High CSF penetration and potent CSF bactericidal activity of NZ2114 a. novel Plectasin variant during experimental pneumococcal meningitis ACCEPTED

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1 AAC Accepts, published online ahead of print on February 00 Antimicrob. Agents Chemother. doi:./aac.0-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Title. High CSF penetration and potent CSF bactericidal activity of NZ a novel Plectasin variant during experimental pneumococcal meningitis Authors. Christian Østergaard, 1* Dorthe Sandvang, Niels Frimodt-Møller, Hans-Henrik Kristensen. Address. 1) Department of Clinical Microbiology, Copenhagen University Hospital Herlev, Herlev, Denmark. ) National Center for Antimicrobials and Infection Control, Statens Serum Institut, Copenhagen, Denmark. ) Novozymes A/S, Bagsværd, Denmark Running title. Plectasin NZ in experimental meningitis. Key words Antimicrobial peptide, meningitis, Streptococcus pneumoniae, NZ, Plectasin 1 Correspondent footnote. Mailing address: Department of Clinical Microbiology, Copenhagen University Hospital Herlev, Herlev Ringvej, DK-0 Herlev, Denmark. Phone: + 0. Fax: +. coa@ssi.dk 1

2 Abstract Plectasin is the first defensin-type antimicrobial peptide isolated from a fungus and has potent activity against Gram-positive bacteria. Using an experimental meningi- tis model, the CSF penetration in infected and uninfected rabbits and the CSF bactericidal activity of the plectasin variant NZ and ceftriaxone against a penicillin-resistant Streptococcus pneumoniae (NZ and ceftriaxone MICs: 0. and 0. µg/ml, respectively) were studied. Pharmacokinetics: There was a significant higher CSF penetration of NZ through inflamed - compared to non-inflamed meninges (AUC CSF /AUC serum : % vs. 1.1%, respectively, P=0.0). Peak concentrations of NZ in purulent CSF was observed ~ hours after an intravenous bolus infusion of either 0 or 0 mg/kg and exceeded the MIC >-fold during a -hour study period. Bactericidal activity: Treatment with NZ (0 and 0 mg/kg at 0 and hours, respectively, n=) caused a significantly higher reduction in CSF bacterial concentrations ( Log CFU/mL) as compared to therapy with ceftriaxone (1 mg/kg at 0 hours, n=) at hours (median:. (interquartile range:.-.) vs..1 (1.-.), respectively, P=0.001), at hours (. (.-.1) vs..1 (.-.), respectively, P=0.01) and at hours (. (.-.) vs.. (.-.0), respectively, P=0.0) after start of therapy as well as compared to untreated meningitis rabbits (n=, P<0.0). Also, significant more rabbits had sterile CSF at and hours, when treated with NZ as compared to therapy with ceftriaxone (% (/) vs. 0% (0/) and % (/) vs. 1% (1/), respectively, P<0.0). Due to its excellent CSF penetration and potent CSF bactericidal activity, the plectasin variant NZ could be a new promising treatment option of CNS infections caused by Gram-positive bacteria, including penicillin-resistant pneumococcal meningitis.

3 Introduction Streptococcus pneumoniae is the leading cause of bacterial meningitis, and pneumococcal meningitis still remains a disease with high mortality and morbidity (). The corner stone in the treatment of bacterial meningitis is the prompt initiation of adequate antibiotic therapy (), and rapid sterilization of the CSF with antibiotic therapy has been associated with a better clinical outcome (). The world-wide emergence of antibiotic-resistant strains of S. pneumoniae still demands develop- ment of new antimicrobial agents for treatment of pneumococcal disease including meningitis. Antimicrobial peptides are evolutionarily ancient effect molecules, widely distrib- uted in plants and animals, and used by the innate immune system to control in- fections. It is a new class of antimicrobial agents, but its mechanism of action has not yet been disclosed. Most antimicrobial peptides are cationic binding readily to the negatively charged bacterial membrane, and they may promote their activity by compromising bacterial membranes (e.g. fatal depolarisation of the bacterial membrane, degradation of the cell wall by hydrolases, or disturbance of membrane function) or may interact with critical intracellular targets (for a review: (1)) Plectasin was the first defensin-like antimicrobial peptide isolated from a black saprophytic ascomycete (Pseudoplectania nigrella) and showed potent in vitro and in vivo activity against Gram-positive bacteria (). NZ was identified in a high through-put mutation and screening campaign aiming for variants of plectasin with improved antimicrobial potency against staphylococci and streptococci (). NZ exhibited improved in vitro activity against staphylococci including Staphylococcus aureus as well as S. pneumoniae and haemolytic streptococcal

4 strains resistant to clinically used antibiotics (Sandvang et al. abstract ICAAC 00 F1-1, Chicago 1-0 sep 00). Little is known about the distribution of antimicrobial peptides into various body tissues including the penetration across the blood-brain barrier, which forms a tight membrane limiting the entry of many antim- icrobial agents into the CNS. Therefore, when introducing a new antimicrobial agent, it is important to study the penetration into the brain as well as the CSF bactericidal activity of the drug. Correspondingly, we studied the CSF penetration of NZ as well as its CSF bactericidal activity against penicillin-resistant S. pneumoniae in an experimental meningitis model.

5 Materials and Methods Test organism. A penicillin-resistant, but ceftriaxone-sensitive Streptococcus pneumoniae, type V originally isolated from the CSF of a year old female, was used for all meningitis experiments. MIC s of NZ, ceftriaxone, and penicillin were 0. mg/l, 0. mg/l, and 1 mg/l, respectively, as determined by the microdi- lution broth method according to the NCCLS/CLSI guidelines (). Antimicrobial agents. The plectasin variant NZ was produced, purified, and formulated at Novozymes A/S, Bagsværd, Denmark. In short, Aspergillus oryzae MT was selected as production host, and fermentation conditions were opti- mized at Novozymes. The fermented broth was pre-treated, filtrated and centri- fuged, before the recovered product was further processed by two chroma- tographic purifications; cat-ion exchanger, and hydrophobic interaction followed by 0K filtration and final Ultra/DIA filtrations. The API was exchanged into the final formulation buffer (mm Na-acetate, 0.% NaCl, ph.0) by DIA filtration and germ filtered before filled in vials. Stock solutions were made by dissolving NZ (Batch PSI01, purity: %) in Kalium-Natrium-Glucose Fresenius Kabi infusion liquid (0C0, Fresenius Kabi, Uppsala, Sweden) to a final concentration of 1. g/l (vehicle ph=). Ceftriaxone (No. C-, Sigma Chemical Co., St. Louis, USA) was dissolved in sterile water to a final concentration of 0 g/l. Antibiotics were administered intravenously as bolus infusion over - min. Determination of NZ concentrations in CSF and serum. NZ concen- trations in CSF and serum were determined by HPLC. Lower detection limit was 0 µg/l.

6 Determination of bacterial concentrations. Bacterial concentrations in CSF were determined by plating 0µL + 0 µl of undiluted CSF and -fold serial dilutions (in duplicate) on % horse blood agar plates (Statens Serum Institute, Copenhagen, Denmark). Thus, the lowest detectable bacterial counts were CFU/mL. Comparison of the bacterial counts in different dilutions of CSF was per- formed to exclude significant carry-over phenomena. Meningitis model. The experimental protocols were approved by the Danish Ani- mal Experiments Inspectorate (Dyreforsøgstilsynet). A rabbit meningitis model previously described in detail was used (). New Zealand white rabbits,.-.0 kg in weight, were anaesthetised with midazolam (Dormicum, F. Hoffmann-La Roche AG., Basel, Switzerland), 0. mg/kg s.c. and a combination of fen- tanyl/fluanisone (Hypnorm, Janssen Pharmaceutica N.V., Beerse, Belgium), 0. ml/kg i.m. and a dental helmet embedding a half turnbuckle was attached to the skull. Rabbits returned to their cages, after buprenorphin (Nycomed, Roskilde, Denmark) 0.1 mg/kg was given as analgesia. Rabbits that were to become infected with pneumococci were hours later reanesthetized with midazolam and fentanyl/fluanisone followed by intracisternal inoculation of the test organism (~1 x CFU, as confirmed by quantitative cultures). Again, buprenorphin was given, before the infected rabbits returned to their cages. After another 1 hours, all rabbits (both infected meningitis rabbits and uninfected controls) were reanesthetized with ethyl carbamate (Urethane, Fluka Kemi AG, Buchs, Switzerland), 1. g/kg s.c. and pentobarbital (Mebumal, Nycomed), mg/kg and immobilised in a stereotaxic frame. A gauge spinal needle was in-

7 troduced into cisterna magna for repetitive CSF sampling. Blood samples were taken from a central ear artery of the right ear, whereas antibiotics and pentobarbital was administered intravenously into the left ear. After testing for bacterial con- centration, CSF and blood samples were centrifuged, and supernatants were im- mediately stored at - 0 C for subsequent analysis. Pharmacokinetics of NZ. Three - (, 0 and 0 mg/kg) and two different doses were tested in uninfected and infected rabbits, respectively (n= for each group). CSF and blood samples were taken at 0, 0., 0., 1, 1.,,,,, and hours after antibiotic challenge. The following pharmacokinetic parameters were determined for each rabbit using Prism Version.01 (GraphPad Software, Inc., La Jolla, CA, USA): C max, t ½ (estimated by the expression -log /β, where β is the slope of the elimination regression line), AUC 0-, and T >MIC. Efficacy of NZ and ceftriaxone in penicillin resistant pneumococcal meningitis. NZ, 0 mg/kg at 0 hours and 0 mg/kg at hours (n=) were compared to ceftriaxone, 1 mg/kg at 0 hours (n=). Seven untreated rabbits were reserved as control group. CSF and blood samples were taken at 0, 1,,,, and hours after start of antibiotic treatment, respectively. The dosing of NZ was chosen to mimic the CSF pharmacokinetic profile after a bolus infusion with ceftriaxone (1 mg/kg), where previous meningitis experiments showed that CSF concentrations remained at concentrations the MIC (~ mg/l) for the test organism at hours after start of therapy (). Statistical analysis. All results are provided as medians and interquartile range. For calculation of the reduction in CSF bacterial concentrations (e.g. Log CFU/mL 0-h ), a CSF concentration under the detection limit was assigned a value of 1 CFU/mL. Comparison between groups was performed by the non-parametric

8 Mann-Whitney test for continuous data and by Fisher Exact test for categorical data. P-values less than 0.0 were considered statistically significant.

9 Results Pharmacokinetics of NZ. CSF and serum concentration-time curves and pharmacokinetic indices with intravenous bolus infusions of NZ are shown in Figure 1 and in the Table, respectively. Whereas the distribution of NZ in the systemic compartment was comparable for both infected and uninfected rabbits (one compartment model with a t ½ of minutes (-)), the CSF penetration (AUC csf /AUC serum ) was significantly higher in meningitis rabbits as compared to un- infected control different (% (-0) vs. 1.1% (0.-1.), respectively, P=0.0) for the current dosing regimens (0 and 0 mg/kg 1, see Figure 1A+B). The C max was 0-0 times higher in meningitis rabbits than in uninfected rabbits (e.g.. mg/l (.-.) vs. 0. mg/l (0.-0.) after a 0 mg/kg bolus infusion) and was observed ~ hours after the bolus infusion. The CSF concentration did not de- crease significantly after the time of C max (t ½ presumably more than times longer in the CSF than in serum) and remained above the MIC during the hours study period for meningitis rabbits, whereas it was below the MIC for uninfected controls most of the time. During treatment efficacy studies (0 and 0 mg/kg at 0 and hours, respectively), NZ concentrations in CSF and blood remained at concentrations above the MIC during the hours study period (see Figure 1C). 0 1 Treatment efficacy of NZ in meningitis caused by a penicillin resistant pneumococcal strain. CSF bacterial concentrations are shown in Figure. Before the initiation of antibiotic therapy (at 0 hours), no significant difference in CSF bacterial concentrations (Log CFU/mL) were observed among the three experimental groups (NZ treated:.0 (.-.0), n=, ceftriaxone treated:. (.-.), n=, and untreated meningitis rabbits:. (.-.1), n=, P>0.0). NZ

10 1 1 1 treated rabbits had significantly lower CSF bacterial concentrations (Log CFU/mL) than ceftriaxone treated rabbits at hours (. (0.-.1) vs.. (.1-.0), respectively, P=0.01), at hours (0. (0.-.) vs.. (.-.), respectively, P=0.00), and at hours after start of antibiotic therapy (0. (0.-1.1) vs. 1. (1.0-.), respectively, P=0.0), whereas antibiotic treated rabbits had significantly lower CSF bacterial counts than untreated meningitis rabbits at all time points (P<0.0, see Figure ). This was a significantly higher CFU reduction ( Log CFU/mL/hrs) for rabbits treated with NZ than for rabbits treated with ceftri- axone (at hours:. (.-.) vs..1 (1.-.), respectively, P=0.001, at hours:. (.-.1) vs..1 (.-.), respectively, P=0.01, and at hours: (. (.-.) vs.. (.-.0), respectively, P=0.0). In addition, significantly more rabbits treated with NZ had sterile CSF as compared with ceftriaxone treated rabbits at and hours (% (/) vs. 0% (0/) and % (/) vs. 1% (1/), respec- tively, P<0.0).

11 Discussion To our knowledge this is the first study to describe the CSF penetration and CSF bactericidal activity of an antimicrobial peptide. The plectasin variant NZ had a CSF penetration of % through inflamed meninges, which resulted in CSF con- centrations above the MIC for most Gram positive pathogens including S. pneu- moniae. Interestingly, this was a higher CSF penetration as compared to most other antimicrobial agents except fluoroquinolones (i.e. ceftriaxone: ~1%, vanco- mycin: 1%, moxifloxacin 1%) (). This indicates that even relatively large and water soluble molecules such as NZ (peptide of 0 amino acids in length, mo- lecular weight:. kda) (), which is a ~ times larger molecular weight than for most other antimicrobial agents (i.e. ceftriaxone: 0. kda) (), readily enters the CSF, when the blood-brain barrier is inflamed. In contrast, the CSF penetration of NZ through intact blood-brain barrier was poor (1.1%) hardly reaching the MIC after an intravenous dose of 0 mg/kg, but still comparable with the CSF penetration of ceftriaxone through non-inflamed meninges (~%) (1). Beside alterations in blood-brain barrier permeability, lipid solubility and molecular weight of the drug, the protein binding and whether it is a substrate of various active influx and efflux pumps of the brain endothelium also influence the CSF penetration of antibiotics (for a review: ()) and peptides (for a review: ()). The passage of NZ across intact blood-brain barrier showed in part saturable features, since the CSF penetration decreased with higher doses, however, this was not the case for inflamed blood-brain barrier. The CSF bactericidal activity of NZ was high against penicillin-resistant pneumococci resulting in a rapid CSF kill rate (~1 Log CFU/mL/hrs) during ex- perimental pneumococcal meningitis in rabbits, which was a significantly higher kill

12 rate than therapy with ceftriaxone - a recommended treatment of pneumococcal meningitis (). Moreover, sterilization of the CSF was rapid and observed for the majority of rabbits within hours of therapy with NZ, and no regrowth was observed during the following hours of therapy. A previous study investigated the in vivo activity of the parent wild type molecule against pneumococci using a mouse peritonitis model and a mouse pneumonia model and found in accordance with the present study excellent kill rates with plectasin, which was comparable to therapy with other antibiotics (). Most antimicrobial peptides are positively charged and bind readily to the nega- tively charged phospholipid of the bacterial cytoplasmatic membrane promoting a rapid kill of microorganisms at concentrations equivalent or close to the MIC, and contrary to the use of conventional antibiotics, the development of resistance with antimicrobial peptides seems to be surprisingly poor. It has been hypothesized that antimicrobial peptides work primarily by compromising bacterial membranes (e.g. fatal depolarisation of the bacterial membrane, degradation of the cell wall by hydrolase, disturbance of membrane functions, for a review: (1)). However, preliminary results showed that the mechanism by which NZ kills bacteria was different than membrane lysis (H-H. Kristensen, personal correspondence). In addition, no cross-resistance to other classes of antibiotics has yet been observed (H-H Kristensen, personal correspondence). Thus, due to the emerging of resistant pneumococci and its potent CSF bactericidal activity, treatment with NZ could be an interesting treatment option for pneumococcal meningitis, warranting further clinical and experimental evaluation. 1

13 In conclusion, NZ could be a new promising treatment option of CNS infec- tions, including penicillin-resistant pneumococcal meningitis, due to its excellent CSF penetration and potent CSF bactericidal activity. 1

14 Acknowledgements The authors thank Jytte Mark Andersen and Flemming Pedersen for skilful techni- cal assistance. 1

15 References 1. Andersen, C. O. 00. Streptococcus penumoniae meningitis. Clinical and experimental studies. Dan.Med.Bull. :1-0.. Andes, D. R. and W. A. Craig. 1. Pharmacokinetics and pharmacodynamics of antibiotics in meningitis. Infect.Dis.Clin.North Am. 1:-1.. Banks, W. A. and A. J. Kastin. 1. Passage of peptides across the blood-brain barrier: pathophysiological perspectives. Life Sci. :1-1.. Lebel, M. H. and G. H. McCracken, Jr. 1. Delayed cerebrospinal fluid sterilization and adverse outcome of bacterial meningitis in infants and children. Pediatrics :-1.. Mygind, P. H., R. L. Fischer, K. M. Schnorr, M. T. Hansen, C. P. Sonksen, S. Ludvigsen, D. Raventos, S. Buskov, B. Christensen, L. De Maria, O. Taboureau, D. Yaver, S. G. Elvig-Jorgensen, M. V. Sorensen, B. E. Christensen, S. Kjaerulff, N. Frimodt-Moller, R. I. Lehrer, M. Zasloff, and H. H. Kristensen. 00. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature :-0.. National Committee for Clinical Laboratory Standards. 1. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically-Fourth Edition; Approved standard., p. -1. In: NCCLS (ed.), NCCLS document M-A. NCCLS, 0 West Valley Road, Suite 100, Wayne, Pennsylvania 10.. Nau, R., F. Sorgel, and H. W. Prange. 1. Lipophilicity at ph. and molecular size govern the entry of the free serum fraction of drugs into the cerebrospinal fluid in humans with uninflamed meninges. J.Neurol Sci. 1:1-.. Østergaard, C., H. B. Konradsen, and S. Samuelsson. 00. Clinical presentation and prognostic factors of Streptococcus pneumoniae meningitis according the focus of infection. BMC.Infect Dis :1-.. Østergaard, C., T. K. Sørensen, J. D. Knudsen, and N. Frimodt-Møller. 1. Evaluation of moxifloxacin, a new -methoxyquinolone, for treatment of meningitis caused by a penicillin-resistant pneumococcus in rabbits. Antimicrob.Agents Chemother :10-.. Raventos, D., O. Taboureau, P. H. Mygind, J. D. Nielsen, C. P. Sonksen, and H. H. Kristensen. 00. Improving on nature's defenses: optimization & high throughput screening of antimicrobial peptides. Comb.Chem.High Throughput.Screen. :1-.. Tunkel, A. R., B. J. Hartman, S. L. Kaplan, B. A. Kaufman, K. L. Roos, W. M. Scheld, and R. J. Whitley. 00. Practice guidelines for the management of bacterial meningitis. Clin.Infect Dis : Zasloff, M. 00. Antimicrobial peptides of multicellular organisms. Nature 1:-. 1

16 Figure legends Figure 1. CSF and serum concentrations of NZ in infected and uninfected rabbits. A and B. NZ was administered as an intravenous bolus infusion over - minutes at 0 hours (arrows, n= for each group). C. NZ was administered at 0 hours (0 mg/kg) and at hours (0 mg/kg), (ar- rows, n=). Data are shown as medians and range (A and B) or interquartile range (C) Figure. CSF bacterial concentrations * after start of antibiotic therapy in experimental pneumococcal meningitis *Data are shown as medians and interquartile range. Significant differences: NZ (n=) vs. ceftriaxone (n=) or untreated controls (n=), and ceftriaxone vs. uninfected controls at, and hours (Mann Whitney test, P< 0.0). NZ and ceftriaxone treated rabbits had sterile CSF in % (/) vs. 0% (0/) at hours and % (/) vs. 1% (1/) at hours (Fisher Exact test, P<0.0). The lower limit of detection of bacteria was CFU/ml. 0 1

17 Table. Pharmacokinetics of NZ in CSF and blood of rabbits with and without pneumococcal meningitis * Dose of NZ With meningitis CSF t ½ Blood 0 mg/kg n.d. min (-) 0 mg/kg n.d. min (-) Without meningitis CSF.0 µg/ml (1.-.). µg/ml (.-.) C max # Blood 0. µg/ml (.-0.) µg/ml (0.-1) CSF. mg h/ml (.-.). mg h/ml (.-0.) mg/kg n.d. min (1-) 0. µg/ml (0.0-0.). µg/ml (.-.) 0. mg h/ml (0.-0.) 0 mg/kg n.d 0 min 0.1 µg/ml. µg/ml 0. mg h/ml (-) (0.-0.1) (.-.) (0.-0.0) 0 mg/kg n.d min 0. µg/ml. µg/ml 1.0 mg h/ml (-) (0.1-0.) (.0-.) (0.-1.) *T >MIC was ~0% and ~0% in infected and uninfected CSF, respectively. # T max was ~1-0 min and ~ hrs in blood and CSF, respectively. Data are shown as medians and ranges. N= for each group. n.d: not determined AUC 0- Blood 1. mg h/ml (0.-.).0 mg h/ml (0.0-). mg h/ml (.-1.) 0.0 mg h/ml (.-.1) 1 mg h/ml (1-1) CSF penetration % (1-) % (-).% (.-.1) 1.% (1.1-.1) 0.% (0.-1.1) 1

18 mg/l mg/l A. CSF concentration Hours B. Serum concentration Hours C. CSF and blood concentrations during treatment of S. pneumoniae meningitis 0mg/kg (menigitis) 0mg/kg (menigitis) 0 mg/kg (uninfected) 0 mg/kg (uninfected) MIC for S. pneumoniae 0mg/kg (menigitis) 0mg/kg (menigitis) 0 mg/kg (uninfected) 0 mg/kg (uninfected) MIC for S. pneumoniae 00 0 CSF Serum mg/l MIC for S. pneumoniae Hours Figure 1 1

19 Log CFU/mL Hours Control (n=) Ceftriaxone (n=) NZ (n=) Lower detection level Figure 1

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