Am pho te ric in B Inha la tio n Po wde r (ABIP) Ac hie ve s Sig nific a nt Pulm o na ry a nd Lo w Syste m ic Am pho te ric in B C o nc e ntra tio ns Ala n R. Kug le r, PhD, Jo na tha n D. Le e , Lo ri K. Sa mfo rd , Ro b e rt J. G e re ty, MD, PhD, a nd Mic ha e l A. Eld o n, PhD Ne kta r The ra p e utic s Sa n C a rlo s, C a lifo rnia , USA The 16th C o ng re ss o f the Inte rna tio na l So c ie ty fo r Huma n a nd Anima l Myc o lo g y Pa ris, Fra nc e June 26, 2006 Am pho te ric in B Inha la tio n Po wde r (ABIP) Active Pharmaceutical Ingredient 2 Amphotericin B  Potent  Broad Spectrum  Fungicidal Formulation Dry Powder  Spray Dried  Highly Aerodynamic  Packaged in Capsules Delivery Dry Powder Inhaler (DPI)  Small Patient-Driven Device  Efficiently Places the Drug in the Deep Lungs Aerosol Powder + Capsule + Dry Powder Inhaler Po wde r Ae ro dyna m ic Pro pe rtie s   Fungal spores are inhaled  cause pulmonary fungal infections Nektar ABIP powder is inhaled  deposits to same sites in lung as spores Fungal Spore Amphotericin B Particle 2 µm 3 Pre ve ntio n o f Pulm o na ry Fung a l Infe c tio ns Using ABIP Prevention hypothesis: pulmonary administration attains and maintains lung tissue amphotericin B concentrations well above the minimum inhibitory concentrations (MICs) for Aspergillus spp. throughout the risk period Concentration (µ g/g) 60 Predicted human lung tissue amphotericin B concentrations following 50 mg loading dose and weekly 10 mg maintenance doses of ABIP for 6 months 50 40 30 20 10 Amphotericin B MIC against Aspergillus spp. 0 0 28 56 84 112 140 168 196 224 Time (day) 4 Prio r Pre c linic a l a nd C linic a l Sum m a ry  Preclinical Studies • Toxicology • Pharmacology • Pharmacokinetics  Single-Dose Clinical Trial • • • •  Multiple-Dose Clinical Trial • • • •  5 Complete 1, 2.5, 5, 10, 25 mg single-doses Safe and well-tolerated Low systemic amphotericin B exposure confirmed Dosing complete Cohort 1: 25 mg loading dose followed by four weekly 5 mg maintenance doses Cohort 2: 50 mg loading dose followed by four weekly 10 mg maintenance doses Bioanalytical (plasma, BAL/ELF) pending Human Dose and Regimen Strategy Sing le - Do se PK C linic a l Tria l O ve rvie w   Eight healthy subjects (5 men, 3 women) received a single 5-mg inhalation dose in this open-label, safety, tolerability, and pharmacokinetic (PK) trial of ABIP Objectives • ABIP safety and tolerability • Amphotericin B pharmacokinetics (especially to support ABIP dose and regimen selection)   Standard safety measurements and pulmonary function were monitored throughout the trial Three bronchoscopies were performed per subject • Two bronchoalveolar lavage samples (BAL; ‘early’ 1-12 hr and ‘late’ 21-32 hr postdose) • Two airway surface biopsy samples (‘early’ 1-12 hr and ‘late’ 14-35 day postdose)  Seven venous blood samples • Predose, 0.5, 1, 2, 4, 8, 24 hr postdose  6 Plasma, airway tissue, and BAL AmB concentrations were determined using validated LC-MS/MS methods. Serum/BAL urea concentration ratios were used to quantify ELF dilution ABIP wa s Sa fe a nd We ll- To le ra te d     7 The majority of the Adverse Events (AEs) were mild in nature and not related to study drug Seven subjects had AEs that were mild and suspected to be related to the bronchoscopy procedure Two subjects had AEs of moderate severity that were suspected to be related to the bronchoscopy procedure No serious, severe, or life-threatening AEs were reported PFT Spiro m e try Re fle c t Effe c ts o f Bro nc ho sc o pie s Change in Pulmonary Function Test Values From Baseline Vs. Week by Test and by Subject Δ FEV1 (%) 30 20 10 0 -10 -20 -30 30 Δ FVC (%) 20 10 0 -10 -20 -30 Δ FEF25-75% (%) 30 20 10 0 -10 -20 -30 0 1 2 3 4 5 17 Week 8 ABIP Dosing at Time 0 and Bronchoscopies on Days 1, 2, and 14-35 C o nfirm e d Lo w Syste m ic Am pho te ric in B Expo sure     1-2 hr lag in amphotericin B appearance in plasma 1,000* 35 Cmax at 8 hr Modest intersubject variability Low maximal systemic amphotericin B concentrations Plasma amphotericin B concentrations were ≤ 4% of those generally associated with toxicity and should translate into improved safety 30 Concentration (ng/mL)  Plasma Amphotericin B Concentration-Time Profiles (Mean ± SD) 25 20 15 10 5 LLQ 0 0 4 8 12 16 Time Post Dose (hr) * 1,000 ng/mL is generally regarded as the plasma amphotericin B threshold for toxicity 9 20 24 28 Epithe lia l Lining Fluid (ELF) Am pho te ric in B C o nc e ntra tio ns    10 Each subject is displayed as one or two points. Thick lines are linear regressions using pooled data Initial pulmonary amphotericin B concentrations greater than MICs even after a single 5 mg ABIP dose Amphotericin B concentrations ~1000-fold higher than plasma amphotericin B concentrations ELF elimination half-life values of 6-10 hr comparable to animal pharmacokinetic studies 50 ELF Aliquot 1 = 6.4 hr half-life 10 Concentration (µg/mL)  ELF Amphotericin B Concentration-Time Profiles 1 0.5 50 ELF Aliquot 2 = 10.4 hr half-life 10 1 0.5 0 4 8 12 16 20 Time Post Dose (hr) 24 28 Airwa y Tissue Am pho te ric in B C o nc e ntra tio ns     11 Each subject is one point Airway Tissue Amphotericin B Concentration-Time Profiles High initial amphotericin B concentrations with all second biopsy samples within an individual subject BLQ Apparent elimination half-life values much quicker than animal pharmacokinetic studies Elimination half-life values in rat, rabbit, and dog consistently long (15-20 day) Sampling artifact of the biopsy (surface sampling of a major airway with mucocilliary clearance) not representative of pharmacokinetics in peripheral lung 10 Concentration (µg/g)  8 6 4 2 0 0 4 8 12 16 20 Time Post Dose (hr) 24 28 C o nc lusio ns      12 This trial demonstrated that inhaled ABIP was well-tolerated while producing pulmonary amphotericin B concentrations significantly above common Aspergillus spp. minimum inhibitory concentrations accompanied by low systemic amphotericin B exposure Maximal ELF amphotericin B concentrations were ~1000-fold higher than plasma amphotericin B concentrations, demonstrating that ABIP pulmonary delivery achieves a localization of amphotericin B exposure to the site of greatest importance for preventing pulmonary fungal infection, the lung Maintenance of pulmonary amphotericin B concentrations in excess of MICs during the entire period of pulmonary fungal infection risk in immunosuppressed patients, while minimizing systemic amphotericin B exposure, appears possible with repeated ABIP dosing Results from this study will be integrated with other preclinical and clinical results to support human dose and regimen selection ABIP is a promising therapy for prevention of pulmonary fungal infections Ac kno wle dg e m e nts  National Jewish Medical and Research Center • Principal Investigator (PI): Sally E. Wenzel, MD • Denver, Colorado, USA  MEDTOX Bioanalytical • St. Paul, Minnesota, USA  Bioanalytical Consultant • Donald Reynolds, PhD  Nektar R&D Pharmaceutical Development • Clark March, PhD • Richard Malcolmson, PhD • Theresa M. Sweeney, PhD • Michael J. Weickert, PhD 13 15 Appe ndix Po dium De ta ils  Proffered Podium (Abstract O-0011)  Symposium 3 • Antifungals: Pharmacokinetics and Pharmacodynamics  15 minute podium with session concluding panel discussion  10:10-10:25 am on M26Jun06  16 www.isham.org Re fe re nc e s       17 Kugler AR, Sweeney TD, Eldon MA. 2005. Prophylaxis of invasive aspergillosis in neutropenic rabbits using an inhaled, novel, dry-powder amphotericin B formulation, Abstr. LB2-31, p. 227. In Program and abstracts of the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). American Society for Microbiology, Washington, DC, USA. Kugler AR, Sweeney TD, Lalonde G, Eldon MA. 2006. Prophylactic administration of Amphotericin B Inhalation Powder (ABIP) prolongs survival of neutropenic rabbits inoculated with Aspergillus, Abstr. P111, pp. 206-7. In Program and abstracts of the 2nd Meeting of Advances Against Aspergillosis (AAA), Athens, Greece. Lee JD, Kugler AR, Samford LK, Gerety RJ, Eldon MA. 2006. Amphotericin B Inhalation Powder (ABIP) is well-tolerated with low systemic amphotericin B exposure in healthy subjects, Abstr. P118, pp. 214-5. In Program and abstracts of the 2nd Meeting of Advances Against Aspergillosis (AAA), Athens, Greece. Weickert MJ. 2006. Unique forms of amphotericin B: Amphotericin B Inhalation Powder (ABIP), invited podium, pp. 99-103. In Program and abstracts of Focus on Fungal Infections 16 (FoFI), Las Vegas, NV, USA. Kugler AR, Sweeney TD, Lalonde G, Perkins KM, Eldon MA. 2006. Prophylactic administration of Amphotericin B Inhalation Powder (ABIP): Proof-of-Efficacy, Abstr. P-027, p. 186. In Program and abstracts of Focus on Fungal Infections 16 (FoFI), Las Vegas, NV, USA. Kugler AR, Lee JD, Samford LK, Gerety RJ, Eldon MA. 2006. Amphotericin B Inhalation Powder (ABIP) achieves significant pulmonary and low systemic amphotericin B concentrations, proffered podium in “Antifungals: Pharmacokinetics and Pharmacodynamics Symposium,” Abstr. O-0011. In Program and abstracts of the 16th Congress of the International Society for Human and Animal Mycology (ISHAM), Paris, France. Disc lo sure s   18 The authors are employees and shareholders of Nektar Therapeutics Individuals acknowledged at the end of the presentation are also employees and shareholders of Nektar Therapeutics Ab stra c t Title : Am pho te ric in B Inha la tio n Po wde r (ABIP) Ac hie ve s Sig nific a nt Pulm o na ry a nd Lo w Syste m ic Am pho te ric in B C o nc e ntra tio ns Ba c kg ro und: ABIP is b e ing de ve lo pe d a s a n e ffe c tive , sa fe , a nd c o nve nie nt the ra py to pre ve nt pulm o na ry fung a l infe c tio ns in im m uno suppre sse d pa tie nts. ABIP is a hig hly re spira b le dry- po wde r, fo rm ula te d to ha ve a n a e ro dyna m ic size to de live r a m pho te ric in B (Am B) to the sa m e a irwa ys a nd a lve o la r surfa c e s o f the lung s o n whic h fung a l spo re s de po sit a nd g e rm ina te . ABIP is a dm iniste re d with a pro prie ta ry, ha ndhe ld, dry- po wde r inha le r de vic e a nd do se s c o nta ining up to 25 m g Am B ha ve b e e n we ll- to le ra te d. Ta rg e te d drug de live ry to the lung is e xpe c te d to m a inta in lung tissue Am B c o nc e ntra tio ns suffic ie nt to pre ve nt o ppo rtunistic pulm o na ry fung a l infe c tio ns in im m uno suppre sse d pa tie nts while a vo iding the do se - lim iting side e ffe c ts a nd drug - drug inte ra c tio ns o b se rve d with syste m ic use . Pro o f tha t ABIP pro te c ts a g a inst Aspe rg illus fum ig a tus infe c tio n, while pro duc ing lo w syste m ic Am B e xpo sure , ha s b e e n de m o nstra te d in a pe rsiste ntly ne utro pe nic e ffic a c y m o de l in ra b b its. In this tria l, hum a n pulm o na ry a nd syste m ic Am B pha rm a c o kine tic s fo llo wing sing le inha le d do se s o f ABIP we re inve stig a te d. Me tho ds: Eig ht he a lthy sub je c ts re c e ive d sing le 5- m g inha la tio n do se s in this o pe n- la b e l, sa fe ty, to le ra b ility, a nd pha rm a c o kine tic (PK) tria l o f ABIP. Sta nda rd sa fe ty m e a sure m e nts, inc luding pulm o na ry func tio n, we re m o nito re d thro ug ho ut the 17 we e ks o f the study. Se ve n ve no us b lo o d (pre do se , 0.5, 1, 2, 4, 8, 24 hr po stdo se ) a nd 2 b ro nc ho a lve o la r la va g e (BAL; ‘e a rly’ 1- 12 hr a nd ‘la te ’ 21- 32 hr po stdo se ) sa m ple s pe r sub je c t we re c o lle c te d a nd use d to c ha ra c te rize syste m ic (pla sm a ) a nd pulm o na ry (e pithe lia l lining fluid [ELF]) Am B pha rm a c o kine tic s. Pla sm a a nd BAL Am B c o nc e ntra tio ns we re de te rm ine d using va lida te d LC - MS/ MS m e tho ds. Se rum / BAL ure a c o nc e ntra tio n ra tio s we re use d to q ua ntify ELF dilutio n. Re sults: No c linic a lly re le va nt c ha ng e s fro m b a se line fo r se rum c he m istry pa ra m e te rs, vita l sig ns, o r spiro m e try (FVC a nd FEV1) we re o b se rve d. Adve rse e ve nts (AEs) we re ra te d a s m ild o r m o de ra te in se ve rity with no re po rte d se rio us AEs. Me a n pla sm a Am B C m a x wa s 21.7 ng / m L (ra ng e : 8.7 to 33.1 ng / m L) a nd o c c urre d a t 8 hr. The se Am B c o nc e ntra tio ns we re we ll b e lo w tho se a sso c ia te d with re na l to xic ity (g e ne ra lly susta ine d pla sm a Am B c o nc e ntra tio ns o f g re a te r tha n 1000 ng / m L) a nd we re la rg e ly c o nsiste nt with la b o ra to ry a nim a l re sults. The m a xim um o b se rve d ELF Am B c o nc e ntra tio n wa s 50.4 µg / m L a nd c o nc e ntra tio ns de c line d with a 6- 10 hr ha lf- life , a lso c o nsiste nt with a nim a l re sults. C o nc lusio ns: This tria l de m o nstra te d tha t inha le d ABIP wa s we ll- to le ra te d a t pulm o na ry Am B c o nc e ntra tio ns sig nific a ntly a b o ve c o m m o n Aspe rg illus spp. m inim um inhib ito ry c o nc e ntra tio ns a nd we re a c c o m pa nie d b y lo w syste m ic Am B e xpo sure . Ma xim a l ELF Am B c o nc e ntra tio ns we re ~1000- fo ld hig he r tha n syste m ic Am B c o nc e ntra tio ns, de m o nstra ting tha t ABIP pulm o na ry de live ry a c hie ve s a lo c a liza tio n o f Am B e xpo sure to the site o f g re a te st im po rta nc e fo r pre ve nting pulm o na ry fung a l infe c tio n, the lung . Ma inte na nc e o f pulm o na ry Am B c o nc e ntra tio ns in e xc e ss o f MIC s during the e ntire pe rio d o f pulm o na ry fung a l infe c tio n risk in im m uno suppre sse d pa tie nts, while m inim izing syste m ic Am B e xpo sure , sho uld b e po ssib le with re pe a te d ABIP do sing . ABIP is a pro m ising the ra py fo r pre ve ntio n o f pulm o na ry fung a l infe c tio ns in pa tie nts a t risk due to im m uno suppre ssive the ra py inc luding tho se re c e iving o rg a n o r ste m c e ll tra nspla nts, o r tre a te d with c he m o the ra py fo r he m a to lo g ic m a lig na nc ie s. C ita tio n: Kug le r AR, Le e JD, Sa mfo rd LK, G e re ty RJ, Eld o n MA. 2006. Amp ho te ric in B Inha la tio n Po wd e r (ABIP) a c hie ve s sig nific a nt p ulmo na ry a nd lo w syste mic a mp ho te ric in B c o nc e ntra tio ns, p ro ffe re d p o d ium in “ Antifung a ls: Pha rma c o kine tic s a nd Pha rma c o d yna mic s Symp o sium,” Ab str. O -0011. In Pro g ra m a nd a b stra c ts o f the 16th C o ng re ss o f the Inte rna tio na l So c ie ty fo r Huma n a nd Anima l Myc o lo g y (ISHAM), Pa ris, Fra nc e . 19 ELF Dilutio n De te rm ina tio ns Using Ure a Ra tio s     A single blood sample was taken immediately before the first and second bronchoscopies with BAL to determine serum urea nitrogen concentration Urea nitrogen concentrations were also determined in the BAL samples Since urea readily diffuses through the body, plasma/serum, and lungs, it was used as an endogenous marker of ELF dilution using simple dilution principles ELF dilution was estimated by the following formula: ELF, Volume (mL) = BAL, Recovered Volume (mL) * [BAL, Urea Nitrogen (mg/dL) / Serum, Urea Nitrogen (mg/dL)]  ELF AmB concentrations were then calculated using the following formula: ELF, AmB Concentration (ng/mL) = BAL, AmB Concentration (ng/mL) * [BAL, Recovered Volume (mL) / ELF, Volume (mL)]  20 Citation: Rennard SI, Basset G, Lecossier D, O’Donnell KM, Pinkston P, Martin PG, Crystal RG. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol. 1986; 60(2):532-538.