The NanoAssemblr™ Platform: Microfluidics-Based ... · 18 Day n aseline 80 60 40 20 0 -20 -40 10 20 30 40 50 100 Treatment (mg/kg) Placebo 0.150 0.050 0.300 0.500 siRNA Dose †

The NanoAssemblr™ Platform: Microfluidics-Based Manufacture of Nanoparticles

Euan Ramsay, Ph.D.

Co-Founder & CSO/COO

July, 2015

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Presentation Overview

1. Introduction to Microfluidics-Based NanoAssemblr™

Platform for Nanoparticle Manufacture

2. Examples of Nanoparticles Manufactured by the

NanoAssemblr™ Platform

3. Scale-up Manufacture using the NanoAssemblr™

Platform

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Platform

Conceptual Nanomedicine

Scale-up API -

NanoparticleFormulation

Manufacturing Process

Robust Manufacturing

Process

Nanomedicine Product

Nanomedicine Development Process

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Scale-up API -




Process



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NanoAssemblr™ Benchtop Instrument


Scale-up API -




Process

NanomedicineProduct


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NanoAssemblr™ Benchtop Instrument Scale-Up Platform


Scale-up API -




Process



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Accelerated Development of Nanomedicines

Scale-Up Platform

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The NanoAssemblr™ Benchtop Instrument

Microfluidic Cartridge


Proprietary microfluidics-based instrument

Manufacture novel nanoparticles

Nucleic acid-lipid nanoparticles

Polymer nanoparticles

Liposomes

Oil-in-water nanoemulsions

Prepare 1.5 mL – 20 mL nanoparticles / run

Operate at 4 mL/min - 20 mL/min

Make > 30 formulations / day

Software controlled

Easy-to-use

Rapid nanoparticle development

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The NanoAssemblr™ Microfluidic Cartridge

DRAFT COPY UOBC-1-53454

DRAFT COPY UOBC-1-53454

Microfluidic Cartridge – BOTTOM VIEW Microfluidic Cartridge – TOP VIEW

Microfluidic Chip

Easy-to-use consumable cartridge

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The Magic is in the Microfluidics

Solvent (water miscible) aqueous

Rapid & Controlled Mixing

Stroock et al., Science 2002

Channel diameter: ~100 µm

Staggered Herringbone

Mixers

Nanoparticles

Laminar fluid flow

Diffusion mixing

Rapid mixing (< 3 ms-1)

Small reaction volumes (~ 14 nL)

Low energy input

Predictable and reproducible mixing

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Microfluidics Process Parameters – Aqueous:Solvent Flow Rate Ratio

• Ratio of the flow rates (mL/min) of the aqueous and

solvent input streams

• Higher aqueous:ethanol flow rate ratios result in

more rapid increases in polarity

• Rapid change in polarity forces the nanoparticle

components to organize into the most

thermodynamically and energetically favorable

structure

Process parameters dictate nanoparticle biophysical characteristics

Solvent (water miscible)

aqueous

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Microfluidics Process Parameters – Total Flow Rate

Process parameters dictate nanoparticle biophysical characteristics

Solvent (water miscible)

aqueous

• The combined flow rates of the aqueous stream and

the solvent stream

• Ranges from 4 mL/min – 20 mL/min

• Total Flow Rate is a surrogate for mixing speed

• Increased flow rate increases mixing speed

• At high Total Flow Rates nanoparticles reach “limit size”

• Limit size is defined as “The smallest achievable lipid

particles compatible with the packing of the molecular

constituents in an energetically stable structure”

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Platform

The NanoAssemblr™ Platform: Manufacture of Novel Nanoparticles

O/W Nanoemulsions

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Lipid Nanoparticles

Liposomes

Polymer Nanoparticles

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Lipid Nanoparticles

Lipid Nanoparticles for the Delivery of RNA

• Package RNA into nanoparticle core

• Protect RNA from degradation

• Facilitate RNA uptake into cells

• Promote RNA release into the cytoplasm

16 Images courtesy of Prof. Pieter Cullis, University of British Columbia

Ionizable Cationic Lipid

Cholesterol

Phospholipid

PEG-lipid

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RNA-Lipid Nanoparticles are Complex

Manufacture of RNA-Lipid Nanoparticles is challenging

RNA

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Day

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Treatment (mg/kg)

Placebo 0.150

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siRNA Dose

† Serum TTR levels were measured in separate Phase I study of ALN-PCS, an RNAi

therapeutic targeting PCSK9, which uses identical LNP formulation as ALN-TTR02 B.U.Med.Center, July 2012

RNA-Lipid Nanoparticles Represent the Current Clinical Gold Standard for RNAi

Patisiran (ALN-TTR02) is Currently in Phase 3 clinical trials for treatment of Transthyretin-Mediated Amyloidosis

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Microfluidics Manufactures “Solid-Core” RNA-Lipid Nanoparticles

Images courtesy of Prof. Pieter Cullis, University of British Columbia

Wasan K. M . et al. (2008) Impact of Lipoproteins on the Biological Activity & Disposition of Hydrophobic Drugs: Implications for Drug Discovery. Nat. Rev. Drug Disc. 7: 84- 99

Molecular model:

Neutral, Solid-Core RNA-Lipid Nanoparticles

Neutral, “Solid-Core” RNA-Lipid Nanoparticles Mimic Endogenous Delivery Systems

Low Density Lipoprotein (LDL):

“Endogenous Lipid Nanoparticles”

Wasan K. M . et al. (2008) Impact of Lipoproteins on the Biological Activity & Disposition of Hydrophobic Drugs: Implications for Drug Discovery. Nat. Rev. Drug Disc. 7: 84- 99

Neutral, “Solid-Core” RNA-Lipid Nanoparticles Mimic Endogenous Delivery Systems

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“Solid-core” RNA-Lipid Nanoparticles Associate with ApoE In Vivo


ApoE

PEG-lipid dissociates and ApoE associates after injection

LDL receptor, scavenging receptor on hepatocytes

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Ionizable Cationic Lipids Mediate Maximum Endosomal Escape


pH reduced below pKa of cationic lipid

Cationic lipids combine with anionic lipids to induce non-bilayer structures and release of siRNA

Brij 98

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0.05 mg/kg

0.1 mg/kg

0.3 mg/kg

0.5 mg/kg

1 mg/kg

EC 50

EC 90

Day

Resid

ual F

VII (

%)

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“Solid-Core” RNA-Lipid Nanoparticles Mediate Sustained Liver Gene Knockdown In Vivo

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Can ApoE-Medicated Targeting be Used for Other Tissues?

LDL receptor family members:

LDLR, LRP1, VLDLR, ApoER2, LRP4, LRP1B and Megalin

Need to design novel nanoparticles to deliver RNA beyond the liver

Microfluidics Enables Rapid Development of Novel RNA-Nanoparticles

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Incumbent Technology

NanoAssemblr™

Rapid

Development

Novel

Nanoparticles

Reproducibility

Ease-of-Use Encapsulation Efficiency

Size Speed

Seamless Scale-Up

Multi-Function Nanoparticles

Compositional Space

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RNA-Lipid Nanoparticle Size Dictated by Manufacturing Process

siRNA-LNP (Cationic Lipid:DSPC:Cholesterol:PEG) Changing Process

RNA-Lipid Nanoparticles reach “Limit Size” at high Total Flow Rates

RNA-Lipid Nanoparticle Size Dictated by Lipid Composition

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“Limit Size” is dependent on RNA-Lipid Nanoparticle composition

0

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%TotalInjectedDose

Time(hr)Time (hr)

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14Blood Liver Spleen

siRNA-LNP In Vivo Behavior

Particle Size Influences siRNA-LNP Pharmacokinetics & Biodistribution

siRNA-LNP Particle Diameter:

Red = 43 nm (5% PEG)

Green = 78 nm (5% PEG)

Blue = 140 nm (5% PEG)

Grey = 78 nm (1.5% PEG)

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30 nm siRNA-LNP Enables Liver Gene Knockdown by Subcutaneous Injection

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Targeted siRNA-LNP

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In Vivo Gene Knockdown in T-Lymphocytes

Microfluidics for Targeted Nanoparticles

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• Step-wise reactions • Post-insertion for targeting • Programmable mixing

• Targeted medicines • Multi-functional agents • Bespoke medicines

Molecular Assembly Line

Manufacture of Multi-Functional Nanoparticles

Sequential Addition of Cationic (XTC) and Anionic (PS) Lipids

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Encapsulation of Gold Nanoparticles

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Liposomes

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Liposome Size Dictated by Manufacturing process

“Limit size” Liposomes are dictated by the Flow Rate Ratio

Liposome Size Dictated by Lipid Composition

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Liposome size and polydispersity is dependent on cholesterol content

POPC:Cholesterol:PEG-DSPE (3%)

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http ://informahealthcare.com/lprISSN: 0898-2104 (print), 1532-2394 (electronic)

JLiposome Res, Early Online: 1–7! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2015.1025411

RESEARCH ARTICLE

Production of lim it size nanoliposomal systems with potential utilityas ultra-small drug delivery agents

Igor V. Zhigaltsev, Ying K. Tam, Alex K. K. Leung, and Pieter R. Cullis

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, Canada

Abstract

Previous studies from this group have shown that limit size lipid-based systems – defined as thesmallest achievable aggregates compatible with the packing properties of their molecularconstituents – can be efficiently produced using rapid microfluidic mixing technique. In thiswork, it is shown that similar procedures can be employed for the production ofhomogeneously sized unilamellar vesicular systems of 30–40 nm size range. These vesiclescan be remotely loaded with the protonable drug doxorubicin and exhibit adequate drugretention properties in vitro and in vivo. In particular, it is demonstrated that whereas sub-40 nmlipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcho-lines cannot be produced, the presence of such lipids may have a beneficial effect on theretention properties of limit size systems consisting of mixed lipid components. Specifically,a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phos-phatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylatedlipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with ahalf-life for drug release of 12 h. These results indicate that microfluidic mixing is thetechnique of choice for the production of bilayer LNP systems with sizes less than 50 nm thatcould lead to development of a novel class of ultra-small drug delivery vehicles.

Keywords

Doxorubicin, herringbone micromixer,limit size nanoparticles, liposome,microfluidic mixing

History

Received 9 December 2014Revised 23 February 2015Accepted 1 March 2015Published online 9 April 2015

Introduction

Important features of lipid nanoparticle (LNP) drug carriersystems include ease of preparation, reproducibility, andefficient encapsulation and retention of the biologically activeagent. However the LNP size is a critically importantparameter. The ability to generate small (5 50 nm) LNP canbe highly desirable to optimize the biodistribution of the LNPcarrier following intravenous (i.v.) injection. In this regard,the vast majority of LNP systems being used as drug deliveryagents have primarily utilized particles of 80–100 nm diam-eter, largely because of the availability of formulationmethods that produce LNP in that size range. To ourknowledge, all clinically approved (to date) nanomedicinesrepresent nanoparticulate carriers larger than 80 nm, examplesinclude DoxilÕ (80-100 nm) (Gabizon, 2002), MarqiboÕ

(100 nm) (Silverman & Deitcher, 2013), and AbraxaneÕ

(130 nm) (Green et al., 2006). It is well established that theenhanced permeation and retention (EPR) effect contribute tothe passive tumor-targeting of nanoparticles with the size of80–100 nm (Maeda et al., 2000). However, on one hand,

numerous works have reported that whereas such ‘‘large’’systems can often accumulate in the adjacent blood vesselsand in the peripheral regions of solid tumors, there is limitedpenetration into tumor tissue itself, thus limiting the potencyof the anticancer agent (Dreher et al., 2006; Jain et al., 2010;Kano et al., 2007; Perrault et al., 2009; Unezaki et al., 1996;Uster et al., 1998). On the other hand, it is widely recognizedthat smaller ( 50 nm) delivery agents may substantiallyimprove penetration and retention within the tumor tissue(Cabral et al., 2011; Chauhan & Jain, 2013; Chauhan et al.,2011; Huo et al., 2013), provided that they are larger than10 nm to avoid renal clearance. As particles in the size rangeof 10–50 nm can be expected to be the most promising carriersystem in accessing extravascular target tissues, the synthesisof such systems is of intense interest for biomedicalapplications. A number of techniques aimed at productionof smaller size vesicular LNP are available for decades, mostof them can be described as ‘‘top down’’approaches based ondownsizing of previously formed larger structures (De Kruijffet al., 1975; Hope et al., 1986; Huang, 1969). Those methods(exemplified primarily by sonication) have many limitations,including, most importantly, lack of scalability. Other tech-niques to produce nanovesicular systems include ‘‘bottomup’’approaches whereby LNP are formed by condensation oflipid from solution rather than by disrupting larger particles

Address for correspondence: Dr. Igor V. Zhigaltsev, Department ofBiochemistry and Molecular Biology, Faculty of Medicine, University ofBritish Columbia, 2350 Health Sciences Mall, Vancouver, Canada. Tel:+ 1 604 822 4955. Fax: + 1 604 822 4843. E-mail: igorvj@ mail.ubc.ca

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Cryo-TEM study

Figure 4 shows representative images of ‘‘empty’’ (A) anddoxorubicin-loaded (B) POPC/DPPC/Chol/DSPE-PEG2000(45/20/35/3) LNPs. As seen, doxorubicin loading at a0.1 mol/mol ratio did not affect the predominantly sphericalshape of the pre-loaded vesicles; a characteristic ‘‘coffee-bean’’ appearance of the internally precipitated drug can beobserved that is similar to the appearance of larger liposomaldoxorubicin formulations such as DoxilÕ . The cryo-TEMmicrographs also provide size information that can be used tovalidate the sizes determined by the light scattering technique.A size analysis based on a sample of 120 particles indicatedmean diameters of 33±4 nm (mean±SD) for both empty andloaded LNPs, in good agreement with the number-weightedsize values determined by DLS.

Long-term stability

In a final area of investigation, a long-term (up to 6 months)stability of doxorubicin-loaded POPC/DPPC/Chol/DSPE-PEG2000 (45/20/35/3) systems stored at 4 C has beenstudied. LNPs were loaded with drug at 0.1 mol/mol drug-to-lipid ratio and concentrated to 10mg/ml total lipid. Nosignificant changes in mean LNP size and drug entrapmentwere observed during the course of the study (results notshown).

Discussion

As pointed out in the ‘‘Introduction’’section, two approachesto formation of ultra-small vesicular LNP systems can betaken, namely ‘‘top down’’ size reduction methods whichusually are harsh procedures requiring high energy input such

Figure 4. Cryo-TEM micrographs of LNP composed of POPC/DPPC/Chol/DSPE-PEG2000 (45/20/35/3) prior to (A) and after (B) loadingwith doxorubicin at a drug-to-lipid ratio 0.1 mol/mol. The bar represents100 nm. For details of sample preparation and cryo-TEM protocols, see‘‘Materials and methods’’section.

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Figure 2. Drug retention in 22 nm POPC/Chol/DSPE-PEG2000 65/35/3(squares) and 33 nm POPC/DPPC/Chol/DSPE-PEG2000 45/20/35/3(diamonds) systems determined in vivo. LNP formulations containingtrace amounts of the tritiated lipid [3H]-CHE were loaded with14C-labeled doxorubicin at a drug-to-lipid ratio 0.1 mol/mol and theninjected intravenously into CD1 mice at a lipid dose of 50mg/kg. Plasmasamples taken at the indicated time points were analyzed for lipid anddrug content by liquid scintillation counting as described in ‘‘Materialsand methods’’ section. Each data point represents mean values±SDfrom each group of mice (n ¼ 4).

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Figure 3. Liposomal lipid levels obtained in plasma of CD1 miceinjected with 22 nm POPC/Chol/DSPE-PEG2000 65/35/3 (squares) and33 nm POPC/DPPC/Chol/DSPE-PEG2000 45/20/35/3 (diamonds) sys-tems. Lipids were quantified as indicated in Figure 2 and ‘‘Materials andmethods’’ section. Each point represents mean values±SD from eachgroup of mice (n ¼ 4).

DOI: 10.3109/08982104.2015.1025411 Production of limit size nanoliposomal systems 5

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Drug Retention in Small Liposomes

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Encapsulation of Hydrophobic Propofol

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Design of Experiment Studies

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Polymer Nanoparticles

CellaxTM Polymer-Drug conjugates

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100nm 100nm

NanoAssemblr TM Vortex

Nanoparticle Size Dictated by Polymer Concentration

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Particle size is dictated by polymer concentration

CellaxTM Polymer-Drug conjugates

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Polymer Composition Dictates Biophysical Characteristics

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Polymer-mediated Anti-Cancer Activity

O/W Nanoemulsions

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Nanoemulsion Droplet Size Dictated by Manufacturing Process

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Part

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siz

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Aqueous/ethanol flow rate ratio

Zhigaltsev, I.V. Et al., Langmuir 2012, 28, 3633−3640

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0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

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Theoretical diameter

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Zhigaltsev, I.V. Et al., Langmuir 2012, 28, 3633−3640

Nanoemulsion Droplet Size Dictated by Emulsion Composition

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Platform

Assessment of the Robustness of the Manufacturing Process

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Stable Results = Robust Process = Scalable Process

Design of Experiment (DoE) variables

– Lipid Concentration

– Flow Rate

– Mixing Conditions

– Lipid:RNA Ratio

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Continuous Flow Pumps

Parallelized Microfluidic Mixers

RNA -Nanoparticles

Aqueous (RNA)

Solvent (Lipids)

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Dilution

Buffer Exchange &

Nanoparticle Concentration

Microfluidics Enables “Seamless Scale-Up”

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RNA -Nanoparticles

Aqueous (RNA)

Solvent (Lipids)

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Dilution

Buffer Exchange &



Continuous Flow Scale-up Manufacture of RNA-Lipid Nanoparticles Using Single Mixer

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Seamless transfer of optimized manufacturing parameters

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RNA -Nanoparticles

Aqueous (RNA)

Solvent (Lipids)

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Dilution

Buffer Exchange &



4x Parallelized Microfluidic Mixer Manifold System

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-0.02

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1X Mixer 2X Mixer 4X Mixer

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Microfluidic Parallelization Enables RNA-Lipid Nanoparticle Scale-Up

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12 mL/min 24 mL/min 48 mL/min

Parallelization of microfluidic mixers enables greater throughput

On-Chip Microfluidic Parallelization Produces Equivalent RNA-LNP

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Multiple options for increased throughput by parallelization

48 mL/min 48 mL/min 4X Mixer Chip

16x Parallelization Enabled Through 4x Mixer Cartridges in 4x Manifold System

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16x Parallelization Enables > 20L Batches in 2 h

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RNA -Nanoparticles

Aqueous (RNA)

Solvent (Lipids)

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Dilution

Buffer Exchange &



High Quality RNA-Lipid Nanoparticle Product

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Amenable to Industry Standard Post-Manufacture Processing

After Buffer Exchange

After RNA Concentration

After Microfluidics

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RNA -Nanoparticles

Aqueous (RNA)

Solvent (Lipids)

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Microfluidic Mixer

Dilution

Buffer Exchange &



Design for GMP Manufacturing

• Continuous flow pumps

• 8X parallelized mixers in disposable manifold

• Disposable fluid path for product contacting materials

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Aqueous Metering Pump

Solvent Reagent Bag

Aqueous Reagent Bag

Solvent Metering

Pump

Dilution Pump

Dilution Reagent Bag

Pinch Valve

To Post-processing

Tee

Tee

Pinch Valve

Sample Switch Waste

288 mL/min

384 mL/min

8X Scale-up System

8 x 12 mL/min

24 mL/min

72 mL/min 96 mL/min

Microfluidic Mixer Array

Design for GMP Manufacturing

8X Scale-Up Instrumentation Produces High-Quality RNA-LNP

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8X Scale-up system processes 5.75 L/hr

GMP Program in Development

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Pumping system selected

GMP-compliant Disposable COC microfluidic cartridges under development

Working with drug development partner to transfer technology to CMO for scale-up and GMP manufacturing

Fully disposable fluid path using USP Class 5/6 materials

Targeting a GMP-ready system by end-of-year 2015

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Summary

1. The Microfluidics-Based NanoAssemblr™ Platform

enables simple, rapid & reproducible manufacture of

novel nanoparticles

2. The NanoAssemblr™ Platform can be used to

manufacture several different types of nanoparticles

3. Process parameters can be used to dictate nanoparticle

biophysical characteristics such as particle size

4. The NanoAssemblr™ Platform enables “seamless”

scale-up by parallelization of microfluidic devices

http://www.nanoassemblr.com/resources/

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Contact Information

Nepa Gene Co., Ltd.

Sales Team

www.nepagene.jp

3-1-6 Shioyaki, Ichikawa, Chiba, 272-0114 JAPAN

phone: +81 47 306 7222 fax: +81 47 306 7333

Extra Slides

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Reproducible RNA-Lipid Nanoparticles Independent of Operator or Site

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Automated Instrumentation Removes Operator Variability

Operator

Documents

The NanoAssemblr™ Platform: Microfluidics-Based ... · 18 Day n aseline 80 60 40 20 0 -20 -40 10 20 30 40 50 100 Treatment (mg/kg) Placebo 0.150 0.050 0.300 0.500 siRNA Dose †