Chemistry for Medicine 1
医用化学１(1st period, Friday)
November 16th, 2018
(Next time: Nov. 22nd)Interaction of particles and biointerface~For control of cellular uptake~ (continued)- Introduction to pharmacokinetics and pharmacodynamics-

Akihiro Kishimura(Associate Professor,
Katayama Laboratory)
kishimura.akihiro.776@m.kyushu-u.ac.jp

???

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2
M. Zhu, et al., Acc. Chem. Res. 46 (2013) 622–631. Fig. 3

Drug distribution at the target tissue
After delivery to the target tissue, infiltration to the tissue& internalization into the cells is sometimes required.

CapillaryPlasma protein

Drug

Cp: drug concentra-on in plasma
Blood cells

Blood flow

Q: Blood ﬂow rate
Endothelial cells

Kp: -ssue to blood concentra-on ra-o
Tissue space

CL: Clearance(Elimina-on)
Tissue cells

V: volume of the -ssue
C: drug concentra-on at the organ

3

Pharmacokinetics & Physiologically based (PB) Model•For clear descrip-on of the movement of drug into, through, and out of the body, we
consider the -me course of its absorp-on, distribu-on, metabolism, excre-on, andbioavailability. （Pharmacokine-cs）•Modeling by connec-ng organs (as compartments) with the bloodstream (as a pipeline)=> Too complicated!!

Dose(intravenous
injec-on)

Lung
Brain
Heart
Liver

KidneyMuscleSkin
AdiposeFor each compartment, we
can set some parameters:
Kp: -ssue to bloodconcentra-on ra-o
C: drug concentra-on at theorgan
V: volume of the organ
Q: Blood ﬂow rate
CL: Clearance
Bioavailability: the frac)on ofan administered dose of
unchanged drug that reaches
the systemic circula)onCompartmental analysis
Simple models based on several compartments.
=> Useful for predic-on of drug concentra-on at any -me.

1-compartment modela rapid intravenous injec-on (IV bolus)

X0
Tissue 2 V2V1 Tissue 1
C1

C2

Fast
Plasma Kp, Cp, Vp

C4
Tissue 4
V4

C3
Tissue 3
V3

X

C

Vd

For drugs to be eﬀec-ve, they need to be able to move
rapidly from blood plasma to other body ﬂuids and -ssues.
X: Total amount of drug
Ci: drug concentration at the tissue
Vi: volume of the tissue
Ki: tissue to plasma concentration ratio = Ci/Cp(i = 1–4)“p” stands for plasma.
X = VpCp + ∑ViCi= VpCp + ∑ViKiCp= (Vp + ∑ViKi)Cp

This term should be small, if the
drug shows slow clearance.
Ideally, ViKi for the target 1ssue
should be maximized.

Vd (Distribution volume) ≡ Vp + ∑ViKi = X/Cp
kel For a single bolus IV injection
The drug concentra-on is uniform in the body compartment at all -mes
and is eliminated by a ﬁrst order process
dX/dt = –kelX(kel : elimination rate constant)
The integral of 1/x is ln(x).

X = X0·exp(–kelt)
Cp = C0·exp(–kelt)(X0 : Dose)(C0 : initial plasma concentration)

Time (t)LN of Plasma conc. (ln C)

Plasma conc. (C)Cp = C0·exp(–kelt)

C0 = X0/Vd
lnCp = lnC0 – kelt

Elimina-on half-life (t1/2):
The -me required for theconcentra-on of the drug
to reach half of its originalvalue.

t1/2 = ln(2)/kelsemi-log plot

Time (t)Plasma conc. (C)AUC (area under the curve):
The integral of the concentration-time curve (after a single dose or in steady state).

Time (t)Y. Anraku, et al., JACS, 2010, 132, 1631.

Example: Pharmacokinetics of PICsome in micePEG(45)-PAsp(75)
O
MeO

O

m

N
H

fPEG ~ 8%

+
O
N
H

H
N

H
N
o O
O–
O
Na+

H
N

H
N

NanoPICsome

H
p'

O
O

R=

n

Na+

H
N

O
NH

p
O–

O

o'

R

H

10 mM PB
with 0 mM NaCl
(pH 7.4)

NHnR

Homo-P(Asp-AP) (82)

NH3
Cl–

1 mg/mLTEM

Cryo-TEM

DLS

100 nmSize: ~100 nm
Thickness: <15 nm

100 nmCollaborated with Terabase Inc.

Unilamellar Structure

Y. Anraku, et al., JACS, 2010, 132, 1631.

Cross-linking of Nano-PICsomes+

10 mM PB
with 0 mM NaCl
(pH 7.4)++

FITC-Dex@Nano-PICsome–––

FITC-Dex(M.W. = 10000)
1-Ethyl-3-(3-dimethylaminopropyl)+ 150 mM NaCl carbodiimide, hydrochloride
Cross-linked

EDC(EDC) MicronSized

Nano-PICsomes(Transformation of -NH2 and-COOH into amide bond)

HOOC
HOOC

COOH
COOH
COOH

PEG-P(Asp)

+

H2N
H2N
H2N

NH2
NH2

Homo-P(Asp-AP)

Particles

EDC

NHOC
NHOC

COHN
COHN
COHN

NHCO
NHCO

Polyamide Bonds

Y. Anraku, et al., Chem Commun, 2011, 47, 6054.

Advantages of Cross-linking (CL)

Plasma Clearnace of
CL-PICsome in mice
CL-Cy5-PICsomes(100 nm)PEG-P(Asp)-Cy5

Cy5-PICsome w/o CL

Plasma Clearance (%)

Plasma Clearance (%)

Advantages of cross-linking (CL)•Salt-resistance•Stability against Concentration•Stability against Freeze-drying•Thermostability•Tunable Permeability•Tunable Deformability•Tunable Cellular Uptake•Long Blood Circulation●: 35.3 nm (micelles)●: 102 nm●: 158 nm●: 197 nm●: 256 nm●: 298 nm

9Plasma concentration change of 100 nm-sized PICsome
with different stabilty
PIC–Au-nanoparticle hybrid

Sample： Cy5-PICsome (100 nm; diﬀerent
crosslinking (CL) degree: 30-90%)(

Animal：BALB c/nude ♀6-week old (n = 3)
Condition：i.v. injection (10 mg/mL, 200 μL)-‐‑‒ ) 51)-‐‑‒ ( .

2 5   

CL degree range: 30–90%( 00

Plasma clearance

Blood Circulation (%)

90%
80%
60%

30%, 45%

CL degree

90%

80%

60%

半減期 t1/2(h)

29.5

18.9

12.4

Time (h)/ 51   

Au PICsome-Cy5.5(ICP)
1.315
Dose(mg/kg)
Cmax(ug/mL)
23.05
AUC∞(ug/mL*h)
53.4
T1/2（β）
6.14
60.6
Vd(mL/kg)
119
Vdss(mL/kg)

Considering the amount of whole bloodof 70 mL/kg, this material can be mostly
distributed to blood compartment.

tumour)theirshouldelimination
be compared toties of most CDCs preclude
more evident Conventional
when CDCs particulatearecarriers
before previous degradation. The later phenerences in the initial circulatakably responsible formost of the bloodization, non-PEGylated CDCs
Table 2
speciﬁc, unintended distribution
nanomaComparative values ofof
organ
weight, bloo
he liver. The induction of cirtotal animal weight
and totalvolum
ger toxicities and side-effects,
andis blood
theref
not affect their clearance.
from reference [25].
The impact of CDC deposition into the var
ed to slow down non-speciﬁc
Human (70beforekg, 4 900 mL)
must be thoroughly
ascertained
estaR
Plasmacelaranceof
peciﬁc interacting antibodies
PICsome
Cycle W
Organ Weight when
Blood ﬂow
ness of a formulation. Similarly,
assesstime ((mL/
ties of a system, the amount(g)of CDC
foundin(min)amin)
tumour) should be compared to that non-s

Lungs 1,000 5,600
0.9
1Kidneys 310
1,240
3.9
2
Fig. 6. Comparison of the pharmacokinetic proﬁles of different long-circulating
formulations
100
nm-Polymersome
Liver1,800 1,450
3.4
1Table 2PEG-poly(ethylethylene)in mice. Data were extracted from image analysis.
Values expressed in %ID/g were multiplied
Spleen 180blood
77ﬂow and
63.6cyc0(40-37)by total blood weight in mice (i.e., 1.75 g of blood if theComparative
weight of mice isvalues
not statedofinorgan weight,
τ1/2 = 16–28
a blood volume
total
animalweightandtotal
givento in
The time it takes forare
the organreexperimental section). NP: nanoparticles, SWCNT: single-walled carbon nanotubes, PLGA:
from reference [25].
volume. The value is calculated as total v
poly(lactic-co-glycolic acid).

Organ

Human (70 kg, 4 900 mL)

Rat (250 g, 17.5

Weight Blood ﬂow Cycle
time
(g)
(mL/

Weight Blood C
(g)
ﬂow t

a 323.
D. E. Discher et. al. J. Control. Release 2003, 90,

Assignment（Due date: Nov 22nd）
1-Compartment model(for constant-rate IV infusion)
Problems）
k0: infusion ratek0
Tissue 2 V2V1 Tissue 1
C1

C2

Plasma Kp, Cp, Vp

C3
Tissue 3V3Xi) Express the total amount of drug X as afunction of t.
ii) From the equation of Cp = X/Vd, find the Cp at
the steady state using kel, k0, and Vd.
iii) Briefly describe the time course of drugconcentration in plasma after stopping
infusion.

C4
Tissue 4
V4

C

Vd
kel

Hints：- Express the mass balance regarding X using a
differential equation.
- differential of ln(x) is 1/x; integral of 1/x is ln(x);integral of 1/(ax+b) is (1/a)ln(ax+b)

i) dX/dt = k0–kelX(kel : elimination rate)

For accumulation in the body, dX/dt = k0–kelX > 0
dX/(k0–kelX)= dt–(1/kel)·ln|k0–kelX|= t + A(A : integral constant）

Considering k0–kelX > 0,k0–kelX = exp{–kel(A + t)}
X = k0/kel – (1/kel)exp{–kel(A + t)}
Considering X = 0 for t = 0,0 = k0/kel – (1/kel)exp(–kelA)

Cssexp(–kelA) = k0
Therefore, X = k0/kel – k0/kel {exp(–kelt)}= (k0/kel)·{1 – exp(–kelt)}ii) According to Cp = X/Vd,
Cp = (k0/kelVd)·{1 – exp(–kelt)}
For t→∞、Cp = k0/kelVd ⬅Steady-state plasma concentration (Css)iii) The situation is the same with the case of bolus iv injection
with the initial concentration of Css.
Cp = Css·exp(–kelt) = (k0/kelVd)·exp(–kelt)

The decay of concentration follows this equation.

13i) dX/dt = k0–kelX(kel : 消失速度定数)

この時、体内に貯まるのであればdX/dt = k0–kelX > 0
dX/(k0–kelX)= dt
両辺積分して、–(1/kel)·ln|k0–kelX|= t + A(A : 積分定数）k0–kelX > 0より、
k0–kelX = exp{–kel(A + t)}
X = k0/kel – (1/kel)exp{–kel(A + t)}
ここで、t = 0で、X = 0より、
0 = k0/kel – (1/kel)exp(–kelA)

Cssexp(–kelA) = k0
よって、 X = k0/kel – k0/kel {exp(–kelt)}= (k0/kel)·{1 – exp(–kelt)}ii) Cp = X/Vdより、
Cp = (k0/kelVd)·{1 – exp(–kelt)}
t→∞で、Cp = k0/kelVd ⬅定常状態の血漿濃度(Css)iii) 定常状態を初期濃度とする単回静脈投与モデルと考えられることから、
Cp = Css·exp(–kelt) = (k0/kelVd)·exp(–kelt)で減衰する。

14

2-Compartment model http://www.gatewaycoalition.org/files/hidden/deliv/ch3/3_5f.htm
Oaen drug does not distribute evenly amongst all the organs. To account for this a two
compartment model is used in which drug disposi-on is biexponen-al. The drug is assumed to
distribute into a second compartment but be eliminated from the ﬁrst compartment only.
Peripheral compartment
D Central compartment(Fast distribution)(Slow distribution)(Mainly, organs/tissues with slow-blood-flow)(Distribution phase)

No Elimination

Fast distribution & elimination

Elimination

dX1/dt = –(kel + k12)X1 + k21X2(Elimination phase)dX2/dt = –k21X2 + k12X1
Solved using the Laplace transform
C1 = X1/V1 = A·exp(–αt) + B·exp(–βt) (α >β)A = D(α–k21)/{V1(α–β)}
B = D(k21–β)/{V1(α–β)}V1 = D/(A + B), V2 = k12·V1/k21
Vss = V1 + V2

Slow distribution &elimination

Example of 2-compartment model
Methotrexate: Antimetabolite (antifolate type).
Used to treat cancers, autoimmune diseases etc.
Protein binding: -50%, Urine excretion：48% (1 h)Fast distribution& elimination
Slow distribution& elimination

16Model-independent approach (moment analysis)

Plasma conc.

It gives us characteristic properties of drugs, but it’s not useful for predicting its
time course.
First moment
Second moment
Zero moment
Mean residence time
Variance of residence time

Time

17

Y. Anraku, et al., Chem Commun, 2011, 47, 6054.

Advantages of Cross-linking (CL)

Plasma Clearnace of
CL-PICsome in mice
CL-Cy5-PICsomes(100 nm)PEG-P(Asp)-Cy5

Cy5-PICsome w/o CL

Plasma Clearance (%)

Plasma Clearance (%)

Advantages of cross-linking (CL)•Salt-resistance•Stability against Concentration•Stability against Freeze-drying•Thermostability•Tunable Permeability•Tunable Deformability•Tunable Cellular Uptake•Long Blood Circulation

Size (nm)

Mean
Residence
Time (h)

38.0

36.2

102

51.2

158

69.2

197

71

256

47.3

298

45
●: 35.3 nm (micelles)●: 102 nm●: 158 nm●: 197 nm●: 256 nm●: 298 nm

18

1-compartment model
IV bolus injec-on

X0

V1
Tissue 1
C1

Tissue 2

V2
C2

Fast
PlasmaTissue 3X

Vd ≡ Vp + ∑ViKi = X/Cp

For single IV bolus injection
dX/dt = –kelX (kel : elimination constant)
X = X0·exp(–kelt) (X0 : dose = Vd·C0)
Cp = C0·exp(–kelt) (C0 : initial plasma concentration)

Kp, Cp,Tissue 4

C

Vd
kel

Clearance: Clearance of a drug from the body may

be expressed as total body clearance (CLtot), clearance
from speciﬁc organs, such as kidney, liver etc… It isdeﬁned as the rate of elimina)on divided by theconcentra)on:
CLtot = (eliminated drug/time)/blood conc.
= (elimination rate)/blood conc.
CLtot = ∑CLorg,i

AUC(area under the curve):

Plasma conc. (C)CLtot = |(dX/dt )/C |= |Vd(dC/dt )/C | = kel·Vd
For Vd(dC/dt ) = –CLtot·C
Integrate 0 to ∞: Vd(C∞ – C0) = –CLtot·AUC
From C∞ = 0 and Vd·C0 = X0 (Dose),
X0 = CLtot·AUC
AUC = X0/CLtot = C0/kel → CLtot = kel·Vd
Time (t)（参考）クリアランスの概念
Drug

Drug

Vol
Conc

Vol
Conc

Elimination

Elimination(Elimination capacity should be the same for both cases.)
全身からの薬物消失速度CLtotは、各臓器における薬物消失速度CLorg,iの和であると考えられる： CLtot = ∑CLorg,i
各臓器でのより本質的な消失能力を考えるために、以下を考慮する必要がある。
臓器抽出率：薬物が臓器を１回通過する際にどれだけの割合が非可逆的に消失を受けるか。
蛋白結合、血球分配：多くの薬物は血漿中のタンパク質に吸着し、血球成分にも分
配される。
20

Liver metabolism
Protein-bound
Drug

Drug

Interaction withblood components

Sinusoid
Endothelial cells

Membranetransport Drug

Space of Disse✓Liver metabolizes toxic compounds,
such as alcohol, drugs, etc. fordetoxification or elimination.
→ Excreted in urine or bile

Liver cell
(hepatocyte)
Internalization
Metabolitebilecapillary

Drug

Metabolite

MetaboInteraction
肝細胞内
lism
with
Protein-boundintracellular
成分との
Drug
components
相互作用

Excreted inbile『生体内薬物送達学』p. 130 を英訳- Lipophilic drugs are converted intohydrophilic compounds.
- Main route is oxidation by cytochrome P450.
(P450 has a lot of isozymes (57 types) having
many variants.)→ Drug efficacy is dependent on personal difference.

this enhanced blood exposure [149]. The impact of the saturation process depends on the extent of MPS capture of the formulation. Only
CDCs that are highly captured by Kupffer cells induce this effect [150].
CDCs designed to evade the MPS (with PEG- or other hydrophilic coat→ The materials passing throughings) do not show enhanced circulation times with increases in dosageof sinusoids(sizecan< 150
nm)
up fenestrations
to 300 mg/kg [147,148].
Two phenomena
explainthe candosebe internalizedby livertime:
cells,
unlesstheyopsonins
dependent
increment in circulation
depletionof plasma[146]
or saturation ofmacrophage
phagocytic capacity [34,151]. Kupffer
encounter
Kupffercells.
cell uptake functions can also be altered with the administration of→ Main internalization pathway is pinocytosis.
CDCs containing cytotoxic drugs that have a deleterious effect on theThen, bile
excretion occurs.
macrophages[152].

Bile excretion/Gallbladder

Bile: bile is produced by the liver, andstored and concentrated in the gallbladder.
After eating, the stored bile is discharged
into the duodenum for the digestion of lipids
in the small intestine. Main component ofgallbladder bile is bile salts and fats(cholesterol, fatty acids, and lecithin), which→ Bile excretion is the only elimination path of can work as an emulsifier.
5.3.non-biodegradable
Targeted delivery to the livermaterials with size > 6 nm.
Cationic materials show higher uptake.
Hepatocytes are functional units responsible for most of the metaBut,
excretion
process
is very(< 48nm)
h for
Liver
bolic
and secretory
activitiesof the liver.
Smallslow
CDCs (b150that
5-10% of dose).
Pinocytosys

Space of Disse

Hepatocyte
Gallbladder（胆嚢）bile capillary

Duodenum(十二指腸)

（Advanced contents）Organ clearance
outinCapillary

Cout

Plasma protein

Qi

Capillary

Flow rate
Endothelial cells

Protein-binding

Interstitial fluid

CB = Cin
drug

CLint,iElimination
enzymes

Intrinsic clearance

CLint,ivel
Organ clearance-

Eorg,i = (QiCin – QiCout,i)/QiCin

CB: Drug conc. in the artery
Elimination rate at organ i vel,i:Eorg,i-

Organ extraction ratio Eorg,i:CLorg,ivel,i = CLorg,i·CB= QiCin – QiCout,i= Eorg,iQiCin
From CB = Cin
CLorg,i = Eorg,i·Qi
1 – Eorg,i ≡ Forg,i

F: Availability
Both Organ extraction ratio & avialabilry can be determined
only by organ clearance CLorg,i & Blood flow rate Qi.
Upper limit of the E value is 1. Therefore, the upper limit of CLorg,ican be determined by the upper limit value of Qi. (Particularly for
rapid elimination cases.)23

（Advanced contents）Drug distribution to blood cells
Hct: Hematocrit(Volume ratio of red blood cells to
the total volume of blood）
1 – Hct

Hct

PlasmaPlasma (55%) Cp
White blood cells &platelet (< 1%)

Free drug

Free drug

PlasmaproteinProteins ofRBC

RBC (45%) C
RBC
Protein-bounddrug
Drug cocn. in blood cell components (Rb)

Rb = CB/Cp (BC distribution ratio)
CB = (1 – Hct)Cp + HctCRBC
Therefore,
Rb = CB/Cp = (1 – Hct) + HctCRBC/Cp
In the case of CRBC = 0, Rb = 1 – Hct

Red blood cells (RBC)

24

Protein-bounddrug

You do not need to consider the
RBC distribution, when you use
drug carriers or water soluble drug.
24Model system for evaluation of PK
As an alternative for animal experiments.
Ex.1) To estimate liver clearance, microsomes prepared from human liver cells areused. （細胞を破砕してできる小胞体が細分化した小胞で、肝細胞由来だと代謝酵素であるシトクロム
P450を含む）

Ex.2) To estimate intestinal absorption behavior, Caco-2 cell layer system is often used.
(to evaluate drug permeation, function of transporter, etc…)
X.-W. Yang, et al.
Journal of Chinese
Integrative Medicine:Volume 5, 2007, p. 637.

TranswellCaco-2 cells
Microvilli
Semipermeable
membraneDrug passing throughthe epithelial cell layer

Drug absorption mechanism
at intenstinal epithelia.
消化管粘膜部位での薬物吸収機構

MucosaTight junction

transporter

Application of microﬂuidic devices as a model system
K. Sato, et al., ANALYTICAL SCIENCES, 2012, 28, 197–199.
- PDMS-based(シリコンゴム) microfluidic

device（1.5 mm-wide）-

GI tract model system + liver metabolism
model
Final target: breast cancer by systemic
circulation
Useful for estimation of PK/PD

Model system for GI tract!26

Pharmacodynamics (PD)
Pharmacodynamics (PD) is the study of how a drug affects an organism, whereas
pharmacokinetics (PK) is the study of how the organism affects the drug. PD places
particular emphasis on dose–response relationships, that is, the relationships
between drug concentration and effect.
Conc.

Target tissue

Admin.

Distribution

EffectiveConc.

Pharmacological
EffectDrug
Absorp.
Blood
Conc.

Elimin.

Drug-binding to receptor
or target site.

Conc. – Time

Time

Drug conc. in plasma

Response – Time

Purpose of PK/PD analysis:

Response

Concentration

Dose
Oral Admin.

Sigmoidal

PK/PD

Metabolism/Excretion
Dose

Drug Response

Blood

ResponseTime

It enables us to discuss timed e p e n d e n t p r o fi l e o f d r u g
response considering biologicalprocesses, which is helpful for
design of regimen, most effective
and safest drug & its formulation
in a practical manner.

Eﬀect compartment model
Sometimes, drug response isdelayed.
Drug Response

Eﬀect compartment
Eﬀect/
response
Drug conc. in plasma
Pharmacology
Drug conc. in plasma＊各コンパートメントは１次の速度論で結合され、薬効コンパートメン
トの分布容積は小さく、全体の濃度変化に影響を与えないと仮定。

Drug conc. in plasma
x-axis：DelayedDrug conc. in plasma

x-axis：Fall in blood pressure(%)

Fall in blood pressure(%)

Drug conc. in plasma

Drug conc. in theeffect compartment

Bunazosin：an alpha 1 antagonist.
Bunazosin was initially developed
to treat benign prostatic
hyperplasia (BPH).

LinearDrug conc. in effectcompartment!28

PK/PD解析の活用

規模拡大
安全性

有効性(少数, 軽度)(i) 医薬品開発の段階から 安全性の予測 を確実なものとするのに有効。
(ii)動物モデルやin vitro試験の薬理作用が臨床的に有用かを科学的に検証。
(iii)臨床での適正使用を可能にする 定量的・統計的(iv)DDS設計の指針を得るのに有効。

な評価手法の構築。
!29

(Review) Control of pharmacokinetics / Targeting
For eﬀective targeting…
What can be a target?
-

Tissues / organ…
Malignant tumor, inflammatory part etc.
Receptors, enzymes, antigens, etc. (specific molecules
overexpressed in the diseased part)

Strategies
Active targeting …Direct interaction with molecules of target sites.
Passive targeting …Full utilization of characteristic property of thetissue or diseased part.
e.g.) structure, micro-environment, etc.
Anti-targeting …Blocking delivery or accumulation of drugs in thespecific part of the body, which causes elimination or side effects.
30

Passive targeting & active targeting
For estimation of navigation function of DDS
Consider the following compartment model:
Drug Uptake to the target: PSinf
Cumulative amount of drug: XR
AUC can be represented using PSinf and XR
D

Circulationcompartment

CB

PSinf

TargetCompartment

XR↓AUC = D/CLtot

XR = PSinf·AUC = PSinf·D/CLtot(標的部位への取り込みは、全身クリアランスに影響を与えないとする。)

CLtot

飽和すると、相対的に標的外でのクリアランスを受けやすくなることに注意。

Active targeting：
Enhancement of XR by increasing PSinf→ In many cases, receptors on cell surface aretargeted.

Passive targeting：
Enhancement of XR by decreasing CLtot→ Use of drug carrier is effective to overcome
various elimination mechanisms.
31

EPR effect: A typical example of passive targeting
In tumor -ssue and inﬂammatory -ssue, permeability of capillary is enhanced,
but reten-on of larger substances is found due to dysfunc-on of lympha-c drainage.
For the cancer treatment...
Long-circulation
Nano-carriers

Tumor
TissueSize is a critical factor!!
EPR (Enhanced Permeability andRetention) effect: Size < 100–200 nm

<12µm

Lung
uptakeNormaltissueLiveruptakeImmature Lymph

Higher drainage
Lowerdrainage

Blood
Stream
RES uptake

KidneySize:
200 nm~3 µm

M.W. < 30,000

Normal lymph: Drug: Nano-medicine

Leaky
Blood Vessel

Tumor tissue

Effectively found for “stealth” particles.

Y. Anraku, et al., Chem Commun, 2011, 47, 6054.

Example: Tumor accumulation of PICsome in mice
Polyanion was labeled with Cy5.
1. 10 eq. CL-Cy5-Nano-PICsome (100-300 nm)
2. Cy5-Nano-PICsome (100 nm, Control)
3. PEG-P(Asp)-Cy5 (45-75) (Control)
BALB c/nude
Female, 6weeks
n=3
200 μL

After 1, 3, 6,
24, 48, 120 h

C-26 Tumor-bearing mice(i. v. administration)

Conc. : 0.1 mg/mL
Buffer: 10 mM PB(150 mM NaCl; pH 7.4)

Centrifugation 2000g,
10 min, 4 °C

Blood

Fluorescence measurement
at Ex/Em = 650/670 nm

PlasmaBiodistribution
In Vivo Imaging System (IVIS)

Lung Liver Spleen Kidney Tumor＊各臓器の重さを計量

3. Biomedical Applications

Y. Anraku, et al., Chem Commun, 2011, 47, 6054.

Cut-off disposition size
Comparison AUC at 240 after administration for PICsomes with various size
→Threshold size is found at around 150 nm
Acuumulation in
C-26 Tumor
38 nm

Accumulation in
Spleen

102 nm298 nm
256 nm
197 nm

158 nm
158 nm38 nm 102 nm
197 nm 256 nm298 nm

Stable nano-formulation with well-deﬁned size can reveal
the clear size eﬀect.

!34

1308

N. Sasaki, A. Kishimura, et al., Anal. Sci., 2016, 32, 1307.

ANALYTICAL SCIENCES DECEMBER 2 0

Alternative for animal experiments
Model device was developedfor evaluation of leakage
from capillary.

ANALYTICAL SCIENCES DECEMBER 2 0 1 6 , VOL. 3 2
m = 1 .7 + 0 .5 α–1 .4
α = h/w

Using h of 6 7 µm and w of 3 0 0 µm, m was calcu lated

Fig . 1 (a) Sch ematic illu stration of nanoparticle permeatio
5 .7 8 . n th rou g h a tu mor v ascu lar wall.
(b) Sch ematic illu stration of permeation test of nanoparticles Usinth ro
h u atioa pnosroshu sown
membranggu geqabov e, e
k sep
canaratin
be estimatedfollowing manner. ∆P is estimated from Eq. (1 0 ) with otwomicroch annels.
SCIENCES DECEMBER 2 0 1 6 , VOL. 3 2

Fig . 21309

normal endoth elial g aps, it is clear th at th e pores of tu mor
v ascu lar walls increase th e permeability. Additionally, we
reported th at nanoparticles with sizes less th an 1 5 0 nm canselectiv ely accu mu late in tu mors,8 su g g esting th at nanoparticle
size is alsoimportant for increased permeability.
Most nanoDDS stu dies rely on animal testing , and alth ou g h
th is approach is popu lar, it is u nable to ev alu ate only th eincreased permeability of v ascu lar walls becau se of th esimu ltaneou s lymph atic drainag e of nanoparticles. Additionally,animal testing sh ou ld be minimized since it is not with ou t
eth ical concerns. Anoth er testing system for nanoDDS isTranswellTM, in wh ich endoth elial cells are cu ltu red as amonolayer on a centimeter-sized porou s membrane separating
top and bottom ch ambers. Th e permeability of th e cellmonolayer can be determined by, for example, introdu cingflu orescently-labeled nanoparticles into th e top ch amber andringmicro
flu orescenintensity from th e bottom ch amber after a
Sch ematic illu strationof fabricationof a membranmeasu
e-integ rated
flu
idic devt ice.
certain period of time. Howev er, nanoparticles permeate th e

experimental resu lts. C is experimentally obtained as dein th e Permeation tests section. Using th e C v alu es, vsestimated from Eq. (8 ). If a linear relationsh ip is ob
between vs and ∆P, Eq. (1 ) is v alid, and th e k/µδ v alu eobtainm thoeparticles
slope of th efo
reg
nDDS.
line.
permeatio
ned
offro
nan
r ressionano
Th e mTh e dependence of vs on ∆P for 1 .0 µm pore membra
nanoparticlen eth rohwnv ascu
lars. 6walls
0 .4 µm p
permeatioore membran
is ushg oin Fig(b) and2
1
clarified,th erefo
ree forco
al (1 .0 pepositiv∆Pnv<ectio
0 .2 2 kn
Paµm
respectivan
elyd. vs was
an
d
∆P
<
1
.1
5
k
Pa
(0
.4
µm
p
o
re).
Ho
wev
er,
a
fu
rth
er
nanoparticles sh ou ld be stu died.
Alth ou g h inin ∆P resu lted in neg ativ e vs v alu es, wh ich indicates tpermeation u sedof inFITC-BSA
inv toeste
is notbeen
appropriate
model
th e prev iou s stu dyh1 4as
is
aro
u
n
dore9
h ydrodynamic
diameterofFITC-BSA
th e conv ectional permeationof nanoparticles th rou g h p
reaso
n
fo
r
th
e
discrep
ancybetween
th
e
mo
del
an
is mu ch smaller th an th e diameter of typical nan
experimental resu lts seems tobe th e definitionof fc.

nanoDDS. In addition, as described in th e
Discu ssion section, ofwe
fou nd permeation
th at th ebased
th eoryCharacterization
nanoparticle
onthethis study
inapprofrom
priatefor nanoparticles for nanoDDS.
Next, we redefined fc as an approximate equ at
experimen
tal resu lts of conv ectional permeationof
convectional permeationderiv ed from nu merical calcu latith rou g h straig h t micropores ag reed with a th e
dynamics, th e relationsh ipbetweenth e nanoparticle
and ph ysical parameters
isp clarified forp th e first pti
pdata will lead torational desig nof nanomedicines
Th estufirstd th epresen
secondt facto
r op
nerimenth e rig h tal
t sideofde
E
In th isdy,anwean exmorepresent th e steric and th e v iscou s effect, respectiv ely. B
permeatio
n of nanoparticles th rou g h straig h t micr
Eq. (1 3 ), clearly, a linear relationsh ip was observ ed betw
5 . (1h).t Th
flu id flow.
u s whmembran
e withstraigmieand ∆PA(Figp.o
6ro
(d)),
ich is consistent with!3
EqLimitation of drug therapy
Intractable disease: Some of diseases are categorized into “intractablediseases” by Japanese government. There is no effective way fortreatment in terms of curing disease other than the symptomatic
treatment.
Why intractable ?
No effective way for dosing drugs: Effective drug has been known, but noeffective way for dosing has not been found, due to its severe side effect,
not satisfactory response, highly frequent dosing, too narrow therapeutic
window, no effective delivery (the issue of biological barrier), large
deviation of pharmacokinetic property among individual patients, etc… .
ex.) 脳梗塞、脳出血、認知症、統合失調症、自律神経障害、エイズ、COPD（慢性閉塞性肺疾患）、肝硬変、慢性腎不全、糖尿病性腎症、糖尿病性網膜症、睡眠時無呼吸症候群などなど。

Drug resistance: Efficacy can be found only at the initial stage of
medication.
ex.) Multidrug-resistant cancer, Multidrug-resistant bacteria, antibody production
for protein drugs, etc…

Limitation of EPR eﬀect
Macroscopic

Without using nano-carriers

Utilization of nano-carriers

Normal tissue

Cancer tissue

Micro-circulation

Cancer tissue

Lower leakage from capillary?

Continuous capillary

5-9 µmMicro-circulation

Intractable cancer tissue

Endothelial cells + pericytes
Pericyte

Normal tissue

Endothelial cellContinuou Insulated bybasementsmembrane
capillBlocking by- Pericytes- Fibrotic stroma

Common character of some intractable diseases
Insulated by pericyte

Capillary
Pericyte
Endothelial

Pancreatic *GliomaScirrhous
Gastric
Tuberculosis

No NP Leak

Endothelial

No Insulation by pericyte

RBC

Gastric*
Colon*
decubitus

Endothelial

Arterial sclerosis

Leak of NPs

Gastric cancer(症例数)80,000Pancreaticcancer(症例数)14,000nt
Patie

60,00012,000enita

10,000P

8,000

40,000

6,000

Death

4,000

5-year survival: 70%

2,000

20,000

Nanotechnology and tumor microcirculation.
Mitsunobu R. Kano, Advanced Drug Delivery Reviews 74 (2014) 2–11.

‘75

80

85

90

95

（年）

00

05

t

a
De

th

5-year survival: 10%
‘75

80

85

90

95

（年）

00

05

Confirmation of pericytes by histological study
Pericyte

Thick pericites
were found.

Red arrow: Blood capillary

40

TGF-β

Promote proliferation of parasites andother mesenchymal cells; suppression
inhibitionof endothelial cells.
Capillary＋rich pericytes
Red： adriamycin (ADR)

ADR alone

TGF-β inhibitor
ADR@micelles

Less parasites;very leaky capillary

ADR@micelles + TGF-β inhibitor

41

Eﬀect of size-tuning

30-nm micelle/70-nm micelle/
Co-localization

Horacio Cabral, et. al., Nat. Nanotechnol. 6 (2011) 815.
Horacio Cabral, Akihiro Kishimura, et al., Advanced Drug
Delivery Reviews 74 (2014) 35–52.

Size tuning is also effective forenhancement of permeability.

3. For pathophysiologic studies

Another Example of Size-dependent Accumulation

Aortal Aneurysm (AA)→ Conventional clinical treatment:
Surgery after mature of AA (No early treatment)→ No eﬀective way of delivery and accumulation ofdrugs to AA part
→ How about Nano-medicine?

Abdominal Aortal Aneurysm（AAA）- Rat Model：Dr. Miura, Prof. Koyama (U. Tokyo)- Mouse Model：Dr. Yokoyama (Yokohama City U.)→ Administration of Cy5-labeled PICsomes.

3. For pathophysiologic studies

Akai, Miura, Koyama, et al. in preparation.

AAA Rat Model
Material :adult male Sprague-Dawley rats (300 - 350g)

Anesthesia : 1.5 - 2.5% inhalational isoﬂurane

After Infusion

Temporary ligatures

2.5 unit of porcine
pancreatic elastase(E-2150, Sigma
Chemical, St Louis, Mo)

Elastase
Infusion

Administration of
PICsomes 7 days after
elastase treatment
Cy5-PICsome(100 nm){Dose: 体重300gのラットに対して濃度10mg/mLのPICsomeを300 µL)

3. For pathophysiologic studies

Akai, Miura, Koyama, et al. in preparation.

Rat Model
1-1. In vivoAAA
imagingsystem (IVIS)
ex vivo ﬂuorescence imaging of AA

TA

Normal AA

AAA
RAPhoto by IVISAAA

7 POD, 3 days after injection

Aortic bifurcation

100-nm-PICsomes are
speciﬁcally accumulated
in the AAA part.

3. For pathophysiologic studies

Akai, Miura, Koyama, et al. in preparation.

Size-dependency (Rat model)
1h

4h

8h

48h

96h

RFU / 100mg Organ

1000

750

500

250

0

40 nm

100 nm

200 nmn=5- 100 nm PICsomes are preferentially accumulated andretained in AAA.
- Larger particles (200 nm) show lower accumulation, andsmaller particles (40 nm) show shorter retention.

3. For pathophysiologic studies

Akai, Miura, Koyama, et al. in preparation.

NP accumulation Mechanism in AAA model
Aneurysm part is a kind of swelling state, due
to high pressure from blood.

大動脈の中には高い圧力（血圧）がかかっており、動脈硬化などの弱った部分で“瘤”ができやすい。血管の壁が薄くなって大きく膨らんでくる病気が動脈瘤。

Maybe, chronically high blood pressure induced theinﬂammation at the diseased part, which facilitates NP
accumulation. Retention of smaller particles might not be
eﬀective, because aneurysm part possesses very leaky
structure.

46

Arteria sclerosis and intimal thickening
http://www.miyake-naika.or.jp/05_health/doumyakukouka/kandoumyaku_puraku.html
Middle layer

Lumen
Monocyte

Outer layer

Atherosclerosis

Endothelial cells
Inner layer
Smooth muscle
cells

Elastic lamina

Fibrous capsule
Macrophage
Inner layer + SMC

粥状硬化は大〜中型血管で起こりやすく、
心筋梗塞や動脈瘤と関係が深い。

Middle layer

47

For the ballon injury modelng Information
itama Medical University, 1981 Kamoda, Kawagoe, Saitama 350-8550,

ngineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo

40-nm particles showed better
infiltration from blood stream
Kyusyu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395,
Japan
comparedto larger particles.

o-oka, Nishi-ku, Fukuoka 819-0395, Japan
dustry Promotion, 66-20 Horikawa-cho, Saiwai-ku, Kawasaki 212-0013,

T: In atherosclerotic lesions, the endothelial barrier against the bloodstream can become compre of the extracellular matrix (ECM) and intimal cells beneath. In theory, this allows adequately so inﬁltrate into the intimal lesion intravascularly. We sought to evaluate this possibility using rat c
intima. Cy5-labeled polyethylene glycol-conjugated polyion
vesicleRyosuke complex
Taniguchi,(PIC)
Yutakamicelles
Miura, andet al.,
200 nm (PICs-40, PICs-100, and PICs-200, respectively)
were intravenously
administered
Adequately-Sized
Nanocarriers
Allowto rat
Sustained
Targetedof Drug
Deliveryry using a balloon catheter. High accumulation andlong retention
PICs-40in theto indu
Neointimal
Lesionsin Rat Arteries.
by in vivo imaging, while the accumulation of PICs-100
andPICs-200
was limited, indicatin
Mol. Pharm., 2016, 13 (6), 2108–2116.
is a crucial factor for eﬃcient delivery. Furthermore, epirubicin-incorporated polymeric micel
hat of PICs-40 showed signiﬁcant curative eﬀects in rats with induced neointima, in terms of
eciﬁc and eﬀective drug delivery to pre-existing neointimal lesions was demonstrated with48adequat

How to observe local environments precisely?
On-site observation
From a satellite photo…

Nanopathophysiology!!

We cannot recognize
significant difference!!
Colon cancer？

Novel on-site
observation isrequired to
explore unknown
mechanism.

If we cannot recognize difference based onconventional methods…

Pancreatic cancer (intractable)

Report
Choose one example of intractable diseases, andexplain briefly what it is, what kind of therapy has
been performed. Also, report why that is
intractable, and state your opinion on the future
effective therapy from the viewpoint of drug
therapy and/or drug delivery system.
＊Please write the impression of this course, if possible.

Report length: 2-3 sheets of A4 paper
Deadline：Dec 7th, 2018.
Please submit your report by e-mail.
kishimura.akihiro.776@m.kyushu-u.ac.jp（otherwise, please visit W3-504 to submit it.）

レポート課題
難治性疾患について一つ取り上げ、病態・現行の治療法について調べるとともに、何が原因で難治となっているか調べよ（原因未解明の場合も何がどこまでわかっているか、あるいは、不明になる理由について述べること）。また、難治性を克服する手段について、主に薬物治療の観点から考察せよ。
難治性疾患/難病：いわゆる不治の病であり、治りにくい病気。
医学的に正確な定義はないが、難病として国に指定されているものがある。
ここでは、治療満足度が低く、有用な医薬品がないものはこの範疇と考えて良い。

分量：A4 2-3枚程度＊この講義全体に関する感想・意見もあれば是非お願いします。(ただし、レポートの分量には入れないこと)〆切：２０１８年１２月７日まで
提出先：メールが望ましい
kishimura@mail.cstm.kyushu-u.ac.jp（ウエスト３号館504号室まで岸村に直接提出しにきても良い。）

Lymph node targeting
Bloodstream

T
i
s
s
u
e

Lympha-c system plays a key role forb a c t e r i a l i n fe c - o n a n d t u m o r
metastasis. Therefore, lympha-csystem is a good target for drug
therapy.
- Macromolecules & par-cles are usefulfor transferring drug to lympha-csystem due to their larger size
compared to smaller drugs.
-

Local injection

Lymphatic
vesselLymph node

『図解で学ぶDDS』p. 48

For Vaccine Development...
Smaller nano-particles
accumulated in lymph nodes.
Smaller particles shows better
dendritic cells internalization.

➡For nanoparticle vaccines,

100 nm25 nm

size control is significant.
M. Swartz, J. Hubbell et al., (from EPFL)
Nat. Biotechnol. 2007, 25, 1159.

Examples of active targeting
Ligand installation onto theparticle surface conjugation
to the polymers are
effective.
Antigens and receptors expressing on thesurface of tumor cells.

抗体により腫瘍細胞に発現する抗原を標的。

『図解で学ぶDDS』p. 103-115

estingly, recent evidence showed that PEG liposomes,
previously considered to be biologically inert, could still
C certain side reactions through activation
DE
induceof theInstallationof ligands,e.g., antibody, sugar,h peptides, etc., is effectivj ecomplement
system33.
f

ver the liposome
omes demonstrate
log-linear kineticsdSurface modiﬁed liposomesiemethod of active targeting.
q
Too many ligands affects the pharmacokinetic
propertiesof drug carriers. k
c
g
D

E

h

j

i

e

s

q

g

Sugar ligands anchored by cholesterol
k

r

p

+

s

An-body

p

o

r

n

+ l
+

m

–

+ l
+

Figure 1 | Evolution of liposomes. A | Early traditional phospholipids+ ‘plain’ liposomes with water soluble drug (a) entrapped into
– the liposomal membrane (these designations are not
the aqueous
drug (b) incorporated into
PEG liposome interior, and water-insolubleonm
repeated on other figures). B | Antibody-targeted immunoliposome with antibody covalently coupled (c) to the reactive(Biocompa-bility)
phospholipids in the membrane, or hydrophobically anchored (d) into the liposomal membrane after preliminary modification with a
118
Peptidesforcellwith
Early traditional
phospholipids
liposomes
with liposome
water soluble
drug(a)entry
entrapped
intopolymer (e) such as『図解で学ぶDDS』p.
hydrophobic
moiety.‘plain’
C | Long-circulating
grafteda protective
PEG, which shields the liposome
r-insoluble
drug (b)incorporated
into theliposomal
membrane(theseare not immunoliposome simultaneously bearing both
surfacefromthe interaction
withopsonizing
proteins(f). designations
D | Long-circulatingtargetedprotective
immunoliposome
covalently
coupled(c) to thetoreactive
polymerwith
andantibody
antibody,
which canbe attachedthe liposome surface (g) or, preferably, to the distal end of the grafted
ophobicallyanchored
(d)
into
the
liposomal
membraneafter
preliminary
modificationwitha be modified (separately or simultaneously) by different
polymeric chain (h). E | New-generation liposome, the surface of whichcanliposome
graftedwith athese
protective
polymer (e)are:suchasattachment
PEG, which ofshields
the liposome
ways.
Among
modifications
theprotective
polymer (i) or protective polymer and targeting ligand, such as
zing proteins (f). D | Long-circulating immunoliposome simultaneously bearing both
antibody (j); the attachment/incorporation of the diagnostic label (k); the incorporation of positively charged lipids (l) allowing for the
an be attached to the liposome surface (g) or, preferably, to the distal end of the graftedcomplexation with DNA (m); the incorporation of stimuli-sensitive lipids (n); the attachment of stimuli-sensitive polymer (o); theiposome, the surface of which can be modified (separately or simultaneously) by different
attachmentof cell-penetrating
peptide polymer(p); the incorporationof viralcomponents(q). In addition to a drug, liposome can loadede attachmentof protective
polymer (i) or protective
and targeting ligand,suchaswith magnetic
particles(r) for magnetic
targetingand/orwithgold
n of the diagnostic
label (k);the incorporationof positively
chargedlipids(l) colloidal
allowing fortheor silver particles (s) for electron microscopy.

ration of stimuli-sensitive lipids (n); the attachment of stimuli-sensitive polymer (o); theV. Torchilin,
ADVANCESWITH
LIPOSOMESAS PHARMACEUTICAL CARRIERSp); the incorporationof viralRECENT
components (q).
In addition to a drug,
liposome
can loaded
argeting
and/orVOLUME
4 with colloidal gold or silver particles (s) for electron microscopy.
www.nature.com/reviews/drugdisc

Nat. Rev. Drug Discovery, 2005, 4, 146–160.

!54

Examples of liposomal formulations on-market『図解で学ぶDDS』p. 111

Examples of liposomal formulations

New Developments in Liposomal Drug Delivery

DOI: 10.1021/acs.chemrev.5b00046
V. P. Torchillin, et al., Chem. Rev. 2015, 115, 10938−10966

Improvement of drug absorption & DDS
Enhancement of “availability” is so important.
Key factors for DDS design considering drug properties
Hydrophobic drug: Solubilization Improvement of absorption (enhancement ofbioavailability & controlled release; targeting
Hydrophilic drug：Renal clearance; permeation across cellular membrane;
targeting
Biopharmaceutics（Nucleic acids/genes, proteins, peptides, cells）：
Escape from immunosystem and metabolism or degradation (keeping originalactivity); Cell internalization; Targeting

Key factors for DDS design considering administration routes
Oral: Absorption at GI tract, escape from metabolism at liver
Plumonary: Inhalation method
Transdermal: Overcoming epithelial barrier
Intravenous injection: Blood criculation; Targeting!57

DDS based on drug carriersSide effectNormalorgan/tissue

Insufficienttherapeutic
efficacy

Target site

No sideeffectNormalorgan/tissue

Sufficienttherapeutic
efficacy
Target site
Release- How to deliver drugs
to the target tissue?
- How to enhance
efficacy selectively?

InactivatedDrug

Biologicalbarriers

Escape from
Biologicalbarriers
Drug in carrier

58

DDS based on drug carriers
Design should be performed considering 1.‒3.
Molecular carrier- Low Molecular weight (MW)- Macromolecuels{

Natural: Serum albumin, polysaccharide
Synthetic: Polyethylen glycol (PEG)(PEG; stealth property.)Par-culate carrier- Low MW components:

Liposomes (cell-membrane mimic),
Emulsions, Lipid microspheres- Macromolecular components: Microspheres,

Nano-capsules (micelles, vesicles),Nano-gels

Bio-based carrier
Red blood cells, antibody, viruses, proteins, etc..

59

K.W. Leong, et al., Advanced Drug Delivery Reviews 65 (2013) 104–120
Liposomes & micelles

Polymer-drug conjugate

Biodegradable materials

Protein-based DDS

R. Duncan, et al., Advanced Drug Delivery Reviews 65 (2013) 60–70

Some examples of the formulations

Polymeric medicine
Paclitaxelpoly(glutamicacid) conjugates

TNP470-HPMA copolymer
TNP-470: 非特異的に血管内皮細胞の増殖、浸潤、管腔形成を阻害する(抗血管新生療法)
ポリHPMA: poly(N-(2-hydoxylpropyl)
methacrylamide)
免疫原性が低く、よく使われる。

Anticancer drug conjugate dextran

SMANCS

Better retention and controlled release of
mitomycin c at the cancer tissue.

Protein-conjugated polymer
Higher hydrophobicity enhancecell killing action.

Mitomycin cEmulsion

Dispersion of Oil (O) or water (W) into W or O using emulsifier.

Lipidmicrosphere
Water

Oil
Wa t e r soluble
drug

Oil
Water
Inner Water Oil Water
Lipophilic
Water-soluble drug
drug
Emulsifier(lecithin)

Oil solution/oil suspension- Oil solution shows longer
retention time at the administeredpart, and effective for depot
formulation.
- For oil suspension, solubilizationof drug solid into oil, andsubsequent transfer from thesolution to water phase are key
steps.

Lipophilic
drug

Soy
oil

Gene therapy & Gene delivery
Gene Therapy: The therapeutic delivery of nucleic acid polymers into a patient's
cells as a drug to treat disease. (Transfer genes to humans to produce specificproteins.)=> An innovative method for congenital diseases (e.g. gene defect), and also
useful for intractable diseases, such as cancer, Parkinson disease, and so on.

Essential things to be developed

Gene delivery routeLocal
injection

Systemic
injection

1. Development of gene vectors for
efficient gene expression.
2. Development of efficient carrier for
effective delivery and cellinternalization.

=> Ethical & social issues are a big hurdle.
=> Use of mRNA is a better option?

Gene therapy & gene delivery: some examples
Virus vector- High gene-transfer efficiency.
- Long-term expression through integration
into chromosome.
- Risk of unpredictable accidents &immunogenicity.

Nonviral vector (plasmid vector)

開Less efficient to viruses. Just transient
expression, but lower risk in terms of safety.
Typical transfection reagent

発

PEGylated polycation(リガンドなど)

Plasmid

Cationic lipid

東大・片岡ら、他。

“Stealth” liposome type

Lipoplex
Cationic polymer

Liposome

北大・原島ら

Hidetaka Akita, Hideyoshi Harashima, et al.,Biomaterials, Volume 30, Issue 15, 2009, 2940-2949

（参考）遺伝子導入を利用したがん免疫療法
Cancer immunotherapy：
がん細胞に対する免疫を誘導し、がん細胞を除去・治療する方法。
DNA vaccine

抗原提示細胞にがんを認識させる抗原を提示させ、がん細胞除去する細胞（細胞傷害性T細胞など）を活性化。
がん細胞に抗原を細胞表面に発現させる手法もある(Allovectin7®など)。

遺伝子改変T細胞療法 (CAR-T)
患者の末梢血のT細胞を採取し、体外で遺伝子導入することによりがん細胞特異的な細胞傷害性T細胞に改変する手法。がん細胞特異的なT細胞受容体や、キメラ抗原受容体遺伝子の導入が行われている。患者体内に戻したあと、抗原認識することで活性化し、がんを撃退可能。今、注目され臨床試験も進む治療法。治療後も体内に残り体内を監視することもあるらしい（再発防止に有効）。

Nucleic acid medicine
Nucleic acid medicine: The nucleic acid which binds to the target gene or protein
specifically, to inhibit the function of the target.

Advantage - Precisely target-specific.
- Suppression of protein production can lead to high and amplified
efficacy.
- Easy to tune through the flexible sequence design.
- Favorable for scale-up production based on chemical synthesis
approach.
- Potentially immunogenic, but not intrinsically non-toxic material.
- More stable compared to proteins in terms of structural robustness.

Concerns- Easy to be degraded by nuclease- Intense negative charges disturb the membrane permeation.
- Off-target effectEﬀective DDS is required.

RNA interference (RNAi) & siRNA delivery
RNAi：二本鎖RNAを 細胞質内 に持ち込んだ時、そのうちの一本鎖がそれと相補的な配列を持つmRNAに結合することによりそのmRNAが 切断・分解 される現象。
Silencing of target mRNARNA duplex (basically fromviruses and other pathogens)
Processing by Dicer

短い二本鎖RNA (siRNA)- ２０数塩基程度の長さ

Formation of RNA-induced
silencing complex
Binding to target mRNA
Cleavage oftarget mRNA

Nobel prize (2006)

Example of carrier
SNALP

Cationic lipid-

遺伝子送達 の手法も活用可能。

Neutral lipid
PEG lipidsiRNA

先に挙げたMENDやPEG-ポリカチオンなど-

まだマーケットに出たものはない。（特に全身投与製剤は未熟。）

Aptamer、LNA
Aptamer：Single stranded nucleic acid which can speciﬁcally and strongly
bind to target material like antibody. 20-50 base length. It is reported that
RNA-aptamer shows higher aﬃnity to protein than DNA type.

Advantages to antibody-

Chemical synthesis is possible.
Small molecules can be a target.
It can bind to the target with entire molecule.
Low immunogenicity.

Ex.) Drug of age-related macular degeneration (AMD)Pegaptanib(Macugen)
LNA (locked nucleic acid)/BNA (bridged -)：Modiﬁed nucleic acids withhighly resistant to degradation.

BNA: 大阪大学名誉教授の今西武先生により開発された人工核酸の総称

http://www.funakoshi.co.jp/contents/686

Protein delivery
Important therapeutic materials to control biological system.

Concerns- Susceptible to digestion/degradation.
- Low permeation across the bio-membrane- Immunogenic & short blood half-life- Renal clearance (for small proteins)

NanogelPEGylation
PEG

Protein

PEG is biocompatible.
&Enhancement ofblood circulation(Effect of increaseof MW, escape fromMPS)
&Low immunogenicity&Suppression of
degradation by proteinase

Effective for improvement ofavailability => Low frequency ofadministration

Useful for vaccine(K. Akiyoshi, Kyoto Univ.)

However,● Potential damage upon chemical modiﬁcation● Loss of protein activity

Peptide/hormone delivery
Similar to proteins, but molecular weight is much smaller.
黄体形成ホルモン放出ホルモン(luteinizing hormone-releasing
hormone; LHRH及びその誘導体)
視床下部ホルモンの一種。
前立腺がんや閉経前乳がん、子宮内膜症の治療に用いられる。
タケダの開発したリュープリン®（Leuprorelin acetate
PLGA microsphere; biodegradable & controlled
release formulation）は、平均粒子径約20 µmの微粒子であり、皮下または筋肉に注射して利用する徐放剤であった。乳酸グリコール酸共重合体(PLGA)からできているため、体内で高分子基材が分解され、4-5ヶ月にわたって安定に薬物がリリースされる。その結果、１〜3ヶ月の間隔の投与が可能となった。

その他のペプチド/ホルモン- 下垂体ホルモン：成長ホルモン、甲状腺刺激ホルモン、
副腎皮質刺激ホルモンなど.
- 視床下部ホルモン：体温・血圧・食欲・睡眠などの調
節ホルモン- インスリン（膵臓）：血糖値/糖尿病- 副甲状腺ホルモン、副腎皮質ホルモン

Low molecular weight drug
Permeability anddistribution
coeﬃcient plays
key role.

ex.) hydrophilic → lipophilic
Drug

Drug

Absorption
enhancer

Cell internalizaiton ofdrugs is correlatedwith drug response.
=> Octanol/water
distribution
coefficient(log P) is
normally used.
Absorption enhancer/
Solubilizer

Drug

Hydrophobic
MoietyCellmembrane

Absorption
barrier
Transporter

Absorption

Absorption

Promotion of premeablity of drugs.
- Solubilizing drugs with extremely low water solubility.
-ex.) Insulin（from GI tract、nose、eyes）
streptomycin（drug for tuberculosis;
from intestine）
Streptomycin

Improvement of absorption by chemical modification (prodrug)
SN-38/Irinotecan

抗悪性腫瘍薬
Active form

ProdrugCamptothecin

Hydrolysis(Low solubility,
sever side eﬀect)

Irinotecan(more soluble)

Vitamin B1/fursultiamine

Lack of V.B1 causes 脚気（beriberi）.

(タケダ、アリナミン)

SN-38(カンプトテシンアナログ)

Hard to be absorbed due tohydrophilicity.

5-FU and its
prodrugs
5-FU: water-soluble anticancer
drug. Short elimination half-life(16 min). Severe side effect.

プロドラッグ

Enzyme Prodrug Therapy
プロドラッグ化の手法は、標的部位特異的な酵素によりプ
ロドラッグが活性化されるシステムを採用すると、ターゲティ
ングの優れた手法にもなる。同時に、投与量を増やし、標的部位で高い薬物濃度を長時間維持するのにも有効である。

(II)
(I)
(V)
(IV)
5-FU

Capecitabine (商品名: ゼローダ)

Metabolism

Liver
Metabolism

Liver orCancercell

Metabolism

Cancercell

http://kusuri-jouhou.com/medi/cancer/capecitabine.html

肝 臓 で カ ル ボ キ シル エ ス テ ラ ー ゼ に よ り 5 ' - d e o x y - 5 fluorocytidine (5'-DFCR) に代謝される。次に主として肝臓や腫瘍組織に存在するシチジンデアミナーゼにより5'-deoxy5-fluorouridine (5'-DFUR) に変換される。更に、腫瘍組織に高レベルで存在するチミジンホスホリラーゼ (TP) により活性体である5-FUに変換され、抗腫瘍効果を発揮する。

For Enzyme Prodrug Therapy (5-FC→5-FU)
1. Delivery of CD@PICsomes totarget site (tumor tissue)
2. Conversion of 5-FC into 5-FU
3. Tumor Treatment

Cytosinedeaminase (CD)@PICsomes
5-FC

CD
Push-in

Mr. A.Goto

5-FU
CD@PICsome

M.W. of CD = ~17,000
Preservation of Activity of CDs (in vitro)

Anti-tumor effect by CD@PICsome & 5-FC
5-FC or 5-FU concentra-on: 10 mg/kg; 200 µL(Administered intravenously at Day 4,8,15)

5-FC
5-FU
0

20

40

60

80

100

Time/min✓ Cell-killing activity was confirmed.
✓ Long blood circulation was confirmed.

Rela-ve Tumor VolumeRela-ve amountC26-subcutaneous model
60
50

PBS
5-FU

Drug or prodrug
administra-on

40
30
20

CD-PICsome+ 5-FC

10
0

1

6

11

16

Day
Admin. of CD@PICsomes

21

26

31

2. Development of new administration route &overcoming absorption barrier
Oral admin.
Pulmonary admin.
Nasal admin.
Intraoral/Sublingual
admin.

Depending on administration route,formulation should be designed
carefully.

For systemic distribu-on, use ofblood ﬂow is common route, but
I.v. injection
there are some concerns…
Transdermal✓ Diges-on at stomach and intes-ne
Subcutaneous✓ Diges-on at liver
Intramuscle

Rectaladmin.

Targetsite
Drug administration
Drug absorption
Drug distribution
Drug elimination

Skin barrier
Mucosal barrier✓ Excre-on✓ Adsorp-on onto proteins✓ Immune response✓ Intrinsically less absorbable (watersoluble compound, macromolecules
etc…)
Necessary to overcome theseissues.
79

From the “Course Overview”-From the viewpoint of chemistry, “medicine” can be understood as collective actions ofmolecular events. Particularly, deep insights on hierarchical structures of body composedof biomolecules and chemical reactions performed in our body facilitate overcoming
unsolved issues in the field of medicine. In this course, we will introduce new approaches,
mainly focusing on Evolutionary medicine (or Darwinian medicine), and nanopathophysiology. In the part of evolutionary medicine, you will learn the mechanism ofsome specific diseases (diabetes and infarction) through chemical and molecular
evolution of diseases by considering intermolecular interaction found in our body. In thepart of nano-pathophysiology, you will learn some basics of drug delivery system andhow to get hints of treatment of intractable diseases based on molecular- and nano-scopic
observation of the diseased part.
- We would like the students to understand evolutionary medicine from the viewpoint ofmolecular chemistry, in other words, to understand the relationship between evolution
and disease from microscopic molecular scale to macroscopic biological scale. Also, byunderstanding biological and physiological events as nano- and submicrometer-scaled
issues, the students will learn the way of thinking for treatment of diseases utilizing theengineering methodology, particularly, through rational design of materials from theviewpoint of chemistry.