 to 38 nucleotides, preferably from 13 to 37 nucleotides, preferably from 14 to 36 nucleotides, preferably from 15 to 35 nucleotides, preferably from 16 to 34 nucleotides, preferably from 17 to 33 nucleotides, preferably from 18 to 32 nucleotides, preferably from 19 to 31 nucleotides, preferably from 20 to 30 nucleotides, preferably 21 to 29 nucleotides, preferably 22 to 28 nucleotides, preferably 23 to 27 nucleotides, preferably 24 to 26 nucleotides, or preferably 25 to 26 nucleotides.

The polynucleotide may comprise a first sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 over at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides of SEQ ID NO: 1. The polynucleotide may comprise a first sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 over at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides of SEQ TD NO: 2.

The polynucleotide may comprise a first sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to SEQ ID NO: 1 and wherein the first sequence comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, optionally wherein the first sequence has fewer than 40 nucleotides. The polynucleotide may comprise a first sequence having at least 80%, 85%, 860/o, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 and wherein the first sequence comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, optionally wherein the first sequence has fewer than 40 nucleotides.

The first sequence may comprise a 3' overhang. The overhang may not increase the percentage identity of the polynucleotide to SEQ ID NO: 1 or SEQ ID NO: 2. The overhang may comprise 1, 2, 3 or 4 nucleotides, preferably 1 or 2 nucleotides. The nucleotides of the overhang may be selected from adenine, thymine, cytosine, guanidine and/or uracil, preferably selected from thymidine, cytosine and/or uracil, or most preferably selected from thymidine and/or uracil.

The polynucleotide may also comprise a second sequence. The second sequence may be substantially complementary to the first sequence. The second sequence may be substantially complementary to the first sequence in that it comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity to the first sequence.

The polynucleotide may comprise a second sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity to the first sequence over all nucleotides of the first sequence.

The polynucleotide may comprise a second sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity to the first sequence and wherein the second sequence comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, optionally wherein the second sequence has fewer than 40 nucleotides.

The second sequence may comprise a 3' overhang. The 3' overhang may not be paired with any nucleotide in the first sequence. The overhang of the second sequence may comprise 1, 2, 3 or 4 nucleotides, or preferably 1 or 2 nucleotides. The nucleotides may be selected from adenine, thymine, cytosine, guanidine and/or uracil, or preferably selected from thymidine, cytosine and/or uracil, or even more preferably selected from thymidine and/or uracil.

A 3' end of the first sequence may be connected to a 5' end of the second sequence by a third sequence. The third sequence may be configured to form a first hairpin loop. The third sequence may comprise more than 3 nucleotides, 3 to 23 nucleotides, preferably 3 to 9 nucleotides. The third sequence may comprise AUG, CCC, UUCG, CCACC, AAGCUU, CCACACC, UUCAAGAGA. Additionally or alternatively, a 3' end of the second sequence may be connected to a 5' end of the first sequence by a fourth sequence. The fourth sequenced may be configured to form a second hairpin loop. The fourth sequence may comprise more than 3 nucleotides, 3 to 23 nucleotides, preferably 3 to 9 nucleotides. The fourth sequence may comprise AUG, CCC, UUCG, CCACC, AAGCUU, CCACACC, UUCAAGAGA. Alternatively, the third or fourth sequence may comprise any sequence that is substantially not complementary to the first or second sequence.

The first sequence of the polynucleotide may comprise any one of the sequences selected from SEQ ID NO: 3 to 44, preferably SEQ ID NO: 3, 23, or 43. Preferably, the polynucleotide may comprise the sequences SEQ ID NO: 43 and SEQ ID NO: 44.

The polynucleotide may comprise a chemical modification. The chemical modification may improve metabolic stability. The chemical modification may comprise one or more of a phosphorothioate linkage, a modification at a 2' position of the ribose sugar comprised in the nucleotide, and/or a chemically modified nucleobase. The polynucleotide may comprise the phosphorothioate linkage in a portion of the sugar/phosphate backbone of the polynucleotide. The modification at the 2' position of the ribose sugar comprised in the nucleotide may comprise a methoxy or methoxyethyl group at the 2' position of the ribose sugar comprised in the nucleotide. The chemically modified nucleobase may comprise one or more of an alkylated nucleobase, an acetylated nucleobase, a hydrogenated nucleobase, and an isomerised nucleobase.

In an aspect the invention provides a method of manufacturing a polynucleotide of the invention. The polynucleotide may be prepared synthetically. The synthesis may comprise solid phase synthesis. The method of synthesis may comprise phosphoramidite chemistry. The method of synthesis may be automated. The method of synthesis may comprise (i) an initial coupling step, (ii) a deprotection step, (iii) a coupling step, (iv) a capping step, (v) an oxidation step and (vi) a cleavage step. Steps 00 to (v) may be repeated as many times as necessary to add the required number of nucleotides comprising the polynucleotide sequence. The person skilled in the art is aware that the polynucleotide may be synthesised by "solid phase oligonucleotide synthesis" or similar techniques that are well known in the art. It is also within the competencies of the person skilled in the art to adapt 'solid phase oligonucleotide synthesis" to synthesise polynucleotides comprising chemically modified nucleotides, phosphorothioate linkages between nucleosides and other chemical modifications. For example, the oxidation step may be replaced with a sulfurization step to provide a phosphorothioate linkage between nucleosides. If the oxidization step is replaced with a sulfurization step the capping step may be performed after the sulfurization step. Thus, the synthesis may now comprise (i) an initial coupling step, (ii) a deprotection step, (iii) a coupling step, (iv) a sulfurization step, (v) a capping step and (vi) a cleavage step. Steps (ii) to (v) may be repeated as many times as necessary to add the required number of nucleotides comprising the polynucleotide sequence.

As will be appreciated by the person skilled in the art, the term antisense oligonucleotide', antisense polynucleotide' or similar is typically used when referring to a single stranded polynucleotide comprising the first sequence. Typically, such polynucleotides comprise, substantially comprise or consist of DNA, and/or may comprise a chemical modification.

As will be appreciated by the person skilled in the art, the term csiRNA', 'small interfering RNA', 'short interfering RNA', 'silencing RNA', "micro RNA", "short hairpin RNA" or similar is typically used when referring to a polynucleotide comprising the first and second sequence, where the complementarity or substantial complementarity between the first and the second strand allows for the formation of a double stranded polynucleotide (when the first and second strands are provided on separate strands) or a hairpin (when the first and second strands are provided on the same strand). Such polynucleotides comprise, substantially comprise or consist of RNA, although some parts of the polynucleotide (for instance, an overhang, if present) may comprise DNA.

In another aspect, the invention provides a polynucleotide of the invention for use as a medicament. In another aspect, the invention provides a polynucleotide of the invention for use in the treatment of an infection. In another aspect the invention provides a method of treatment of an infection in a patient comprising administering a polynucleotide of the invention to the patient. The infection may be bacterial, viral or fungal.

In another aspect, the invention provides a composition comprising the polynucleotide. The composition may comprise a delivery agent. The delivery agent may comprise a liposome or a viral vector. The viral vector may comprise an adeno-associated virus. The composition may comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may comprise a salt, a surfactant, or a buffering agent. In one aspect, the invention provides a composition of the invention for use as a medicament. In one aspect, the invention provides a composition of the invention for use in the treatment of an infection. The infection may be bacterial, viral or fungal. In an aspect, the invention provides a method of treating an infection in a patient comprising administering a composition of the invention to the patient. In an aspect the invention provides a method of manufacturing a composition of the invention.

In another aspect, the invention provides a conjugate comprising the polynucleotide of the invention that is conjugated, covalently or non-covalently, to a delivery agent. The delivery agent may comprise an antibody, cholesterol, a cellular penetration peptide, a nanoparticle, a liposome, or a vitamin. The cellular penetration peptide may comprise the ROD peptide, the TAT peptide, penetratin or transportan. In one aspect the invention provides the conjugate for use as a medicament. In one aspect the invention provides the conjugate for use in the treatment of an infection. The infection may be bacterial, viral or fungal. In one aspect, the invention provides a conjugate of the invention for use as a medicament. In one aspect, the invention provides a conjugate of the invention for use in the treatment of an infection. The infection may be bacterial, viral or fungal. In an aspect, the invention provides a method of treating an infection in a patient comprising administering a conjugate of the invention to the patient. In an aspect the invention provides a method of manufacturing a conjugate of the invention.

In another aspect, the invention provides a method of treating an infection in a subject comprising administering a therapeutically effective amount of the polynucleotide of the invention to a subject in need thereof The therapeutically effective amount may range between 0.1 mg/kg and 10 mg/kg, preferably 0.2 mg/kg and 9 mg/kg, preferably 0.3 mg/kg and 8 mg/kg, preferably 0.4 mg/kg and 7 mg/kg, preferably 0.5 mg/kg and 6 mg/kg, preferably 0.6 mg/kg and 5 mg/kg, preferably 0.7 mg/kg and 4 mg/kg, preferably 0.8 mg/kg and 3 mg/kg, or preferably 0.9 mg/kg and 2 mg/kg, i.e, according to the kg body weight of a patient. The infection may be bacterial, viral or fungal. A therapeutically effective amount comprises the amount of the polynucleotide that raises the HGA concentration in the target tissue, system, organ, or body in the range from 2 uM to 50 04, from 4 uM to 40 uM, from 6 04 to 30 uNI, from 8 tiM to 20, or from 10 uM to 15 MM. The therapeutically effective amount may be administered for a duration until the infection has been eliminated from the subject. The duration may be at least one week, preferably at least two weeks and even more preferably at least four weeks. The therapeutically effective amount may be administered once daily, once every two days, once every three days, once every four days, or once every week.

The polynucleotide may be administered orally, subcutaneously, topically, peritoneally or intravenously, preferably subcutaneously.

For administration, the polynucleotide may be prepared with a pharmaceutical carrier such as a monosaccharide (such as glucose), a disaccharide (such as lactose or sucrose), a polysaccharide (such as starch, gum arabic, cellulose or dextran), a paraffin, a fatty acid glyceride, water, an alcohol, a salt, a stabilizer, a wetting agent, an emulsifier, a lubricant, a binder, or any other conventional additive. The pharmaceutical carrier may aid in modifying the release of the polynucleotide. This may be particularly useful when the polynucleotide is administered subcutaneously. For instance, the pharmaceutical carrier may comprise a cationic polymer such as poly(ethyleneimine). The polynucleotide may form a complex with the cationic polymer. If such a complex is for instance subcutaneously administered, the slow release of the polynucleotide from the complex may be desirable. For instance, the pharmaceutical carrier may comprise a dendrimer such as poly(amidoamine), which may form a complex with the polynucleotide, again slowing the release therefrom. For instance, the pharmaceutical carrier may comprise microparticles or nanoparticle, onto which the polynucleotide, or which encapsulates the polynucleotide. The release from and/or gradual degradation of the micro-or nanoparticle may provide for a slow release of the polynucleotide. Suitable material for such particulates may be selected from a poly(alkylcyanoacrylate), a poly(lactide-co-glycolide), a poly(lactide), and a poly(isobutylcyanoacrylate). For administration, the polynucleotide may be prepared as a solid such as a tablet, granule, powder, or capsule. The polynucleotide may also be prepared as a liquid such as an emulsion, suspension, or a solution. The polynucleotide may be prepared as a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be a polynucleotide-sodium salt, a polynucleotide-potassium salt, or a salt of the polynucleotide and any other pharmaceutically acceptable counter ion.

Inhibitors of HGD have been described (Veldhuizen et al, Biochem. J (2005), 386, 305-314), and these inhibitors may be used to inhibit HGD to treat an infection. When administered to a patient, by inhibiting the patient's endogenous HGD, the natural substrate of HGD, i.e. HGA, will accumulate over time in a patient's body, thereby treating an infection in the patient. Thus, in one aspect, the invention provides an inhibitor of HGD for use as a medicament. In another aspect, the invention provides an inhibitor of HGD use in the treatment of an infection. In an aspect, the invention provides a method of treating an infection in a patient comprising administering an inhibitor of the invention to the patient. The inhibitor has the following structure: wherein: Ri is H, OH, or a halogen; R2 1S H, OH, CI to C4 linear or branched alkyl, or a halogen; Ft1 is FI, OH, or a halogen; R3 1S H, OH, or a halogen; and Xis -Cl-I2-or -CH2CH2-.

RI preferably is OH. R2 is preferably methyl or Cl. It; is preferably H. R4 is preferably OH. X is preferably -CH2-. The inhibitor may be synthesised by following or adapting synthetic routes previously described (Veldhuizen el al, Biochem. J (2005), 386, 305-314).

In another aspect, the invention provides a pharmaceutical composition comprising the inhibitor and a pharmaceutically acceptable excipient. The inhibitor may be prepared as a pharmaceutically acceptable salt such as a sodium salt, a potassium salt, or as a salt with any other pharmaceutically acceptable counter ion. In one aspect, the invention provides the pharmaceutical composition for use as a medicament. In one aspect the invention provides the pharmaceutical composition for use in the treatment of an infection. In one aspect, the invention provides an inhibitor of the invention for use as a medicament. In one aspect the invention provides an inhibitor of the invention for use in the treatment of an infection. In another aspect, the invention provides a method of treating an infection in a subject comprising administering a therapeutically effective amount of the inhibitor of the invention to a subject in need thereof.

The infection may be bacterial, viral or fungal. A therapeutically effective amount comprises the amount of the polynucleotide that raises the HGA concentration in the target tissue, system, organ, or body in the range from 2 MM to 50 pM, from 4 MM to 40 [NI, from 6 jtM to 30 gM, from 8 gIVI to 20, or from 10 AI to 15 RM. The therapeutically effective amount may range between 0.1 mg/kg and 10 mg/kg, preferably 0.2 mg/kg and 9 mg/kg, preferably 0.3 mg/kg and 8 mg/kg, preferably 0.4 mg/kg and 7 mg/kg, preferably 0.5 mg/kg and 6 mg/kg, preferably 0.6 mg/kg and 5 mg/kg, preferably 0.7 mg/kg and 4 mg/kg, preferably 0.8 mg/kg and 3 mg/kg, or preferably 0.9 mg/kg and 2 mg/kg, i.e. mg per kg body weight of a patient.

The therapeutically effective amount may be given for a duration until the infection as has been eliminated from the subject. The duration may be at least one week, preferably at least two weeks and even more preferably at least four weeks. The therapeutically effective amount may be administered once daily, once every two days, once every three days, once every four days, or once every week. The inhibitor may be administered orally, subcutaneously, topically, peritoneally or intravenously.

The pharmaceutical composition comprising the inhibitor and a pharmaceutically acceptable excipient may comprise an excipient selected from a monosaccharide (such as glucose), a disaccharide (such as lactose or sucrose), a polysaccharide (such as starch, gum arabic, cellulose or dextran), a paraffin, a fatty acid glyceride, water, an alcohol, a salt, a stabilizer, a wetting agent, an emulsifier, a lubricant, a binder, or any other conventional additive. The pharmaceutical composition may as a tablet, granule, powder, or capsule. The pharmaceutical composition also be provided as a liquid, an emulsion, suspension, or a solution.

It will be appreciated that HGA can be administered to a patient undergoing treatment with the polynucleotide of the invention or the HGD inhibitor to treat an infection. The HGA administration may be simultaneous, separate or sequential. When the patient is being administered the polynucleotide or the HGD inhibitor, the amount of HGA in the body will increase naturally, but according to this aspect of the invention, the amount of HGA can be increased rapidly by administering FIGA to the patient who is undergoing treatment with the polynucleotide of the invention or the HGD inhibitor to treat an infection. Thus, in an aspect, the invention provides HGA for use in the treatment of an infection in a patient, wherein the patient is undergoing treatment with a polynucleotide of the invention, or a HGD inhibiting compound of the invention. In another aspect, the invention provides a kit comprising HGA and the polynucleotide. The HGA may be administered orally, subcutaneously, topically, peritoneally or intravenously. The polynucleotide may be administered orally, subcutaneously, topically, peritoneally or intravenously. For administration, the polynucleotide may be prepared with a pharmaceutical carrier such as a monosaccharide (such as glucose), a disaccharide (such as lactose or sucrose) a polysaccharide (such as starch, gum arabic, cellulose or dextran) a paraffin, a fatty acid glyceride, water, an alcohol, a salt, a stabilizer, a wetting agent, an emulsifier, a lubricant, a binder, or any other conventional additive. For administration, the polynucleotide may be prepared as a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be a polynucleotide-sodium salt, a polynucleotidepotassium salt, or a salt of the polynucleotide and any other pharmaceutically acceptable counter ion.

For administration, the HGA may be prepared as a solid such as a tablet, granule, powder, or capsule. The HGA may also be prepared as a liquid such as an emulsion, suspension, or a solution. For administration, the HGA may be prepared as a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be an HGA-sodium salt, an HGA-potassium salt, or a salt of FICA and any other pharmaceutically acceptable counter ion.

In one aspect, the invention provides nitisinone for use in reversing 1,2 di oxygenase inhibition therapy. Nitisinone is used to treat hereditary tyrosinemia type-1, by inhibiting 4-4-Hydroxyphenylpyruvate dioxygenase (HPD) (see Figure 1) in the phenylalanine/tyrosine metabolic pathway. Hereditary tyrosinemia type-I is thought to result from a mutation in the FAH gene that encodes fumarylacetoacetate hydrolase. As a result of this deficiency, the substrate fumarylacetoacetic acid (or the breakdown product succinyl acetone) accumulates to toxic levels in the body, resulting in damage to kidneys and the liver. By inhibiting BPD, which is an enzyme involved in the earlier upstream stages of the phenylalanine/tyrosine metabolic pathway, levels of ffillflarylacetoacetic acid is accordingly reduced.

HPD is the enzyme in the phenylalanine/tyrosine metabolic pathway that catalyses the conversion of 3-(4-hydroxyphenyl)pyruvic acid into ITGA. By inhibiting HPD, the production of endogenous HGA can accordingly be reduced. This can be useful when reversing the effects of the polynucleotide or the HGD inhibitor of the present invention in a patient, if, for instance, the patient's infection has been successfully treated. Alternatively, reversing the effects of the polynucleotide or the HGD inhibitor of the present invention in a patient may be useful when the physiological effect of the polynucleotide is no longer desired, since, for instance, sepsis may cause renal impairment, which itself leads to a decrease in HGA excretion. Therefore, in one aspect, the present invention provides nitisinone for use in reversing homogentisate 1, 2 dioxygenase inhibition therapy. In another aspect, the present invention provides nitisinone for use in reversing homogenti sate 1, 2 dioxygenase inhibition therapy in a patient suffering from sepsis. In another aspect, the invention provides a method of reversing homogentisate 1, 2 dioxygenase inhibition therapy comprising administering a therapeutically effective amount of nitisinone to a subject in need thereof The therapeutically effective amount may be between 1 and 20 mg nitisinone, 2 and 15 mg nitisinone, 4 and 12 mg nitisinone, or 6 and 10 mg nitisinone. The therapeutically effective amount of nitisinone may administered once daily. The therapeutically effective amount may be preferably administered orally. The nitisinone may be administered as an aqueous suspension or preferably as a capsule. The nitisinone may be administered with a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may comprise pre-gelatinised starch, magnesium stearate, lactose or a lubricant.

In one aspect the present invention provides a method of screening for an inhibitor of homogentisate 1, 2 dioxygenase comprising: providing a plurality of compounds; providing a mammalian homogentisate 1, 2 dioxygenase; contacting each of the plurality of compounds and the mammalian homogentisate 1, 2 dioxygenase; and identifying a compound that inhibits the mammalian homogentisate 1, 2 dioxygenase.

The plurality of compounds preferably exhibit diversity in their physical characteristics and structure. The plurality of compounds may be provided as a 'library'. The plurality of compounds may be comprised in a library of peptides of diverse or random sequences, which may optionally be displayed on bacteriophage. The step of identifying a compound that inhibits the mammalian homogentisate 1, 2 dioxygenase may comprise an assay that is configured to determine how much one of the plurality of compounds reduces the rate of conversion of HGA by HGD. The assay may comprise a measurement of consumption of 09 by HGD in the presence or absence of one of the plurality of compounds compared to a control. Preferably, the assay comprises an oxygen electrode assay. Alternatively, or additionally, the assay may comprise measuring the conversion of HGA to maleylacetoacetic acid by homogentisate 1, 2 dioxygenase in the presence and absence of one of the plurality of compounds compared to a control, preferably using mass spectrometry. Preferably, the assay may comprise measuring the breakdown of HGA through the action of HGD by tandem liquid chromatograph and mass spectrometry (LC/MS) in the presence and absence of one of the plurality of compounds compared to a control Preferably, the assay may comprise measuring the production of maleylacetoacetic acid by LC/MS in the presence and absence of one of the plurality of compounds compared to a control. More preferably the assay comprises spectrophotometrically measuring production of maleylacetoacetate by HGD in the presence and absence of one of the plurality of compounds compared to a control Skin and wound infections present a significant problem, especially considering the rise of antibiotic resistance. If these infections are localised to the skin, they do not typically require systemic treatment. A topical composition comprising an antibiotic can deliver high levels of an active compound to a local area on the skin. Topical antibiotic therapy can prevent the unnecessary exposure of the gut flora to an antibiotic, thereby avoiding the exertion of selection for resistance; it can exert high, local concentrations of the drug to overcome any mutational resistances, and is less likely to cause side effects compared to systemic therapy. Moreover, due to the antibiotic being applied directly to site of need, i.e. the infected skin or wound, the antibiotic is not exposed to metabolic enzymes and has increased bioavailability.

Thus, in one aspect the invention provides a topical antibiotic composition comprising HGA and a pharmaceutically acceptable excipient. In another aspect, the invention provides a topical antibiotic composition comprising HGA, a pharmaceutically acceptable excipient and an additional active compound. In another aspect, the invention provides the use of HGA in a topical antibiotic composition. In another aspect the invention provides the topical antibiotic composition for use as a medicament. In another aspect the invention provides the topical antibiotic composition for use in the treatment of an infection. In another aspect the invention provides HGA for use in the treatment of an infection of the skin or wound of a patient. In another aspect the invention provides a method of treating an infection of the skin or wound of a patient, comprising administering HGA to the infected skin or wound. The infection may be bacterial, viral or flingal.

The HGA concentrations in the topical antibiotic composition may range from 0.1-50% (w/w), preferably 0.2-40 % (w/w), preferably 0.4-30% (w/w), preferably 0.5-20% (w/w), preferably 1-15 °A (w/w), preferably 2-10% (w/w), preferably 3-8% (w/w), and preferably 5-10% (w/w) of the composition. The acceptable pharmaceutical excipient may comprise any of the following, alone or in combination a buffering agent, an antioxidant, a humectant, a preservative, a wax, an aqueous solvent, or a hydrocarbon. The concentration of the active compound in the composition may range from 0.1-50 % (vv-/w), preferably 0.2-40 94) (w/w), preferably 0.4-30% (w/w), preferably 0.5-20% (w/w), preferably 1-15 % (w/w), preferably 210 % (w/w), preferably 3-8 % (w/w), and preferably 5-10 % (w/w) of the composition. The additional active compound may comprise any of the following, alone or in combination: an antibacterial compound, an antifungal compound, or an antiviral compound. The antibacterial compound may comprise amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin or any other antibacterial compound. The antifungal compound may comprise clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole, amphotericin, or any other antifungal compound. The antiviral may comprise inosine pranobex, lamivudine, ribavirin, stavudine, zidovudine, acyclovir or any other antiviral compound. The topical composition may comprise a gel, cream, ointment, foam, emulsion, lotion, or any other form intended for topical application. The composition may also comprise an additional agent such as a steroid. The steroid may comprise an anti-inflammatory steroid to reduce inflammation of an infected area to aid in wound healing and pain reduction. The steroid may comprise a glucocorticoid. The glucocorticoid may comprise any one of hydrocortisone, cortisone, dexamethasone, or prednisone. The concentration of the steroid may range from 0.005-10% (w/w), preferably 0.01-5% (w/w), preferably 0.02-4% (w/w), preferably 0.05-3% or even more preferably 0.1-2.5% (w/w) of the composition. The composition may also comprise an HGD inhibitor, which is described in further detail later. The HGD inhibitor may be present in an amount ranging from 0.1-50% (w/w), preferably 0.2-40 % (w/w), preferably 0.4-30% (w/w), preferably 0.5-20% (w/w), preferably 1-15 % (w/w), preferably 2-10 % (w/w), preferably 3-8 % (w/w), and preferably 5-10 % (w/w) of the composition.

Bacterial colonisation of orthopaedic implantable devices, such as endoprosthesis or vascular prosthesis, following implantation is a significant problem, especially considering the increase in antibiotic resistance. To establish infection, invading bacteria may first adhere to and then proliferate on the surface of the implanted device. During this process, bacteria can form a biofilm which can have an increased resistance to the host's immune system and antimicrobial therapy. As a result, even high local concentrations of antibiotics may not completely eradicate bacteria in biofilms. It is therefore important to prevent bacterial adhesion to implantable devices to prevent biofilm formation. This may be achieved through applying an antibiotic coating to the device prior to implantation. Typically, these antibiotic coatings are comprised of an active agent to inhibit bacterial growth and a binding agent to adhere the active agent to the device. Similar to a topical composition, the antibiotic coating has a local, rather than systemic effect Thus, in an aspect the invention provides an antibiotic coating for a surface comprising HGA. In another aspect the invention provides a use of HGA in an antibiotic coating. In another aspect, the invention provides a method of providing an antibiotic coating a surface, comprising coating the surface with HGA. The surface may be of an implantable device. An "implantable device" may be that which is for implantation in a patient, in other words, an orthopaedic device. In an aspect the invention provides an implantable device coated with an antibiotic coating comprising HGA. The coating may comprise a binding agent. The binding agent may bind the HGA to the surface contently or non-covalently. The HGA coating may advantageously inhibit bacterial attachment to the surface and prevent proliferation of the bacteria, whilst promoting osteoblastic attachment and growth. In a preferred aspect the invention provides an antibiotic coating for an implantable device comprising HGA, optionally wherein the coating further comprises a binding agent. Further, the oxidation of at least a portion of the HGA of the HGA-coated surface of the implantable device, which leads to a darker col ouration thereof, may advantageously inhibit bacterial attachment to the surface and prevent proliferation of the bacteria thereon. It is thought that bacteria attach to surfaces and form biofilms more readily to lighter coloured or white surfaces compared to darker surfaces. Thus, in an aspect, the invention provides oxidised HGA on the surface of an implantable device. HGA may be applied to the implantable device and allowed to oxidise through exposure to oxygen in the air or through exposure to other oxidizing agents such as hydrogen peroxide, nitric acid, sulfuric acid, sodium hypochlorite or sodium perborate.

The binding agent may be a surfactant or a polymer. The surfactant may comprise benzalkoni um chloride, tri dodecylmethylammoni um chloride, or any other suitable surfactant. The polymer may comprise poly-L-lactic acid, poly-DL-lactic acid, poly-DL-lactide-coglycolide, poly-L-lysine, polyethylene glycol, poly-lactic-co-glycolic acid, or any other suitable polymer. The HGA concentrations in the antibiotic coating may range from 0.1-50 % (w/w), preferably 0.2-40 % (w/w), preferably 0.4-30% (w/w), preferably 0.5-20% (w/w), preferably 1-15 % (w/w), preferably 2-10% (w/w), preferably 3-8 % (w/w), and preferably 510 % (w/w) of the coating. The binding agent concentrations in the antibiotic coating may range from 0,1-50% (w/w), preferably 0.2-40% (w/w), preferably 0.4-30% (w/w), preferably 0.5-20% (w/w), preferably 1-15 (w/w), preferably 210 °.//. (w/w), preferably 3-8 % (w/w), and preferably 5-10 % (w/w) of the coating. Alternatively, the coating may comprise HGA in an amount in the range of 0.1 mg/cm' to 100 mg/cm', 0.5 mg/cm' to 80 mg/cm2, 1 mg/cm2 to 60 mg/cm", 2 mg/cm" to 40 mg/cm2, 3 mg/cm" to 20 mg/cm", or 5 mg/cm" to 10 mg/cm" of the area of the surface. The thickness of the coating may be in the range of from 10 nm to 100 pm, 20 nm to 80 pm, 50 nm to 40 pm, 100 nm to 20 pm, 200 nm to 10 pm, 500 nm to 5 pm, 1 pm to 2 pm. The antibiotic coating may further comprise a pharmaceutically acceptable excipient. The acceptable pharmaceutical excipient may comprise any of the following, alone or in combination: a buffering agent, an antioxidant, a humectant, a preservative, a wax, an aqueous solvent, or a hydrocarbon.

It is thought that HGA metabolism occurs primarily in the kidney and liver. Thus, the topical composition and the antibiotic coating of the present invention allows for the delivery of HGA directly to a site of infection of a patient in need thereof Topical administration or coating on a medical implant may mean that HGA is not rapidly metabolised and excreted, has anti-infective action, and allows for a high concentration of HGA to be achieved at a site of infection.

It will be appreciated that the description above is given by way of example only, and does not limit the scope of the invention. The invention may be better understood with reference to the following non-limiting Examples. The invention may be modified within the scope of the appended claims.

Examples

Example 1

E. Coll cultures were incubated on a shaker 37°C for 24 hours in standard growth medium and in the presence of varying concentrations of HGA. Control cultures (with no HGA), when grown on plates without HGA, resulted in a lawn of bacteria which covered the plate. Cultures grown in standard growth medium and in the presence of 10 mNI HGA, and then plated onto plates with 10 mM HGA, exhibited significantly inhibited bacterial growth (Figure 2). Where cultures were incubated in standard growth medium and in the presence of 20 mM FICA, and then plated with 20 mNIHGA, no bacteria grew on plates at all (data not shown).

Example 2

Urine and plasma from 48 BALB/c Hgd-/-mice were analysed by LC/MS using an Agilent Triple Quadrupole LC/MS system This shows that HGA levels in AKU mice urine were above 20 mM so sufficient to completely abolish the growth of E. col (Figure 3).

Example 3

300,000 HUH7 cells were placed into a 6 well plate and incubated overnight in 2 mL DIVIEM with 10% FCS and no antibiotic until 50% contluency was reached. To each well, 7.5 ME RNAi MAX (Thermofisher Scientific) containing (i) 25 picomoles of siRNA polynucleotide that binds to HGD mRNA (having the sequences SEQ ID NO 43 and SEQ ID NO 44) ('knockdown'), (ii) 25 picomoles of a 'scrambled' siRNA polynucleotide control (i.e. having a random sequence that is not able to bind to HGD mRNA), or (iii) a vehicle 'control' (just RN Ai M AX) was added and the cells incubated for 48 hours. After this time, the control cells were 90% confluent and the polynucleotide treated cells were 80% confluent. The medium was removed, and the total mRNA was extracted from the cells. The level of mRNA corresponding to HGD and a house keeping gene (beta-actin) was determined using RT-PCR. The level of expression of the HGD gene was normalised to the expression of the house keeping gene (Figure 4, top). The cells treated with the siRNA polynucleotide that binds to HGD mRNA (SEQ ID NO: 43 and SEQ ID NO: 44) had an 86% knockdown of HGD mRNA compared to the scrambled polynucleotide control (Figure 4, bottom).

Example 4

BALB/c Hgd-/-mice were treated with (i) an antisense oligonucleotide targeting 4HPD mRNA, (ii) a scrambled oligonucleotide control (i.e, having a random sequence that is not able to bind to 4HPD mRNA) or (iii) vehicle (Phosphate buffered saline (PBS)) for 20 weeks. Following this time, the mice were sacrificed, the livers removed and the total mRNA from the liver extracted. The level of 41-IPD mRNA was determined using RT-PCR. A roughly 5-fold decrease in the level of 4HPD mRNA was observed in mice treated with an antisense oligonucleotide targeting 4HPD mRNA compared to control, showing that antisense oligonucleotides efficiently target mRNA of liver enzymes in vivo (Figure 5). Results are expressed as (l'o of average control (PBS). Note: "Ctrl ASO" in the Figure = treatment with the scrambled oligonucleotide; n = 12 per group.

Example 5

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disease, caused by genetic loss of homogentisate 1,2 dioxygenase (HGD). HGD is expressed predominantly in the liver and the kidney. In human subjects with HGD deficiency, homogentisic acid (HGA) production is dramatically increased systemically and possibly also at very high local concentrations. The daily amount of HGA eliminated from the body in urine in AKU is 30376+837 mon, compared to 1.94+0.12 pmol/L in non-AKU subjects. Despite a massive overproduction of HGA (4.86+0.13 g/day approximately), a circulating concentration of HGA 30.4+0.8!mon (mean + SD) is achieved in AKU subjects, compared to 2.24+0.25 pmol/L in non-AKU subjects, showing only a moderate increase as a result of extremely efficient renal clearance of HGA.

Nitisinone 2 mg daily has been used off-label in the United Kingdom National Alkaptonuria Centre since 2012. Administration of nitisinone 10 mg daily to patients suffering from AKU has been investigated in a clinical trial to investigate the suitability of nitisinone in alkaptonuria study. All patients in this trial suffered from AKU.

Nitisinone was shown to significantly lower the levels of FICA in serum ("s-HGA") and urine (u-HGA24-, i.e. the amount of HGA in urine collected over a 24 hour period) over the course of treatment (Figure 6).

The nitisinone-treated group exhibited increased infections and infestations when compared to the no-nitisinone control group (Figure 7). There was a significantly higher incidence of infections and infestations (also termed adverse events, or "AE", also termed events, or "E") in this systems organ class (SOC) in the nitisinone-treated group; 56 AEs were reported for 27 patients in the nitisinone-treated group. On the other hand, in the no-nitisinone control group there were only 24 AEs reported for 11 patients (Figure 7). Pneumonia and bronchitis were among the AEs more commonly reported in the nitisinone-treated group than in the nonitisinone control group. In addition, viral, bacterial and fungal infections were all generally more commonly seen in the nitisinone-treated group (Figure 8). Thus, all patients receiving nitisinone developed more infections.

The following analysis of Figure 8 also reveals that the increased levels of HGA have broad spectrum activity. In particular, a comparison of the total number of subjects (n) between the untreated (no-nitisinone) group and the nitisinone group reveals that the subjects in the untreated group exhibited substantially fewer viral, bacterial or fungal infections/infestations, as demonstrated in the Table below.

Untreated (N=69) Nitisinone (N=69) Vi rail 8 15 Bacterial' 7 20 Fungal' 1 5 Bronchitis; Nasopharyngitis; Influenza; Viral upper respiratory tract infection; Herpes zoster; Ophthalmic herpes simplex; Pharyngitis; Rhinitis; Oral herpes; Respiratory tract infection, Varicella Urinary tractinfection; Pneumonia; Tooth abscess; Conjunctivitis; Erysipelas; Breast abscess; Cellulitis; Cystitis; Ear infection; Folliculitis; Kidney infection; Pulpitis dental; Tonsillitis; Hordeolum; Lower respiratory tract infection; Wound infection Candida infection; Fungal infection; Onychomycosis; Vulvovaginal candi di asi s; Vulvovaginal mycotic infection; Tinea versicol our Nitisinone treatment decreases HGA and increases tyrosine through inhibition of phydroxyphenylpyruvate dioxygenase (see Figure 1). Nitisinone has been used at much higher doses for the treatment of hereditary tyrosinaemia 1 (HT 1) and no increased infection risk has been described either in the literature or during post-marketing surveillance, which suggests that neither the post-nitisinone tyrosinaemia nor the nitisinone itself (or other metabolites such as hydroxyphenylpyruvate or hydroxyphenyl-lactate) are causing the infections observed in the nitisinone-treated group in Figures 7 and 8, particularly since the doses of nitisinone used to treat HT 1 are substantially higher, whilst also suggesting that the increased levels of HGA causes the reduced infection events observed in the no-dtis.none control group. Note HGA is only raised in AKU but not in HT I Thus, the marked elevated levels of HGA in AKU patients left untreated by nitisinone is associated with a reduced infection. Therefore, the AKU metabolic state, characterised by serum levels of at least approximately 21 iimol/L HGA and urine levels of HGA of at least approximately 20000 iimol HGA, reduces infections.

Claims

CLAIMS: 1 A polynucleotide which binds to mRNA encoding mammalian homogentisate 1, 2 dioxygenase and at least partially inhibits translation thereof 2. The polynucleotide according to claim 1, wherein the mRNA encodes human homogentisate 1, 2 dioxygenase.3. The polynucleotide according to claim 1 or 2, wherein the polynucleotide comprises RNA and/or DNA.4. The polynucleotide according to any one of claims 1 to 3, comprising a first sequence having at least 80% identity to at least a portion of SEQ ID NO: 1 or SEQ ID NO: 2.5. The polynucleotide according to claim 4, wherein the first sequence comprises 5 to 60 nucleotides.6. The polynucleotide according to claim 4 or 5, wherein the first sequence comprises a 3' nucleotide overhang, optionally wherein: a. the overhang comprises 1, 2, 3 or 4 nucleotides, or b. the overhang comprises nucleotides selected from thyMdine and/or uridine.7. The polynucleotide according to any one of claims 4 to 6, wherein the polynucleotide comprises a second sequence that is substantially complementary to the first sequence 8. The polynucleotide according to claim 7, wherein the second sequence comprises 5 to nucleotides.9. The polynucleotide according to claim 7 or 8, wherein the second sequence comprises a 3' nucleotide overhang, optionally wherein: a. the overhang comprises 1, 2, 3 or 4 nucleotides, or b. the overhang comprises nucleotides selected from thymidine and/or uridine.10. The polynucleotide according to any one of claims 7 to 9, wherein: a. a 3' end of the first sequence is connected to a 5' end of the second sequence by a third sequence configured to form a first hair pin loop, and/or b. a 3' end of the second sequence is connected to a 5' end of the first sequence by a fourth sequence configured to form a second hair pin loop.11. The polynucleotide according to any one of claims 4 to 10, wherein the first sequence comprises a sequence selected from any one of SEQ ID NO: 3-43, optionally wherein the sequence is selected from SEQ ID NO: 3, 23 or 43.12. The polynucleotide according to any one of claim 1 to 11, wherein the polynucleotide comprises a chemical modification, optionally wherein the modification is selected from a group consisting of: a. a phosphorothioate linkage between two adjacent nucleotides comprising the polynucleotide; b. a modification at a 2' position of a ribose sugar comprising the polynucleotide; and, c. a chemically modified nucl eobase.13. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 12, wherein the composition further comprises a delivery agent and/or a pharmaceutically acceptable excipient, optionally wherein the delivery agent comprises a liposome or a viral vector.14. A conjugate comprising the polynucleotide according to any one of claims 1 to 12 and an antibody, cholesterol, a cellular penetration peptide, a nanoparticle, a liposome, or a vitamin, which is optionally coval ently linked.15. The polynucleotide according to any one of claims 1 to 12 for use as a medicament.16. The polynucleotide according to claim 15 for use in the treatment of an infection, wherein the infection is bacterial, viral or fungal.17. An inhibitor of HGD for use as a medicament, wherein the inhibitor has the structure: wherein: RI is H, OH, or a halogen, 16 is H, OH, CI to C4 linear or branched alkyl, or a halogen; RI is H, OH, or a halogen, R3 is H, OH, or a halogen; and Xis -CI-12-or -C1-12CH2-, optionally wherein RI is OH; R2 is methyl or Cl; R3 is H; R4 is OH; or X is -CH2-, optionally wherein the inhibitor is for use in the treatment of an infection.18. An antibacterial topical composition comprising homogenti sic acid and a pharmaceutically acceptable excipient.19. Homogentisic acid for use in the treatment of an infection in a patient, wherein the patient is undergoing treatment with the polynucleotide according to any one of claims 1 to 12.20. A method of screening for an inhibitor of homogentisate 1, 2 dioxygenase comprising: providing a plurality of compounds; (ii) providing a mammalian homogenti sate 1, 2 dioxygenase; (iii) contacting each of the plurality of compounds and the mammalian homogentisate I, 2 dioxygenase, and (iv) identifying a compound that inhibits the mammalian homogentisate 1, 2 dioxygenase.21. Nitisinone for use in reversing homogenti sate 1, 2 dioxygenase inhibition therapy.22. A polynucleotide of claims Ito 12 or a composition of claim 13 for use in the treatment of an infection in a patient, wherein the polynucleotide or composition is co-administered simultaneously, subsequently or separately with HGA.23 HGA for use in the treatment of an infection in a patient, wherein the HGA is co-administered simultaneously, subsequently or separately with a polynucleotide of claims 1 to 12 or a composition of claim 13.24. A kit comprising a polynucleotide according to any one of claims 1 to 12 and HGA.25. An antibiotic coating for an implantable device comprising HGA, optionally further comprising a binding agent