SOME HEALTHY CYNICISM REGARDING NANOMETER-SIZED DRUG AND ANTIGEN DELIVERY SYSTEMS

SOME HEALTHY CYNICISM REGARDING NANOMETER-SIZED DRUG AND ANTIGEN

DELIVERY SYSTEMS

Amit Misra

Pharmaceutics Division, Central Drug Research Institute, CSIR, Lucknow

Vaccine Delivery: Vinod Labhasetwar, Rajeev Raghuvanshi (NII).

Pulmonary Delivery: Rolee Sharma, Pavan Muttil, Awadh Yadav, Rahul Verma Amit Singh (CDRI); Jayanthi Suryakumar, Vineeth Raghavan, Rakesh Sinha, Vijay Modak, (Lupin), Swati Subodh (TCGA).

GLUT targeting: K Bhargava, Sraddhanjali Mohapatra, Mukesh Kanojiya, V Sasikiran, Heikham Kajal Devi, Saman Habib (CDRI)

Male Contraception: Ritu Malik, Gopal Gupta (CDRI) Shailesh Tondwal, Kripa Shankar, KS Venkatesh (IIT-K)

Moliere, The Bourgeois Gentleman; Act Two, Scene Four (Adapted by Timothy Mooney)*…MONSIEUR JOURDAIN: ... Before you go, I must confide to you a secret. I am in love with a ladyof great rank and quality, and wish to ask your help in writing her a notewhich I intend to drop most casually at her feet.…PHILOSOPHY MASTER: Oh, certainly. A verse you'd like to jot?J: No, no, no verse for me.PM: So you want prose?J: No, neither.PM: Well, I think we must suppose/ It's one or its the other.J: Why?PM: I guess/ That those are all the options to express.…PM: To make the point most terse./ What isn't verse is prose, and what's not prose is verse.….J: And this, the way I speak. What name would be applied to the --PM: The way you speak?J: Yes.PM: Prose.J:Well, what do you know about that! These forty years now, I've been speakingin prose without knowing it! How grateful am I to you for teaching me that!-----------------Amit: Well, what do you know about that! These twenty years now, I’ve been making colloidal drug delivery systems without knowing that I’ve been doing nanomedicine! How grateful am I to you for having invited me to speak today!

*http://moliere-in-english.com/bourgeois.html

PLGA (50:50, 48 KDa) Particles incorporating tetanus toxoid (TT)

AlPO4-Al(OH)3 inorganic microparticles with surface-adsorbed TT

CASE 1: VACCINE DELIVERY

Groups:

A. 2 Lf TT (7.5 μg) adsorbed on alum; X2, 3wk (conventional)

B. 1 Lf TT in NP + 1Lf on alum, single injection (combination)

C. 2 Lf TT in NP, single injection (single dose NP)

D. 2 Lf TT on alum, single injection (singled dose MP)

E. Blank NP + 2Lf TT, single injection (NP adsorbed)

A: TT on Alum (Conventional)

B: Combination

C: NP Single Dose D: MP Single Dose

Inhalable microparticles containing anti-tuberculosis drugs

•Giving drugs by mouth: convenience versus non-target tissue bioavailability and toxicity

•Pulmonary delivery, targeting alveolar macrophages: inhalation apparatus

•Preparation of dry powder inhalation: industrially scalable– spray dryingshelf stability– 2 yrsat least 2 drugs (resistance)– isoniazid and rifampicin / rifabutintargeting to macrophagessustained releasebiodistributionserum and tissue PK/PDinhalation safety/toxicity

•M tuberculosis induces ‘alternative activation’ of host macrophage: can microparticle phagocytosis rescue infected macrophages to classical activation ?

•Free radicals, Th1 versus Th2 cytokines, apoptosis versus necrosis

•Genome-wide expression profiling by microarray, validation by real-time PCR

•Inter-individual differences in responses to microparticles:polymorphism at the HLA and VDR loci?

Co-localization of MP and H37Ra Confocal Microscopy

Early Efficacy Latency/Recrudescence

Oxidative Radicals Nitric Oxide

0

5

15202530354045

N NMP I ISD IMP IBMP

Groups

NO

(M

)

Apoptosis

N NMP I ISD IMP IBMP6

8

10

12

14

16

18

20 No inhibitor Caspase 3 inhibitor

Ann

exin

V+ c

ells

(%

)

0 2 4 6 80

300

600

900

1200

1500

1800

Me

an

Flo

ure

sce

nce

Time (min)

NMP; k = 148.59; r2= 0.976

I; k = 117.08; r2= 0.984

ISD; k = 143.72; r2= 0.991

IMP; k = 186.55; r2= 0.980

IDMP; k = 137.93; r2= 0.983

1 2 3 4 5 6 7 M

- - + - - + - M

M 8 9 10 M 11 12 13 14 15 16

M + - +ve M - - - + - +ve

Amplified PCR product

1 2 3 4 5 6 7 M

- - + - - + - M

M 8 9 10 M 11 12 13 14 15 16

M + - +ve M - - - + - +ve

Amplified PCR product

DRB1*1501 allele in healthy volunteers

M 1 2 3 4 5 6

2000bp

825 bp

265 bp

M 1 2 3 4 5 6M 1 2 3 4 5 6

2000bp

825 bp

265 bp

VDR Polymorphisms in Apa1, Bsm1and Fok1 fragments

M 1 2 3 4 5 6

M PCR bb BB Bb BB bbproduct

M 1 2 3

M FF ff Ff

825 bp650 bp

265 bp196 bp

M 1 2 3 4 5 6

M PCR bb BB Bb BB bbproduct

M 1 2 3

M FF ff Ff

825 bp650 bp

265 bp196 bp

I ISD IMP IBMP0

50

100

400

800

1200

1600

CF

U

Groups

I ISD IMP IDMP05

101520

100

200

300

400

CF

U

I ISD IMP IBMP

50100150200

500

750

1000

1250

CF

U

Groups

Donor 4 Donor 1 Donor 1

Human diversity, not particle reproducibility, influences outcome of treatment

B

CASE 3: Receptor-mediated targeting of nanoparticles containing anti malarial agents to RBC via mammalian + parasite Glucose Transporters

Uninfected RBC Infected RBC

Mammalian GLUT-1 PfHT-1 Nanoparticles displaying free glucose moiety should be targeted to infected rather than uninfected RBC through mammalian GLUT as well as plasmodial hexose transporter

101 102 103

FL1-H101 102 103

FL1-H 101 102 103

FL1-H

RBC separated in FACS (A, blue region) show dose-dependent fluorescence (B) when whole blood is incubated with FITC-labelled NP. NP uptake by RBC in whole blood is inhibited by GLUT-blocker phloretin (C), and is competed out by natural GLUT ligand, glucose (D).

A B C D

Nanoparticles bearing free glucose on surface

CASE 3: Setting a Pulse to Catch a Pulse: Non-invasive Episodic Intervention in the Hypothalamo-Pituitary-Testicular Axis for Male Contraception

Hours

A C

D

E F

TUNEL labeling, Hoechst counterstain of anterior pituitary sections on Day 71. (A, B): Untreated control, (C,D): Sham-treated, (E,F): pulsatile transdermal testosterone

No. of days

Control Sham Treated

Mating frequency/ animal

Deliveries/mating

Mating frequency/ animal

Deliveries/matings

Mating frequency/ animal

Deliveries/matings

0-30 (n=6)

0.78 0.84 0.75 0.33 0.3*# 0.25*

30-71(n=6)

0.61 0.66 0.3 0.88 0.2* 0.5#

71-81(n=4)

0.15 1 0.45 1 0.1 0*#

81-136(n=2)

0.69 0.6 1.65 0.25 0.97# 0.142*Kruskal Wallis ANOVA on ranks (P < 0.05) followed by post –hoc Tukey test(P<0.05). *Significant difference (P<0.01) from untreated control, # Significant difference (P<0.01) from sham control.

Behaviour and Fertility

Cynicism, Skepticism and Hope

Cynical about

enhanced permeability, efficacy and toxicity (nano-size fits all)

policy, funding agency, civil society and media priorities

Skeptical about

size-specific effects, amenability to scale-up, adaptation to shopfloor

Hopeful about

“…being the right size” (Haldane, 1926)