Brain Re.~earch.

3 0 8 ( 1 9 8 4 ) 2 0 1 - 2 1 4 2{/1
El sevi er
BRE 101S4
Research Reports
Theoretical Interpretation of Extraction (in Brain) of Peptides
Including Concentration Variations
R. R. S HAR MA and R. L. P. VI MAL
Department of Physics, University o[ lllinois at Chicago, Chicago, IL 60680 (U.S.A.
( Ac c e pt e d De c e mb e r 20t h, 1983)
Key words: ext r act i on - - pe pt i de s - - b l o o d - b r a i n bar r i er br ai n upt a ke - - di f f us i on, f aci l i t at ed
The t r ans por t pr ope r t i e s of s ever al pc pt i de s acr oss b l o o d - b r a i n bar r i er ( BBB) have be e n i nves t i gat ed t heor et i cal l y in t e r ms of si m-
ple di f f usi on and f aci l i t at ed di f f usi on pr oces s es . Co mp a r i s o n of t he cal cul at ed r es ul t s f r om t he si mpl e di f f usi on and t hc e x p e r i me n t a l
dat a r eveal s t he pr c s e nc e of t he f aci l i t at ed di f f usi on of t hes e s ubs t a nc e s whi ch we have concei ved of as a c a r r i e r - me di a t e d pr oces s . The
val ues of t he par t i t i on coef f i ci ent s f for t hes e pe pt i de s wer e in t he r ange 7 x l(I -a ~< f ~< 200 10 4. Th e cal cul at ed f val ues gave per -
meabi l i t i es. P,. in lipids be t we e n ll)- r ~< p, ~< 14 x 10 ~ cm/ s. The s e val ues wer e t he n us ed to e s t i ma t e t he ext r act i on for pe pt i de s f r om
si mpl e di f f usi on al one whi ch var y f r om 0. 3 t o 3. 5% c ompa r e d wi t h t he e xpe r i me nt a l ext r act i on ( 0 . 4 - 1 2 %) i ndi cat i ng t he i na de qua c y
of t he si mpl e di f f usi on al one to expl ai n t he e xpe r i me nt a l dat a. As for t he c a r r i e r - me di a t e d f aci l i t at ed di f f usi on pr oces s we have us e d
t he act i vat ed- compl ex t heor y. The ext r act i on in t hi s case d e p e n d s on t he ma xi ma l r at e of t r a ns por t (Tmax) f and t he r eci pr ocal of t he af-
fi ni t y c ons t a nt K, for t he t r a ns por t of pe pt i de s t hr ough BBB. We have de duc e d t hat (Tmax) f ~ 0. 46 10 3 p mo l / g ' s and & ~ 0. 35 nM
for Me t - e nke pha l i n ( Me t - E NK) , Le u - e n k e p h a l i n ( L e u - E NK) , gl ut a t hi one , c a r nos i ne , a - MS H and MI F and (Trade) f ~ 10 10 3
pmol / g' s and K, - 7 nM for AVP, t i LT, fi E and a E t o expl ai n t he obs e r ve d r esul t s. We have al so obt a i ne d t he quant i t at i ve var i at i on of
ext r act i on wi t h c onc e nt r a t i on of pe pt i de s in t he br ai n- capi l l ar y and have es t abl i s hed t hat t he ext r act i on de c r e a s e s wi t h i ncr eas i ng con-
cent r at i on of pcpt i des , t e ndi ng to a smal l c ons t a nt val ue at hi gh c onc e nt r a t i ons . It has be e n i nf er r ed t hat c a r r i e r - me di a t e d f aci l i t at ed
di f t usi on is i mpor t a nt for t he t r a ns por t of pe pt i de s acr oss BBB.
I NT R ODUC F I ON
The knowledge of the transport of peptides across
blood-brain barrier (BBB) has been of considerable
interest~ :3 in recent years. Some peptides effect cen-
tral nervous system (CNS) pharmacologically and
exert behavioral changes in animals and in menS,14,1~.
It has been reported ~,> that in bacteria and mamma-
lian intestines, the transport of peptides is via satu-
rable carrier systems.
in recent years, several measurements of the
transport of peptides across BBB have been report-
ed. The experimental data show large variations with
respect to concentration and nature of the peptides.
Some investigators~2,~3 have reported that the pep-
tides permeate readily across BBB with brain-uptake
index (BUI) values lying between 10-15%. On the
other hand, there have been observations 5 which re-
port that the peptides are less permeable in BBB with
BUI values between 2. 4-3. 6%. In particular,
Greenberg et a1.12 have employed lower concentra-
tions of peptides in their investigations and have re-
ported BUI values of peptides much higher than the
values deduced by Cornford et al. 5 who have used
higher concentration of peptides.
A close analysis of the experimental resultsS, 12
shows that, in general, the extractions of peptides de-
crease with the increase of concentration of the pep-
tides approaching for large concentrations to a
(somewhat) small constant value characteristic of
saturation effect. These results suggest peptide-car-
tiers for the transport of peptides across the BBB.
Since the asymptotic value of the experimental ex-
traction (E) in various cases does not exactly ap-
Corres7)ondence: R. R. Sha r ma , De p a r t me n t of Physi cs, Uni ver s i t y of Il l i noi s at Chi cago, Chi cago, IL 60680, LI. S. A.
0006-8993,' 84/$I)3.00 1984 El s evi er Sci ence Publ i s her s B. V.
202
pr oach t o zer o, one is l ead t o pr edi ct t he exi st ence of
anot her process whi ch coul d give a const ant , t hough
small, cont r i but i on t o E par t i cul ar l y at hi gh concen-
t rat i ons. I n fact , our pr esent i nvest i gat i ons show t hat
t he si mpl e di ffusi on pr ocess whi ch depends on t he
lipid sol ubi l i t y of pept i des ( par t i t i on coef f i ci ent ) con-
t ri but es t o t he small cons t ant val ue of t he ext r act i on
obser ved at high concent r at i ons .
The aim of t he pr esent paper is t o exami ne t he t wo
i mpor t ant mechani sms, namel y, t he car r i er - me-
di at ed faci l i t at ed di ffusi on and t he si mpl e di ffusi on
which t oget her coul d be r esponsi bl e f or t he t r ans por t
of t he pept i des across t he BBB. For si mpl e di ffusi on
processes one needs t o t ake i nt o account appr opr i -
at el y t he var i at i on of t he sol ubi l i t y of var i ous pep-
tides in lipids. In t he fol l owi ng sect i on a t heor y rele-
vant t o t he t r anspor t of pept i des i ncor por at i ng ex-
plicity t he par t i t i on coeffi ci ent s appr opr i at e t o pep-
tides in lipids has been gi ven. The expr essi on f or t he
per meabi l i t y f or mal l y comes out t o be t he same as
t he one wr i t t en down phenomenol ogi cal l y by var i ous
aut hor s in t he l i t er at ur e, and depends on t he concen-
t r at i on of t he subst ance in t he br ai n- capi l l ar y and on
t wo ki net i c const ant s, ( T ~ ) f , t he maxi mal r at e of
t r anspor t and K,, t he Mi c ha e l i s - Me nt e n cons t ant
r epr esent i ng t he reci procal of t he affi ni t y o f bi ndi ng
of t he subst ance t o a carri er. We have used this ex-
pressi on t o est i mat e t he ext r act i on due t o t he car r i er -
medi at ed faci l i t at ed di ffusi on and have f ound t hat it
can expl ai n in conj unct i on wi t h t he cont r i but i on f r om
si mpl e di ffusi on t he obs er ved exper i ment al dat a at
var i ous concent r at i ons .
THEORY
A gener al expr essi on f or cal cul at i ng t he ext r act i on
f or compl ex cases i nvol vi ng var i ous di ffusi on pr oc-
esses has not been avai l abl e in t he l i t er at ur e. Howe v-
er, a basic f or mul a whi ch connect s t he per meabi l i t y
(P) and ext r act i on ( E) is gi ven by:
P = - ~ In ( l - E )
wher e F is t he rat e of bl ood fl ow and S is t he sur f ace
ar ea of t he br ai n capi l l ary. We have der i ved else-
where25 an expr essi on f or E by sol vi ng mat hemat i cal
r el at i ons descr i bi ng t he t empor al and spat i al vari a-
t i ons of t he concent r at i on of a subst ance in t he br ai n
capi l l ary and ext r avascul ar space of t he bl ai n st ar t m#
f r om a general f or ma l i s m The expr essi on ~ comc~
out t o be:
E = l - e x p - ( c , ; + K ; i I
wher e P, (cm/s) is t he per meabi l i t y of t he subst ance
in t he b l o o d - b r a i n bar r i er due t o si mpl e di f f usi on: S
is in cmZ/g of br ai n; F is in cmL"g s: c~ ( nM) is t he con-
cent r at i on of t he l abel ed pept i de in t he bol us of single
i nj ect i on t ype, ( Tma x ) f (in pmol / g' s : mM = umol / cm 3)
is t he maxi mal rat e of t r anspor t due t o faci l i t at ed dill
fusi on and K, (in mM) is t he Mi ct i ael i s - Ment en con-
st ant r epr esent i ng t he reci procal of t he af f i ni t ) of a
pept i de t o its car r i er in t he b l o o d - b r a i n bar r i er
(BBB).
Eqn. 1 f or g is appr opr i at e l o t he t r anspor t o/ a
subst ance f or a single t ransi t of a bol us t hr ough brai n-
capi l l ary when bot h t he si mpl e and faci l i t at ed diffu-
sion pr ocesses are significant. The br ai n- upt ake in-
dex is r el at ed t o E by t he expressionZ ,~.
E
BUI - EHOI_t (2)
wher e E HO H st ands f or t he ext r act i on: of wat er.
For penet r at i on of a pept i de mol ecul e via si mpl e
di ffusi on t hr ee possi bl e bar r i er s may be cons i der ed
t o be encount er ed by t he mol ecul e as have been dis-
cussed by Dani el l i 7. The first bar r i er (see Fig. 1) con-
si dered is at t he wa t e r - me mbr a ne i nt erface with a
barri er energy/ ~a f or t he mot i on of a mol ecul e f r om
BLOOD-BRAIN BARRIER
[
A
EXTERNAL a e o. INTERNAL
/ I POTENTIAL BARRIER T':, i I a
I
AQUEOUS
MEDIUM MEDIUM
(BLOOD) . , ~ I A PEPTIDE MOLECULE " ~ (BRAIN ECF:)
t
B D
F . . . . . .............. 4
Fig, 1. Schematic diagram of a blood~brain barrier with the
curve representing the potential barrier encountered by a pep-
tide molecule. AB is the membrane interface for diffusion from
water to the lipid medium With barrier-energy u~;/~c represents
the intermittent barrier height with:n inaxima, CD is the mem-
brane interface for the diffusion from lipid to water medium
with barrier-energy #b' 2 is the wavelength (distance between
two maxima) and d is the thickness of the blood-brain barrier.
ECF stands for extracellular fluid
t he ext ernal wat er - medi um ( bl ood side) t o t he inter-
nal l i pi d- medi um in t he me mbr a ne . The second bar-
rier effect i vel y i nvol ves ma ny successi ve bar r i er s in
t he i nt eri or of t he me mbr a ne with bar r i er ener gy/ , ~.
The t hi rd bar r i er is l ocat ed at t he ot her side of t he
me mbr a ne (agai n, me mb r a n e - wa t e r i nt erface) with
a barri er ener gy ,t4~. The expressi on f or t he per mea-
bility P, of a membr ane to a mol ecul e due t o simple dif-
fusion can be wri t t en a s 7 ,
a e
P ~ - n b + 2 e (3)
wher e
- - ZmVl RT
e=y~, ~exp -RT
b = , b V2~M exp -
( 4 )
(5)
( 6 )
wher e 7 is a const ant , q:',,, q:'c, ebb, are t he probabi l i -
ties f or penet r at i on of a pept i de mol ecul e across t he
barri er of energy/ ~a, f~o and ,Ub, respect i vel y; n is t he
number of barri ers in t he i nt eri or of t he membr ane.
R is t he st andar d gas const ant ; M is t he mol ecul ar
wei ght of t he di ffusi ng mol ecul e and T is t he absol ut e
t emper at ur e. The ener gy ,u~, is expect ed t o be nearl y
equal t o t he bar r i er e ne r gy/ 4, f or a mol ecul e pen-
et rat i ng t he l i pi d- wa t e r i nt erface. Accor di ngl y, f r om
Eqns. 5 and 6 with t he as s umpt i on: @~ = q<,,
b = e (7)
The numbe r of maxi ma n can be appr oxi mat el y writ-
t en as,
a
n - ~ ( 8 1
wher e d is t he t hi ckness of t he BBB and 2 is t he aver-
age wi dt h of t he i nt er mi t t ant bar r i er inside t he BBB.
Maki ng use of d -~ 78 A (ref. 26) and ~. -~ 1
A (ref. 7) we get 11 = 78 whi ch implies f or t he BBB
that,
nb >> 2e (9)
Consequent l y, f r om Eqn. 3 one obt ai ns.
203
Ps _ f e (10)
wher e
a (11)
f -
b
The f act or f is t he par t i t i on coeffi ci ent 7 r epr esent i ng
t he rel at i ve sol ubi l i t y of t he di ffusi ng mol ecul e in t he
lipid membr ane, t hat is, t he f r act i on of mol ecul es in
t he me mbr a ne c ompa r e d t o t hose in wat er. Fr om
Eqns. 5 and 10 we have,
f
P~= K/ ~ ~ (12)
with K as t he pr opor t i onal i t y const ant def i ned by:
7_~ ' e 2~ exp - (13)
K
In t he above K depends on t he charact eri st i cs of t he
bar r i er syst em, part i cul arl y, on t he bar r i er hei ght ~"c
and t he me mbr a ne t hi ckness ( pr opor t i onal t o n). As
f~e does not vary si gni fi cant l y: ( f r om one subst ance t o
t he ot her ) K is assumed t o be t he same f or t he pep-
tides consi der ed. In or der t o est i mat e t he val ue of K
f or pept i des we have adopt ed an i ndi rect met hod
based on sucrose as t he r ef er ence mat eri al , as ex-
pl ai ned in Cal cul at i ons and Resul t s.
In Eqn. 13, Dani el l F has ar gued t hat , besi des t he
f act or exp ( - / ~j RT) , t he f act or 7g'~ coul d be t emper -
at ure dependent . In fact, t he t emper at ur e depen-
dence of t he pr oduct 7gk, exp ( - u j R T ) is deci ded by
t he syst em under consi der at i on. We shall assume
here t hat t he pr oduct },0 exp (-,tk,/RT) is a slowly
varyi ng f unct i on of t emper at ur e.
In our case t he par t i t i on coeffi ci ent f r epr esent s t he
solubility of a pept i de mol ecul e in t he lipid medi um
associ at ed with t he BBB c ompa r e d t o t he medi um
( assumed aqueous) out si de t he BBB. It has been
f ound m t hat t he benzene rings and CHe- gr oups of a
mol ecul e i ncrease t he val ue of t he par t i t i on coeffi-
cient f, wher eas t he gr oups such as, - OH, and
- CH2 CONH decr ease its val ue.
An equat i on useful for t he eval uat i on of f may be
wri t t en as4:
lgm fpcp = logm frcf + ni bi (14)
i
where fpep is t he par t i t i on coeffi ci ent of a pept i de, and
fre~ is the par t i t i on coeffi ci ent of a r ef er ence sub-
204
FABLE I
b, c o n t r i b u t i o n s
List of b i contributions of various single groups (components~
constituting the peptide solute to log m f where f is the parti-
tion coefficient of the solute. The b, value of the - OH group is
not known accurately and therefore the relevant uncertamt~
has been given.
Gr o u p i n a s o l u t e ( i ) b i o f s o l u t e
- CH 2 (with terminal methyl group) 0.49
Benzene ring 1.40
- OH -0. 60 + 0.04
- CH2CONH or - CHRCONH -0. 84
s t a nc e ; n i r e p r e s e n t s t he n u mb e r of t he i th g r o u p pr es -
e n t in a p e p t i d e ; b i is t he e f f e c t o f a s i ngl e i th g r o u p on
l og m fp~p. T h e a p p r o x i ma t e v a l u e s 4 o f b i of v a r i o u s
g r o u p s r e q u i r e d in l og m fp~p a r e c o mp i l e d i n Ta b l e 1
f or e a s y r e f e r e n c e . F o r t h e r e f e r e n c e s ol ut e gl yc i ne
( H- C HC OOHNH2 ) Co h n a n d Ed s t a l l 4 ci t e l og m fCy
= - 3 . 3 9 1 . Eq n . 14 t h e n gi ve s ,
lOgl0 fpep = - 3 . 3 9 1 + X n i b i ( 15)
I
Egns . 12 a nd 15 a r e us e f ul f o r e s t i ma t i n g t o t he e x-
t r a c t i o n f r o m s i mp l e di f f us i on.
As f or o b t a i n i n g t he c o n t r i b u t i o n t o E f r o m f aci l i -
t a t e d di f f us i on it is e v i d e n t f r o m Eq n . 1 t ha t o n e
n e e d s t o k n o w t h e p a r a me t e r s (Tmax) t a nd K, whi c h
d e p e n d on t h e i n t e r a c t i o n b e t we e n t h e s u b s t a n c e a no
t he c a r r i e r s in t h e b l o o d - b r a i n ba r r i e r . At p r e s e n t ~t
is n o t pos s i bl e t o c a l c u l a t e t h e s e p a r a me t e r s f r o m
f i r s t - pr i nc i pl e s , a nd t h e r e f o r e we r es or t t o an r e d i r e c t
me t h o d o f e s t i ma t i n g (Tin,x) t a nd K, in c o n l u n c n o n
wi t h t he a v a i l a b l e d a t a f or v a r i o u s p e p t i d e s . Fo r t hi s
p u r p o s e o n e e x p r e s s e s Eq n . 1 in t he f o l l o wi n g f or m.
Th e r i ght h a n d s i de o f t he a b o v e e x p r e s s i o n d e p e n d s
onl y o n t he q u a n t i t i e s pl a yi ng par t m t he f a c i l i t a t e d
di f f us i on. Thi s e x p r e s s i o n wi l t be us e d b e l o w t o ana-
l yze t he e x p e r i me n t a l d a t a f or E f or v a r i o u s p e p t i d e s
a nd t o e xt r a c t i n f o r ma t i o n as t o t he c o n s t a n t s ( T ~ , , ) ~
and K t.
CALCULATI ONS AND RESULI S
To c a l c ul a t e t h e e x t r a c t i o n E due t o s i mp l e di f f u-
si on a l o n e o n e n e e d s t he p e r me a b i l i t y P~ whi c h c a n
be e s t i ma t e d f r o m Eq n s . 12 a n d 15. As yet t h e r e is no
f i r s t - pr i nc i pl e s me t h o d a v a i l a b l e f or e s t i ma t i n g K
a nd t h e r e f o r e we a d o p t an i n d i r e c t me t h o d t o es t i -
ma t e i t by e mp l o y i n g t he k n o wn p e r me a b i l i t y P~ o f
s uc r os e . We h a v e c h o s e n s uc r os e as a r e f e r e n c e s ub-
s t a nc e s i nce its t r a n s p o r t pr oper t i es6, 22 a r e k n o wn t o
be ma i nl y d u e t o s i mp l e di f f us i on s o t ha t t h e d e d u c e d
P, va l ue is e x p e c t e d t o c o r r e s p o n d t o s i mp l e di f f u-
TABLE II
L i s t o f t he v al ue s o f v a r i o u s c o n s t a n t s u s e f u l f o r t he c a l c u l a t i o n q f e x t r a c t i o n o ! p e p t i d e s i n t he t e x t a l o n g wi t h t he r e l e v a n t r~j erenct ' ~
Co n s t a n t s Value.~
Surface area S 240 cm2/g of brain
Molecular weight of glycine, Mot ~ 75
Molecular weight of a-MSH, MMS n 1681
Molecular weight of AVP, MA; . v 1083
Molecular weight of MIF, MMn. 301
Molecular weight of sucrose, MsLr ~ 342
Molecular weight of Me t - ENK, MMv:N k 573
Molecular weight of carnosine, MCA R 226
Molecular weight of glutathione, MGt,T 307
Molecular weight of tiLT 1000
Molecular weight of fiE 3426
Molecular weight of aE 1826
Partition coefficient of sucrose, fsLc 1.8 10 ~
Rate of blood flow, F 9.33 10- 3 cm,~/s.g of brain
Permeability of sucrose 0.26 10 -7 cm/s
Extraction of water Eno n 8(I 5':~
Thickness of BBB 78 A
Molecular weight of Le u- ENK, Mt.~:NK 591
Refs.
Lund-Andersen ~
Merck Index v~
Merck Index w
Merck Index v'
Merck Index >
Merck Index >
Merck Index ~"
Merck Index >~
Merck Index ~'
Rapoport et al.:~
Rapoport et al. -q
Rapoport et a12 ~
Rapoport et al. ~z
Lund-Andersen ~
Rapoport et al, :2
Lund-Anderscn ~
Lund-Andersen t and Sharma and Vimal 2~
Merck Index ~'~
205
TABLE 1II
L i s t o f t he n u m b e r o f c o n s t i t u e n t g r o u p s i n a mi n o a c i d a n d n i
val ues f o r M e t - E N K ( 7 ) , r - G l y - G l y - P h e - M e t )
Gr o u p (i) N u m b e r o f e f f e c t i v e g r o u p s in n i
a mi n o a c i d par t s o f M e t - E N K
T y r Gl v Gl y P h e Me t
-CH~ 1 - 1 3 5
Benzene ring 1 - - 1 - 2
OH 1 - - 1
- CHR( ' ONt t 1 1 1 1 4
s i on. Th i s al s o s u b j e c t s o u r p r o c e d u r e t o s e l f - c ons i s -
t e n c y as f a r as t h e me c h a n i s m ( s i mp l e d i f f u s i o n ) is
c o n c e r n e d . Al t e r n a t e l y , o n e ma y d e c i d e t o c h o o s e
gl yci ne as a r e f e r e n c e s u b s t a n c e b u t gl yc i ne , b e i n g a n
a mi n o a c i d, is i n a d e q u a t e as i t s t r a n s p o r t ma y n o t
p u r e l y b e s i mp l e d i f f u s i o n . Th u s , t a k i n g t h e P~ v a l u e
of s u c r o s e x2 as 0. 26 10 -7 c m/ s we o b t a i n K = 1.5 x
10 ~ c m/ s X/ ~ f r o m E q n . 12 u s i n g mo l e c u l a r we i g h t
M of s u c r o s e l~ = 342, T = 310 K a n d e x p e r i me n t a l f
of s u c r o s e 22 = 1. 8 10 a ( s e e T a b l e I I ) .
Ne x t , t h e p a r t i t i o n c o e f f i c i e n t s f of p e p t i d e s a r e
c a l c u l a t e d u s i n g E q n . 15 a n d t h e v a l u e s of n i a n d b i as
l i s t e d i n T a b l e s I I I a n d V- X f o l l o wi n g t h e d e t a i l s
TABLE IV
gi ve n a b o v e . Wi t h t h e a b o v e e s t i ma t e d K a n d T a b l e
II f or t h e mo l e c u l a r we i g h t s r e q u i r e d i n Eq n . 12 o n e
t h e n e s t i ma t e s t h e p e r me a b i l i t i e s P~ f o r B B B t o p e p -
t i d e s d u e t o s i mp l e d i f f u s i o n ( a l s o l i s t e d i n T a b l e I V) .
I f o n e a s s u me s t h a t f or p e p t i d e s o n l y t h e s i mp l e di f f u-
s i on p r o c e s s is i mp o r t a n t , o n e ma y c a l c u l a t e t h e c or -
r e s p o n d i n g E v a l u e s f r o m E q n . 1 by t a k i n g (Tm~) t =
0, t h e P~ v a l u e s l i s t e d i n T a b l e I V, a n d S a n d F
v a l u e s 17 f r o m T a b l e I I . As i t wi l l be e v i d e n t i n s ec-
t i o n s A t o J t h e c a l c u l a t e d E v a l u e s f or v a r i o u s p e p -
t i d e s d u e t o s i mp l e d i f f u s i o n a l o n e a r e t o o l ow t o ex-
p l a i n t h e e x p e r i me n t a l r e s ul t s .
T h e t h e o r e t i c a l a na l ys i s of E c o n s i d e r i n g b o t h t h e
s i mp l e d i f f u s i o n a n d f a c i l i t a t e d d i f f u s i o n is a l s o pos s i -
bl e ( s e c t i o n K) f r o m E q n . 16 by e v a l u a t i n g t h e l ef t
h a n d s i de o f E q n . 16 wi t h t h e c a l c u l a t e d v a l u e s of P~
f r o m T a b l e I V a n d t h e a v a i l a b l e e x p e r i me n t a l d a t a
f or E. Th i s p r o c e d u r e is us e f ul f or e x t r a c t i n g i n f o r -
ma t i o n as t o t h e r i ght v a l u e s of t h e k i n e t i c c o n s t a n t s
(Tmax) f a n d K t t h a t c o u l d e x p l a i n c o n s i s t e n t l y t h e o b -
s e r v e d E v a l u e s . T h e d e t a i l s f or v a r i o u s p e p t i d e s c o n -
c e r n i n g t hi s a r e g i v e n i n s e c t i o n s A t o K.
( , 4 ) M e t - E N K - - r n e t h i o n i n e - e n k e p h a l i n
Me t - E NK is a p e n t a p e p t i d e ( T y r - Ol y - Gl y -
P h e - Me t ) wh i c h act s as a n i n h i b i t o r y n e u r o mms mi t -
Ta b u l a t i o n o f t he c a l c u l a t e d p a r t i t i o n c o e f f i c i e n t (f), p e r me a b i l i t y (P, ) d u e t o s' i mpl e d i f f u s i o n , t he c o n t r i b u t i o n t o t he e. t t r act i on d u e t o
s i mp l e d i f f u s i o n al one ; a n d e x p e r i me n t a l e x t r ac t i on ( E) o f p e p t i d e s
The calculated E due to simple diffusion al one is significantly lower t han t he experi ment al E indicating the possible existence of peptidc
carrier in the bl ood- br ai n barrier.
Pe pt i de s Par t i t i on c o e f f i c i e n t Pe r me a b i l i t y d u e t o Cal c ul at e d e x t r ac t i on E x p e r i me n t a l e x t r a c t i o n
f ( i n 10 4) s i mp l e d i f f u s i o n P, d u e t o s i mp l e E (in (' ()
(in 10 r c m/ s ) d i f f u s i o n a l o n e (in % )
Me t - ENK 7' )_+7 8. 7_+08 2. 3_+0. 2 12+ 1' 2.5_+11.Y ~*
Le u- ENK 7 9 + 7 8 . 5 + 0 8 2. 3_+0. 2 2. 7_+0. 3 2.7_+1).4 b,~
Gl ut at hi onc 7 1 0.3 0.6 + {L5 0.4 + ().1 h~,~'
Carnosine 17 3 0.8 1. I h
~t-MSH 212 ,+ 57 13.7 _+ 3 7 3.5 + 1 7.7 + 1.S,
MIF 63 9.6 2.5 11 = 1.2'
AVP 7`) + 7 6.3 + 0 6 1.6 ,+ 0.2 1(I.5 * 2.9 ~
tiLT (fl lipotropin) 120 III 2.5 3.53 + 1.5':
f i e ( f i - endor phi n) 130 5.9 1.5 ').5 _~.2.9 ~
aE 0~-endorphin) 17 I. 1 (}.3 5.75 + 31
* High and low limits are due to effect of concent rat i on c~ (see also Tabl es XI and XII).
~' Calculated from BUI value from Kastin et al. 14
b See Cornford et al / ' .
Calculated from BUI values from Gr eenber g et al. 12
a Calculated from measured total permeabi l i t y P - (14 _+ 6) x 10 7 cm/s (ref. 21).
~ Calculated from measured total permeabi l i t y P = (39 _+ 12) 10 7 cm/s (ref. 21/.
f Calculated from measured total permeabi l i t y P = (23 + 13) x 10 : cm/s (ref. 21 ).
206
t er or n e u r o mo d u l a t o r in t he cent r al ne r vous s ys t em.
I t i nhi bi t s t he a dr e ne r gi c , chol i ner gi c and dopa -
mi ne r gi c t r ans mi s s i on. The pa r t i t i on coef f i ci ent of
Me t - E NK is c a l c ul a t e d t o be f = (79 + 7) x 10 '*
f r om Eqn. 15 usi ng Ta bl e s I - I I l . Th o u g h in Met - -
ENK t he sul f ur gr oup ( - S ) is al so pr e s e nt in t he
s t r uct ur e, i t doe s not c ont r i but e t o bi si nce its ef f ect is
nul l i f i ed by t he H of t he CH3- gr ou p a t t a c he d t o i t , in
a na l ogy wi t h t he nul l i f i cat i on of t he ef f ect of - S in
cys t ei ne ( Cys) gr oup as di s cus s ed in ref. i 0. The per -
me a bi l i t y of BBB t o Me t - E NK due t o s i mpl e di f f u-
si on is t hen ( f r om Eqn. 12 and Ta bl e I I ) P~ = ( 8. 7 _+
0. 8) x 10 -7 cm/ s at 37 C whi ch wi t h (Tm,~0 f = 0
yi el ds t he e xt r a c t i on EMENK = 2. 3 --+ 0. 2% ( see al so
Ta bl e I V) due t o s i mpl e di f f usi on a l one f r om Eqn. 1.
Al t h o u g h t hi s val ue a gr e e s wi t h t he e x p e r i me n t a l E
( 2 - 3 %) r e p o r t e d by Co r n f o r d et al.6 f or t he bol us
c onc e nt r a t i on c; of 2 1 - 2 7 0 nM, it is si gni f i cant l y l ow-
er c o mp a r e d t o t he e xt r a c t i on 12 -+ 1% ( c a l c ul a t e d
f r om Eqn. 2 wi t h o b s e r v e d 13 BUI = 15% and EH6~H
= 80 + 5% f or c onc e nt r a t i on c~ = 0. 12 nM) .
( B) L e u - E N K - - l e u c i n e - e n k e p h a l i n
L e u - E NK is a p e n t a p e p t i d e ( T y r - Gl y - Gl y -
P h e - L e u ) whi ch act s as an i nhi bi t or y n e u r o t r a n s mi l -
t er in t he cent r al ne r vous s ys t em. The pa r t i t i on coef -
f i ci ent of L e u - E NK c ome s out t o be f = (79 _+ 7)
10 -4 f r om Eqn. 15 usi ng Ta bl e V. The p e r me a b i l i t y
of BBB t o L e u - E NK due t o s i mpl e di f f usi on is t hen
( f r om Eqn. 12 a nd Ta bl e I I ) p, = (8. 5 + 0. 8) x 10 -~7
cm/ s at 37 C whi ch wi t h (Tmax) f = 0 gi ves t he ext r ac-
t i on ELENK = 2. 3 + 0. 2% due t o s i mpl e di f f usi on
al one f r om Eqn. 1. The c a l c ul a t e d ELENK is cl ose t o
t he e x p e r i me n t a P val ue 2. 7% ( see al so Ta bl e I V) as-
s oci at ed wi t h t he c onc e nt r a t i on c~ in t he s a t ur a t i on
TABLE V
List of the number of constituent groups in ami no acid residues
and n i values f or L e u - E NK ( T y r - Gl y - Gl y - Ph e - L e u )
Group ( i ) Number of effective groups in n i
ami no acid parts o f Le u - ENK
Tyr Gly Gly Phe Leu
1 - 1 3 5
-CH~
Benzene ring
1 - 1 - 2
- OH 1 . . . . I
-CHRCONH ! 1 1 1 4
TABLE VI
List of the number o f constituent group.~ m atnino acid residues"
and the corresponding n, values f , r Jut at hi one cG/u ~('v>
Gly)
In Cys-group we have not included the contribution from suilur
(S) since its effect is nullified bv the adlaccnl hvdroeen, ~ di.~-
cussed in ref 5
Group i~ Number oj et}e,-'m e groups i~t
amine acid part~ of ghaathione
Glu ( w ( ;h'
- CH-
-CHRCONH .... :
r ange 22- 550 nM. Thus at l ar ge c onc e nt r a t i ons t he
cal cul at ed c ont r i but i on t o E due t o s i mpl e di f f usi on
agr ees wi t h t he e x p e r i me n t a l val ue as in Me t - E NK
( Ct Gl u t a t h i o n e
Gl ut a t hi one ~s a t r i pe pt i de ( Gl u - C y s - Gl y ) f ound
in ani mal and pl a nt t i ssue as a c a r r i e r of oxygen. It
cont ai ns gl ut ami c aci d r e s i due j o i n e d in an unusual
pe pt i de l i nkage i nvol vi ng F- car boxyl i ns t e a d of t he
usual a - c a r boxyl gr oup. The pa r t i t i on coef f i ci ent f of
gl ut a t hi one is c a l c ul a t e d t o be f - 7 t 0 -`4 f r om
Eqn. 15 usi ng Ta bl e VI.
It mus t be r e ma r k e d t hat becaus e of t he pe pt i de
l i nkage i nvol vi ng y- car boxyl in t hi s case t he gr oup
- C HR OC ONH be t we e n Gl u and Cvs c ont r i but e s
di f f er ent l y t o b i val ue t han t he c ont r i but i on ar i si ng
f r om t he n o r ma l a - c a r b o x y l gr oup as we have d i s c m-
er ed by e xpl a i ni ng t he e x p e r i me n t a l val ue 4 of Iog,~
folu whi ch is - 2. 992. It is be c a us e of t hi s r eas on t hat
we have as s i gned n i - 0. 5 for t he l i nkage be t we e n
Gl u and Cys in Ta bl e VI . The pe r me a bi l i t y of BBB
t o gl ut a t hi one ~s e s t i ma t e d f r om Eqn. 12 and Ta bl e
1I t o gi ve P, = 10 -7 cm/ s al 37 ~C. Eqn. 1 wi t h (Tm.x)t
- 0 t hen yi el ds t he e xt r a c t i on Eo~.~ = 0. 3% due t o
si mpl e di f f usi on al one. One not es t ha t t he c a l c ul a t e d
EGLT due t o s i mpl e di f f us i on al one ~s cl ose t o t he ex-
pe r i me nt a l 5 val ues whi ch var i es t r om (1.6 t o 0. 4% [or
high c onc e nt r a t i ons c~ f r om 1000 t o 10.000 nM ( see
al so Ta bl e I V) .
( D) Ca r n o s i n e
Ca r nos i ne ts a d i p e p t i d e of a l a ni ne and hi s t i di nc
( Al a - Hi s l occur r i ng m mus cl es of ani mal s and me n.
One f i nds t he pa r t i t i on coef f i ci ent of c a r nos i ne f = 1"
207
FABLE VII
List of the number o! constituent groups in amino acid residue.~
and the corresponding n i values f or carnosine ( Al a- Hi s)
Group ~l) Number c)f effective groups in n,
amino acid parts of carnosine
A la His
-CH, 1 2 3
-CHRCONtt 1 - 1
10 4 from Eqn. 15 (see also Tabl e Vl I). The per-
meability of BBB to carnosi ne due to simple diffusion
alone is est i mat ed by using Eqn. 12 and Tabl e II to
obt ai n P~ = 3 10 7 cm/s at 37 C which from Eqn. 1
with the condi t i on (Tm~,x) f = 0 gives the extraction
Ec;,a = 1t.8% (due to simple diffusion alone)
agreeing with the experi ment al value 1.1% for high
concentration ca = 38000 nM as in Me t - ENK, Leu-
ENK and gl ut at hi one (see also Tabl e IV).
( E) a - MS H - - me l a n o o , t e - s t i mu l a t i n g h o r mo n e
The structure I~ of a-MSH consists of first 13 (see
Tabl e VIII) ami no acids of adrenocort i cot ropi c hor-
mone ( ACTH) and an acetylated NH: -t ermi nal on
one side and an ami dat ed CO2H-t ermi nal ami no acid
on the other side. It is one of the most pot ent mel ano-
cyte-stimulating hormones and has an i mport ant
physiological pi gment ary function. It has been estab-
lished that the i nj ect i on of synthetic a- MSH im-
proves the performance in the Bent on Visual Ret en-
tion test and increases significantly the somat osenso-
ry cortical evoked responses.
The partition coefficient of a- MSH comes out to
be f = (212 _+ 57) 10 ~ from Eqn. 15 using Tables I
and VIII. The permeabi l i t y of a-MSH at 37 C is
then (from Eqn. 12 and Tabl e I1) P~ = (13.7 _+ 3.7) x
10 -7 cm/s yielding, with (T ..... )~ = (), the extraction
EMS H = 3.5 + 1% due to simple diffusion alone which
is much lower t han the experi ment al ~: extraction 7.7
_+ 1. 8~ deduced from BU112 = 9.6 _+ 2.3% using
Eqn. 2 and EHo H = 80 + 5% (see also Tabl e IV).
(F) MI F - - MS H- r e l e a s e i n h i b i t i n g . f act or
MIF is a hypot hal ami c hormone which controls the
release of MSH from the pituitary and produces neu-
romodul at ory effects~9. It is a tripeptide with ami no
acid (AA) sequence Pr o- Le u- Gl y- NH> In this
case partition coefficient of MIF is f = 63 x 10 ~ from
Eqn. 15 and Tables l and IX and P~ = 9.6 10 -7 cm/s
at 37 C (from Eqn. 12 and Tabl e II) which with
(Tm,,~)f = 0 gives from Eqn. 1 the extraction EMW =
2.5% due to simple diffusion, a value much lower
t han the experi ment al value of extraction 11 _+ 1.2%
deduced from BU112 = 13.7 _+ 1.6~4 (see also Table
IV) correspondi ng to c~ = 0.14 nM and Euo u = 80 +
5%. We note that c~ correspondi ng to the experi men-
tal E is very low and, in compari son, the calculated
extraction EMW (due to simple diffusion alone) is sig-
nificantly lower t han experi ment al extraction.
( G) A V P - - a r g i n i n e - v a s o p r e s s i n
AVP is antidiuretic hor mone v~ which is extracted
from the posterior lobe of the pituitary of healthy do-
mestic animals. It produces neur ot r ansmi ner - l i ke ef-
fects when admi ni st ered at high concent rat i on in the
body and neuromodul at or-l i ke effects at low concen-
tration. It has 9 ami no acids (nanopept i de) and its
chemical formul a is C4e, Ho5NIsOIeS 2. The structural
formula of AVP indicating ami no acid sequence is
TABLE VIII
List of the number ~I' constituent groups in amino acid residues and the corresponding n~ values invoh' ed in Eqn. 15 .lor (*-MSH
( CH~( ' O- Ser- f y r - Ser Met - Gl u- Hi s - Phe - A rg Tr p- Gly Lys' - Pr o- Va l - NH 2)
Group (i) Number of groups in various amino acid parts of ~- MSH n,
Ser Tyr Ser Met Glu His Phe Arg Trp Gh' l.v~ Pro Val
-CH, 1 1 1 3 2 2 1 2 1 - 2 - 2 18
Benzene ring 1 - - 1 -- 1 1 4
OH 1 1 1 - - 3
CHRCONH 1 1 1 1 1 1 1 1 1 l 1 1 1 1 13
208
TABLE IX
List o f the number of constituent groups in ami no acid residuc~
and the corresponding n i values f or MI F ( Pr o- Leu- Gl v- - Nt t ei
Group (i) Number of effective groups /i
in ami no acid parts in MI F
Pro Leu Glv
-CH~ 3 3.
Benzene ring 1 - !
-CHRCONH 1 1 2
gi ven by
C y s - T y r - P h e - Gl u - As n - C y s - P r o - Ar g - Gl y - NH 2
We have e s t i ma t e d t he pa r t i t i on coef f i ci ent f of
AVP f r om Eqn. 15 and Ta bl e I I a nd f ound it t o be
(79 + 7) x 10 .4 as l i s t ed in Ta bl e X. Eqn. 12 and
Tabl e II t hen gi ve t he p e r me a b i l i t y of AVP P~ = (6. 3
+ 0. 6) x 10 -7 cm/ s at 37 C. Thi s val ue wi t h (Tmax) f =
0 c or r e s ponds t o t he e xt r a c t i on EAV e = 1.6 + 0. 2%
due t o s i mpl e di f f us i on al one at 37 C ( usi ng Eqn. 1
and Ta bl e I I ) . The c a l c ul a t e d E due t o s i mpl e di f f u-
si on is ver y l ow c o mp a r e d wi t h t he e x p e r i me n t a l ex-
t r act i on 10.5 + 2. 9% d e d u c e d f r om t he e x p e r i me n t a l
BUI >" = 13 + 2. 8% ( f r om Eqn. 2 and usi ng EHOH =
80 + 5%) c o r r e s p o n d i n g t o c; = 6. 7 nM, a l ow val ue
of c onc e nt r a t i on ( s ee al so Ta bl e I V) .
( H) f l L T - - f l - [ D- Al a2- 14C- homoar gOg] l i pot r opi n61_69
t i LT is a s ynt het i c p e p t i d e whi ch is an a na l og of t he
na t ur a l p e p t i d e fl-lipotropin6~_69 de r i ve d f r om t he pi -
t ui t ar y h o r mo n e f l - l i pot r opi n. I t is r e p o r t e d 2~ t o e xe r t
cent r al ef f ect s ( such as opi a t i c ef f ect ) when i nj e c t e d
syst emi cal l y. Be c a us e t he s t r uc t ur e of t i LT is not
known def i ni t el y we ar c unabi c !~.~ cal cul al e it,; parlJ-
t i on coef f i ci ent usi ng our me t h o d gi ven ill t he t heor ~
sect i on. Fo r t u n a t e l y . t he part t t ~on coef f i ci enI in t hi s
case is known e x p e r i me n t a l l y - ' , whi ch i~ t -:. 12fi x
10 4 The pe r me a bi l i t y of BBB t o fil_-l' due t o s i mpl e
di f f usi on at 37 C is t hen P. -: !J.l > ll) " cm, s l t r om
Eqn 12 and Ta bl e I1). Thi s g~vc, ~i l h ( l'm,,~)t ~ (i and
Eqn. 1 E~l t = 2. 5% clue t o ~m~pte di f f usi on al one.
The e xpe r i me nt a l val ue of t he e xt r a c t i on for i l l / 1 is
not avai l abl e for c ompa r i s on. Ho we v e r , t he experi--
ment al 21 pe r me a bi l i t y P is I14 t~l x 1(1 " cm/ s. Thi s
me a s u r e me n t c or r e s ponds t o l hc a nl ounl ol /~LT
r angi ng f r om 2 t o l (I, uCi i nj e c t e d in t he br ai n capi l -
l ar y, t he speci f i c act i vi t y of/;~L I " 58 mCi / mM and
t he f r act i on r = 0. 84 of t i LT u n b o u n d t o bl ood pl as-
ma. Thes e val ues e na bl e us t o c s t i ma t e c~ t r om
a mo u n t of dr ug r e j e c t e d
c~ = r x speci f i c act i vi t y of t he dr ug / 17}
yi el di ng G = 87 nM f or t he a ve r a ge val ue o u Ci ot
t i L T i nj ect ed. It me a ns t hat t he me a s u r e d P val ue ( 14
_+ 6) x 10 -7 cm/ s c or r e s ponds t o ci; - 87 ri M. Thi s P is
t he t ot al p e r me a b i l i t y and t h e r e f o r e it y i e l d s t h e t ot al
e xt r a c t i on E = 3.53 ~_ 1.5c/r ( f o r f i LT at ca = 87 nM) .
whi ch is hi gher t han our c a l c ul a t e d val ue of t he ex-
t r act i on 2. 5% ar i si ng f r om s i mpl e di f f usi on al one
(see al so Ta bl e I V) .
(1) f i e f l - l D- Al a 2 - t 4 C- h o mo a r g V] e n d o r p h i n
f i E is a s ynt het i c 21 opi oi d pe pt i de , an a na l og of t he
na t ur a l p e p t i d e f l - e ndor phi n de r i ve d f r om t he pi t u-
i t ar y h o r mo n e f l - l i pot r opi n. It c ont a i ns t he s a me ami -
no aci d s e que nc e T y r - D- Al a - Gl y - P h e - Me t as
t i LT and a E. It s pa r t i t i on coef f i ci ent f has be e n meas -
ur ed 21 t o be 130 x 10 -4. We have us ed t hi s val ue o f f
TABLE X
List of the number of constituent groups in amino acid residues and the corresponding n i values f or A VP ( ):~'-, 71yr- Ph e - Gt n - Ash ....
Cys - Pro - A rg- Gly - NH2)
Group (i) Number o f effective groups in various acid parts of A VP ,!.
- CH z
Benzene ring 1
- OH 1 . . . .
-CHRCONH between amino acids 1 1 1 1
Cys Tyr Phe Glu Asn Cys Pro Arg
1 1 1 2 1 1 - - 2
1 . . . . . I I
1 i 1 I
GI)
~ 3
3
2119
TABLE XI
List of the calculated values of the total extraction (due to simple &ffusion and carrier-mediated facilitated diffusion) and experimental
extraction for various concentrations c~ of Met - ENK. Le u- ENK, glutathione, carnosine, a- MSH and MIF, using the maximal rate of
transport ( Tm,,Q f - O. 46 x 10 --~ pmol/g.s and the reciprocal of affinity K t (= 0.35 nM) of peptide to its carrier which have been e~'timated tLs-
ing experimental extractions f or c; equal to 0.12 nM and 50 nM of Met - ENK, and calculated extraction E, = 2.3~'4 of Me t - ENK due to
fimple diffusion (see Table IV)
Though the values of the parameters (Tmax) f and K, explain all the experimental results within uncertainties, the K~ values may actually
be different for different substances.
Peptides Concentration c~ (in nM) Extraction* Icalculated) (in ~ ) Extraction (experimental) (in C~ )
Met - ENK 0.12 12 _+ 1 12 +1 ~'
21 2.5 + 0.2 2.7 _+ 0.3 ~'
25 2.5_+0.2 3 _+1 h
50 2.4 + 0.2 2.4 (!.3 ~'
270 2.2 +_ 0.2 " "S + I "~1,
Leu- ENK 22 2.4 + 1).2 2.7 + 0.3 t'
1311 2.2 _+ 0.2 2.7 _+ 0.3 t'
550 2.2 + 0.2 2.7 + 0.4 ~'
Glutathione 10()ll 0.3 11.6 + 1!. 5 h
10000 0.3 0.4 _+ 0.1 h
Carnosinc 38000 0.N 1.1 h
a-MSH 0.583 g.4 _+_1 7.7 + 1.g c
MIF 0.14 11.8 l l +_1.2"
* The uncertainties in the calculated E values are due to uncertainties in calculated P~ values.
Calculated from BUI value from Kastin et al. t3.
h Calculated from BUI values from Greenberg et a1.12.
c See Cornford et al. 5.
t o c a l c ul a t e t he p e r me a b i l i t y of BBB t o f i E d u e t o
s i mpl e di f f us i on whi c h c o me s o u t t o be P~ = 5. 9 x
10 7 cm/ s at 37 C ( f r o m Eq n . 12 a nd Ta b l e I I ) . On e
t he n c a l c ul a t e s ( f r o m Eq n . 1 wi t h (Tmax)f = 0) t he ex-
t r a c t i on Ef~ E = 1. 5% ( s ee al so Ta b l e I V) , a v a l u e
l o we r t ha n t he e q u i v a l e n t e x p e r i me n t a l e x t r a c t i o n
9. 5 _+ 2 . 9 % wi t h t h e e xpe r i me nt a l 21 t ot a l p e r me a b i l i -
ty P = (39 _+ 12) 10 .7 cm/ s f or E c o r r e s p o n d i n g t o c;
= 6. 8 n M e s t i ma t e d f r o m Eq n . 17 wi t h t he e xpe r i -
me n t a l p a r a me t e r s r = 0. 2, a mo u n t o f f i E i n j e c t e d =
2/ ~Ci a nd t he s peci f i c a c t i vi t y 58 mCi / mM.
i t y 58 mCi / mM.
(J ) a E - - a- [ D- A l a2- 14C- homoar gJ] endor phi n
t e e is a s ynt he t i c 21 o p i o i d p e p t i d e r e p r e s e n t i n g t he
na t ur a l p e p t i d e a - e n d o r p h i n d e r i v e d f r o m t he pi t u-
i t ar y h o r mo n e f l - l i pot r opi n. Th e me a s u r e d p a r t i t i o n
c oe f f i c i e nt of E is f = 17 x 10 4 whi c h yi el ds t he as s o-
c i a t e d p e r me a b i l i t y d u e t o s i mpl e di f f us i on a l o n e as
P~ = 1.1 10 -7 cm/ s at 3 7 C ( us i ng Eq n . 12 a nd
Ta b l e 11) a nd t he c o r r e s p o n d i n g E = 0 . 3 % ( f r o m
Eq n . 1 wi t h (Tma) f = 0). Th e exper i ment al l y21 me a s -
ur e d t ot al p e r me a b i l i t y in t hi s cas e is P = (23 + 13)
10 .7 cm/ s whi c h gi ves t he e q u i v a l e n t e x p e r i me n t a l
e xt r a c t i on E = 5. 75 _+ 3 % f or c; = 11 n M ( e s t i ma t e d
us i ng Eq n . 17 wi t h r = 0. 34, a mo u n t i n j e c t e d = 2 # C i
a nd t he s peci f i c act i vi t y = 58 mCi / mM) . We n o t e t hat
t he c a l c ul a t e d E ( 0 . 3 %) due t o s i mpl e di f f us i on a l o n e
is mu c h l ess t ha n t he e x p e r i me n t a l E ( 5, 75 +_ 3 %)
( see al so Ta b l e I V) .
( K) Cal cul at i on o f (T, , ~x)f and K,
Ta b l e I V c o n t a i n s t he e x t r a c t i o n s f or va r i ous pe p-
t i des c a l c ul a t e d a b o v e f r o m s i mpl e di f f us i on a l o n e ,
whi ch ar e s i gni f i cant l y l o we r t ha n t he e x p e r i me n t a l
va l ue s i ndi c a t i ng t ha t t he me r e s i mpl e di f f us i on pr oc -
ess c a n n o t e xpl a i n t he t r a n s p o r t o f p e p t i d e s t h r o u g h
BBB. Fu r t h e r , t h e e x p e r i me n t a l e x t r a c t i o n s a r e
f o u n d t o d e p e n d on t he c o n c e n t r a t i o n s of p e p t i d e s in
t he bol us ( see Ta b l e s I V, XI a nd XI I ) . Wi t h t he in-
21(1
TABLE XII
List of the calculated values of total extraction using ( T, na~) f = l O :
pmol/g.s and K t = 7 nM and experimental extractions/br vari-
ous concentration c~ of A VP, t i LT, fiE and aE
(Tmax) f and K t have been estimated from extractions due to sim-
ple diffusion and experimental extraction of tiLT and aE.
Peptides Concentration Extraction Extraction
c~ (in nM) (calculated) (experimental)
(in %) (in %)
AVP (11.7) 6.7 9 10.5 _+ 2.9 ~
tiLT 87 3.53 3.53 _+ 1.5 b
fiE 6.8 (20.6) 8.9 9.5 _+ 2.9 ~'
aE l l (35) 6 5.75 _+ 3 u
Calculated from BUI values of Greenberg et al. J2.
Calculated from permeability values from Rapoport et al.:~.
t ent of d e t e r mi n i n g t he bas i c me c ha ni s m i n h e r e n t in
t he t r a n s p o r t of p e p t i d e s we now s how in Fi g. 2 t he
poi nt s r e pr e s e nt i ng t he l ef t ha nd si de of Eqn. 16 f or
r e l e va nt l og m c; , t a ki ng f or E t he known e x p e r i me n -
t al e xt r a c t i ons f or di f f e r e nt pe pt i de s . Fi g. 2 shows
t hat t he l ef t ha nd si de of Eqn. 16 whi ch r e pr e s e nt s
t he e xt r a c t i on wi t hout s i mpl e di f f us i on ( s ee t he or y
sect i on) de c r e a s e s wi t h c onc e nt r a t i on c~ a p p r o a c h i n g
a s ympt ot i c a l l y t o z e r o i ndi c a t i ng a s a t ur a t i on ef f ect
2O
o~
15
10
',i 4
5 q
o I I "' " ~-'~-.-~, + $ # - - 4 - ~ '.
1 3 4 5 6 7 8 9
Iog~o C O ( C o i n p M )
Fig. 2. The semilogarithmic plot of I-[(1-E)/ exp(-PsS/ F)] vs
log m c~ for Met -ENK (liD), Leu-ENK ( T) , glutathione
(O), carnosine (X), a-MSH (O), MIF (@), AVP (zS),
tiLT (&), fiE ( V) and aE (11). Dotted curve is the theoret-
ical plot of
/ ] v s
[ (Kr+c~)F)I lgl c~l
with (Tmax) ~ = 0. 46xi 0 -3 pmol/g.s and K t = 0.35 nM, which
satisfy approximately the experimental points corresponding to
Met -ENK, Leu-ENK, glutathione, camosine, a - MSHa nd
MIF.
and hence t he pr e s e nc e of p e p t l d e c a r r i e r s for trans.-
por t .
As p o i n t e d out in t he t he or y sect i on it is not possi -
bl e t o cal cul at e di r ect l y (Tmax) ~ and K t at pr e s e nt .
For t una t e l y, it is pos s i bl e t o d e t e r mi n e ( T . . . . )f and K,
i ndi r ect l y by usi ng any t wo e x p e r i me n t a l E val ues f or
di f f er ent c onc e nt r a t i ons in Eqn. 1. Ac c or di ngl y, we
ut i l i ze t he E val ues of Me t - E NK in Eqn. i f or t wo
concent r at i ons c; equal t o 0.12 nM and 50 nM and ob-
t ai n t wo s i mul t a ne ous e qua t i ons whi ch yi el d ( T ..... )t
= 0. 46 I O3 p mo l / g ' s and h, = 0. 35 nM ( usi ng
Tabl es I I , I V and XI f or r at e of b l o o d f l ow ( F) = 9.33
x 10 -3 cm3/g. s, S = 240 cm: / g, P, = 8. 7 10 7 cm/ s,
E = 12% at c a = 0 . 1 2 n Ma n d E = 2 . 4 % f o r c ; - - 5 0
nM) . Knowi ng (Tmax) f and K, wc now a t t e mp t t o cx-
pl ai n t he obs e r ve d E val ues of p e p t i d e s f or di f f er ent
concent r at i ons . For t hi s p u r p o s e we have al so pl ot t e d
in Fi g. 2 t he r i ght ha nd si de of Eqn. 16 ver sus l og> el;
( shown as a d o t t e d cur ve) . We fi nd t hat t he d o t t e d
cur ve fal l s cl ose t o al l t he ~xpe r i me nt a l poi nt s
( Fi g. 2) except t o t hos e f or AVP, t i LT, f i E and <zE
whi ch r e qui r e a di f f er ent set of K: and ( T ..... )~ val ues.
In f act , t he me t h o d a na l ogous t o t he one de s c r i be d
a bove l e a ds t o K t = 7 nM and (Tmax) t = 1(1 ](} a
pmol / g' s a p p r o p r i a t e t o AVP , t i LT, f i E and a E ( see
al so Ta bl e Xl l ) .
Wi t h t he d e d u c e d val ues of (Tmax) f and K, it is in-
t er es t i ng to cal cul at e E f or var i ous p e p t i d e s f r om
Eqn. 1 usi ng t he r e q u i r e d p a r a me t e r s f r om Ta bl e s 11
and I V. Ta bl e XI l i st s t he c a l c ul a t e d E f or Me t -
ENK, L e u - E NK. gl ut a t hi onc , c a r nos i ne , a - MS H
and MI F f or di f f er ent c o n c e n t r a n o n s a nd c o mp a r e s
wi t h t he e xpe r i me nt a l dat a. As for t he t ot al E f or
AVP, t i LT, f i E a nd a E we have l i st ed t he m in Ta bl e
XI I as o b t a i n e d f r om Eqn. l usi ng P, t r om Fabl e
I V, (Tmax) I = 10 x 10 -3 p mo l / g s and K. = - n M.
Ta bl e XI I al so l i st s t he e x p e r i me n t a l E f or c o mp a r i -
son.
If one i ncl udes t he e x p e r i me n t a l e r r or s in t he
me a s u r e me n t of t he e xt r a c t i ons , one f i nds l ar ge er -
rors in t he above val ues of i T ...... )~ and K,. Expl i ci t l y K,
= 0. 35 + 0.31 nM a nd (Tmax) f = (0. 46 - 0. 29) x 10 -3
pmol / g' s f or t he fi rst gr oup of p e p t i d e s ( Me t - E NK,
L e u - E NK. gl ut a t hi one , car os i ne, a - MS H and MI F )
and K t = l l _ 11 nM. (Tmax) I = (15 - 15) x 10 -~
pmol / g' s for t he s e c ond gr oup ( AVP , t i LT. ~E and
a E ) of pe pt i de s . The l ar ge e r r or s in (Tm~xJf and K, de -
ma nd mo r e pr eci s e me a s u r e me n t s of E val ues .
The vari at i ons of t he total cal cul at ed ext r act i on E
( f r om bot h simple and faci l i t at ed di ffusi on) with con-
cent r at i on c~ are depi ct ed in Fig. 3 f or Me t - E NK
and L e u - E NK (curve I), gl ut at hi one (curve 1I), car-
nosine (curve 11I), (z-MSH (curve I V) , and MI F
(curve V) by using appr opr i at el y P~ f r om Tabl e IV, S
and F f r om Tabl e I I , K~ = 0.35 nM and Tma x = 0. 46 x
10 -3 p mo l / g s in Eqn. 1. In Fig. 3 t he exper i ment al
ext ract i ons for all t he pept i des consi der ed are also
shown for compar i son. The curves in Fig. 3 give t he
right behavi or of E vs l og> c; and are i mpor t ant f or
predi ct i ng t he E val ues f or any concent r at i on of pep-
tides.
I)IS(USSION AND PREDICTION OF NORMAL CON-
CENTRAT1ON OF PEPTIDES
We have est i mat ed above t he par t i t i on coeffi ci ent s
of wmous pept i des in et hanol with gl yci ne as a refer-
ence. Assumi ng t hat t he cal cul at ed val ues of t he par-
tition coeffi ci ent s appr oxi mat e cl osel y t he val ues ap-
pr opr i at e for pept i des in b l o o d - b r a i n barri er, we
have cal cul at ed first t he ext r act i on val ues due t o sim-
ple di ffusi on al one f or Me t - E NK, L e u - E NK, gl ut a-
t hi one, car nosi ne, a - MSH, MI F, AVP, t i LT (/:~-
[D-Ala-' -IaC-homoarg""]lipotropin~,l ~,~), t i e ( / :
7 ~ 2 C - -
u J
Z 15 : - ~ - T _ ~ - - . .
d, 1 0
I
h~ 0 1 2 3 4 5 6 7 8 9
, o g ) C o ,C o in pM)
F i g . 3. ] ' h e s e m i l o g a r i t h m i c p l o t o f t h e v a r i a t i o n o f t h e e x t r a c -
t i o n ( i n % ) o f M e t - E N K , L e u - E N K , g l u t a t h i o n e a n d c a r n o -
s i n c w i t h c a p i l l a r y - c o n c e n t r a t i o n ( l o g [ . c~. c~ i n p M ) . C u r v e I , is
a theoretical extraction E for Met-ENK and Leu-ENK as a
function of c~ (see parts A and B in the calculations and results
section): curves If, Ill, IV and V are glutathione, carnosine
(z-MSH and MIF. respectively. { T m a x ) f = 0.46 x 10 3 pmol/g's
and /~, 0. 35 nM have been used. The exper i ment al points arc
indicated by ) , x, O. '~. , , ,/~, ~ and ~\ for Met-
ENK, Lcu-ENK, glutathione, carm)sine, c~-MSH, MIF, AVP,
dE, ~zE and/4ELL respectively.
211
[D-Al a2-14C-homoargg]endorphi n) and a E ( a-
[D-Al a2-14C-homoarg]endorphi n) in t he cal cul a-
tions and results sect i on. Our t heor et i cal est i mat es
of ext ract i ons are 2. 3, 2. 3, 0. 3, 0. 8, 3. 5, 2.5, 1.6,
2.5, 1.5 and 0. 3% (see Tabl e IV) for Me t - ENK, Le u -
ENK, gl ut at hi one, car nosi ne, (z-MSH, MI F, AVP,
t i LT, t i E and a E, respect i vel y, due t o simple diffu-
sion al one.
Explicitly, t he cal cul at ed E due t o simple di ffusi on
al one f or Me t - E NK is 2. 3% whi ch is much l ower
t han t he val ue of 12 + 1% (cal cul at ed f r om t he ex-
per i ment al BUI = 15% obs er ved by Kastin et al.H).
The value f or E f or Me t - E NK, namel y 3% quot ed
by Cor nf or d et al.S appear s t o be f or hi gher concen-
t rat i on c; ( 21- 270 nM) t han t hat (0.12 nM) used by
Kastin et al.~4 so t hat the ext ract i on value for Me t -
ENK is high for low concent r at i on and low for high
concent r at i on. This is t he phe nome na anal ogous t o
t he sat urat i on effect l eadi ng t o t he specul at i on of t he
domi nance of car r i er - medi at ed process f or t he t rans-
port of Me t - E NK. Similar assert i on has also been
made f or ot her pept i des by pl ot t i ng t he left and right
hand sides of Eqn. 16 as a f unct i on of l og> c~ in
Fig. 2 as di scussed in par t K above.
Since t he E val ues f r om si mpl e di ffusi on are t oo
low t o explain t he exper i ment al dat a, we have calcu-
lated E not onl y f r om si mpl e di ffusi on but also f r om
t he combi ned simple di ffusi on and car r i er - medi at ed
facilitated diffusion. The ki net i c const ant s, t he recip-
rocal of t he affinity of pept i de t o its carri er K, and
maxi mal rat e of t r anspor t (T . . . . )~, which are needed
t o cal cul at e E, have been est i mat ed by an i ndi rect
met hod as expl ai ned above. The val ues we have ob-
t ai ned are (Tm,,x)f = 0.46 x 10 ~ p mo l / g s and K r =
0.35 nM appr opr i at e t o explain all t he avai l abl e ex-
per i ment al dat a f or Me t - E NK, L e u - E NK, gl ut a-
t hi one, car nosi ne, a - MSH and MI F. For expl ai ni ng
t he dat a for AVP, t i LT, # E and (hE we have deduced
(Tm~x)f = 10 x 10 s pmol / g-s and K, = 7 nM. This dif-
ference in (Tm~,)f and K~ val ues f or t he t wo gr oups of
pept i des appear s t o be part l y due t o t he di fference in
t he nat ur e of pept i des r equi r i ng di f f er ent carri ers and
part l y due t o t he uncer t ai nt i es in t he exper i ment al
dat a. Tabl es XI and XI I compi l e t he cal cul at ed E
values f or vari ous concent r at i ons of t he pept i des
al ong with t he obser ved values.
Incl udi ng t he exper i ment al uncer t ai nt i es in t he
measur ed ext r act i ons and uncer t ai nt i es in P~ val ues,
212
we have f ound l ar ge uncer t ai nt i es in (Tmax) f and K t so
t hat (Tmax) f = (0. 46 _+ 0. 29) x 10 -3 pmol / g. s and K t =
0.35 + 0.31 nM f or Me t - E NK, L e u - E NK, gl ut a-
t hi one, car nosi ne, a - MSH and MI F and (Tm~xh =
(15 + 15) X 10 -3 pmol / g' s and K t = (11 + 11) nM f or
t he AVP, t i LT, t i e and ctE.
Re ma r ka bl y, it is possi bl e t o expl ai n all avai l abl e
exper i ment al dat a f or di f f er ent pept i des by assumi ng
onl y t wo sets of val ues of (Tmax) f and K t. In fact, one
may expect di f f er ent (Tm:,x)f and Kt f or di f f er ent pep-
tides, par t i cul ar l y, because di f f er ent car r i er s may be
i nvol ved wi t h di f f er ent pept i des. Howe ve r , owi ng t o
t he l arge exper i ment al uncer t ai nt i es in t he obs er ved
E val ues, at pr es ent it is not possi bl e t o make out dif-
f er ences in t he (Tmax) f and Kr val ues f or var i ous pep-
tides. It coul d however be r e ma r ke d t hat , if t he na-
t ur e of all pept i des in a gr oup (as well as t he nat ur e of
carri ers i nvol ved in t he gr oup) is t he same, a single
set of (Tmax) f and K, val ues is per t i nent t o t hat gr oup.
Knowi ng t he val ues of t he par amet er s i nvol ved in
t he expr essi on f or E ( Eqn. 1) we have pl ot t ed t he
cal cul at ed E as a f unct i on of t he concent r at i on c; in
t he br ai n- capi l l ar y and c ompa r e d wi t h t he obs er ved
E val ues (Fig. 3) f or var i ous pept i des, Me t - E NK
etc. (usi ng t he mos t appr opr i at e val ues (Tmax)f = 0. 46
t 0 -3 pmol / g' s , Kr = 0. 35 nM and t he P~ val ues f r om
Tabl e I V) . One not es t hat t he exper i ment al E val ues
can be expl ai ned by our pr esent t heor et i cal t r eat -
ment . Al s o, f or c~ < < K t t he E val ues are hi gh and do
not decr ease fast wi t h c; as expect ed because pept i dc
mol ecul es havi ng l ow concent r at i on c; e nc ount e r
mor e t han suffi ci ent n u mb e r of car r i er uni t s facilitat-
ing t he t r ans por t at i on. Near c; ~ Kr t he ext r act i ons
st art decr easi ng ver y fast i ndi cat i ng t hat mor e and
mor e carri ers are i nvol ved exhaust i vel y f or t he t rans-
por t of pept i des as c; i ncreases. For hi gher concen-
t r at i ons, c~ > > Kr, t he ext r act i on becomes al most in-
dependent of t he concent r at i on of pept i des wi t h t he
as ympt ot i c val ue appr oachi ng t he one f r om si mpl e
di ffusi on. Thi s signifies t hat t he carri ers i nvol ved are
sat ur at ed at hi gh concent r at i ons t r ans por t i ng t he
pept i des wi t h ma xi mum capaci t y.
Fig. 3 shows t he shar p var i at i on of E vs logl0 c; in
t he r ange 102 < c; < 104 (in pM) . Thi s is i nt er est i ng as
it can be used t o ext r act i nf or mat i on as t o t he pept i de
concent r at i on t hat shoul d be pr es ent nor mal l y in t he
br ai n- capi l l ar y f or t he effi ci ent wor ki ng of t he BBB
in nat ur e. Since t he var i at i on of t he ext r act i on with
concent r at i on is large near t he poi nt of inflccuor~
(Fig. 3) we deduce t he nor mal c onc e mr a t i on ~: o[ a
pept i de f r om t he expr essi on
[ J E l
. . . . y =( ) (18)
[d(l og, o co)' ] c~ = C:
whi ch wi t h Eqn. 1 gives,
- 2F + Kr 1+ 2FK, (19)
(Tmax)l
- K t f or 2~FK~- <<1 12t/)
Usi ng (Tmax) f = 0. 46x l() -~ pmol / g' s and K; = 0,35
nM f or Me t - E NK et c. . and F = 9 . 3 3 x t 0 -3 cm3/~'~
we obt ai n
(Tm"x)' - 0.07 << 1
2 F Kt
which means t hat e ~ K r = 0.35 nM and. hence, t he
nor mal concent r at i on of t he pept i des in t he br ai n is c,,
~- Ee ~ EK, ~ ( 8%) (0.35 n M) = 0. 03 nM. The im-
por t ant i nf or mat i on whi ch we have deduced, name-
ly, e ~- K t is usual l y t aken f or gr ant ed and const i t ut es
a good appr oxi mat i on, as poi nt ed out by Gr een-
berg ~l, t hough no valid j ust i fi cat i on f or it appear s t o
have been given previ ousl y.
As i nferred by Gr e e nbe r g e~ al.~2 f r om synapt o-
some exper i ment s t he pept i des ct -MSH. MI F and
AVP do not have specific bi ndi ng in t he maj or re-
gions, f or ebr ai n, cer ebr al cor t ex and hypot hat amus ,
of the brai n. However . it is known ~:,2~ t hat pept i des
exert effect (such as pr oduc e d bv ne ur omodul a t or s
or opi at i c effect , et c. ) in t he cent r al ner vous syst em
i ndi cat i ng t he exi st ence of s ome r egi ons of cent ral
ner vous syst em ( CNS) wher e pept i des may have spe-
cific bindings~L The possi bl e areas II f or specific bind-
ings of pept i des ar e l ocus coer ul eus, r aphe nucl eus,
br ai nst em ret i cul ar f or mat i on, t hal amus and limbic
system. In t hese par t i cul ar ar eas~ concent r at i on of
pept i des may be hi gh even t hough t he t ot al a mount m
t he whol e br ai n- vol ume may be muc h l ower.
It coul d be possi bl e t hat t he pept i des n do not get
met abol i zed in t he non-speci fi c regi ons of t he br ai n
aft er i nj ect i on so t hat t hey may st ay in t he neur onal
cel l - membr anes (in br ai n) f or 2--3 r ai n (unl i ke wat er
for which this t i me is in seconds) and subsequent l y
get r el eased f r om t he cel l - membr ane and r et ur n t o
bl ood. Thi s specul at i on seems valid as acut e i nj ect i on
of some peptides cures l: Parkinsonism only for a
short duration after which it (Parkinsonism) comes
back to its original symptom 11. This implies that for
gaining continuous pharmacological effect one
should infuse the peptides continuously rather than
administer acutely. This can explain why the acute
peptide administration in some experiments (intra-
peritoneally or intraventricularly) does not produce
significant pharmacological effects.
Rapoport et al.:l have concluded that the trans-
port of peptides across blood-brain barrier (BBB) is
a function of the solubility of peptides in BBB com-
pared to aqueous medium. According to our present
investigation, the transport of peptides is mainly due
to the carrier-mediated facilitated diffusion: the sim-
ple diffusion, though dependent on the solubility of
the peptides in BBB, contributes only about 1()-2()/( ,
(Table IV) of the total extraction.
As for the actual carrier-mediated process the pep-
tide-carrier complex (activated complex) may be
conceived of diffusing (or rotating in certain cases of
transport - - not of present concern) in BBB from
blood-side to brain-side, relieving the peptide on the
brain-side and subsequently returning to blood-side
to repeat the process. Greenberg et a1.12 and Kastin
et al.l~ have observed that peptides cross BBB read-
ily, as opposed to conclusion of Cornford et al. 5 who
obtained low extractions of peptides. This apparent
contradiction can be resolved easily in view of our
present theoretical calculations (Fig. 3) which pre-
dict that at low concentrations the extraction is high
and at high concentration the extraction is low. In
fact, if c~ ~< K r, one expects to observe high E as
against low E if c~ ~> K,. Furthermore, the order of
magnitude of K, (~< 22 nM) conjectured by Cornford et
al.5 is consistent with our estimates of K t. The varia-
tions of E with log m ca for various peptides given in
Fig. 3 are important for predicting the E values for
any concentration of peptides.
CONCLUSION
In the present paper we have studies comparative-
ly the simple diffusion (of peptides) which depends
on the solubility of peptides in lipids, and the carrier-
mediated facilitated diffusion to estimate the extrac-
tion (Eqn. I ) of peptides across blood-brain barrier
(BBB).
213
The extraction due to simple diffusion depends on
the permeability P~ of BBB to peptides which has
been expressed in terms of the partition coefficient f
(Eqns. 12 and 15) representing the solubility of a
peptide in BBB compared to water. The partition co-
efficient f and permeability P~ have been calculated
above for various peptides and fall in thc range 7
10 -a~<f~<212x 10 4andl 0 -7~<P~< 14 11) 7cm/s
(Table IV). The P~ values along with the data in
Table II have been used for estimating extractions E
due to simple diffusion alone for Met -ENK, Leu-
ENK, glutathione, carnosine, a-MSH, MIF, AVP,
tiLT (fi-[D-Ala2-14C-homoargg]endorphin), [;~E (fi-
[D-AlaZ-laC-homoarg9]endorphin) and c~E ((z-
[D-Alae-14C-homoarg3]endorphin). The E values
due to simple diffusion alone come out to be in the
range of 0.3 to 3.5% (Table IV) which are significant-
ly lower than the corresponding observed values
which approximately vary from 0.4 to 12% (Tables
IV, XI and XI1) necessitating the investigation of
the other possible mechanism, the carrier-mediated
facilitated diffusion.
To obtain E from carrier-mediated facilitated dif-
fusion we have used our expression deduced using ac-
tivated complex model, which requires information
as to the rate of transport (Tm~,~)t and the reciprocal
of the affinity of a peptide to its carrier (Kt). From the
present theoretical treatment we have been able to
determine (Tm~,)) and K~ values in the calculations
and results section which explain the experimental
extractions for various peptides (Tables XI and XiI,
Figs. 2 and 3). We have obtained (T ...... )~ ~ 0.46
10 ~ pmol/g's and K~ = 0.35 mM for Met -ENK, Leu-
ENK, glutathione, carnosine, (z-MSH and MIF
and ( T m a x ) t ~ 10 x 10 3 pmol/g' s and K, ~ 7 nM for
AVP, t i LT, t i e and aE.
In addition, we have studied the variation of ex-
traction with concentration of peptides (Figs. 2 and 3
and Tables XI and Xll) in brain-capillary and have
concluded that the extraction decreases with increase
in the peptide concentration approaching to a small
constant value for large concentrations, characteris-
tic of a facilitated diffusion process with saturation ef-
fects. In contrast, the simple diffusion does not lead
to any concentration dependence of the transport of
peptides across BBB.
We have found that the carrier-mediated facili-
tated diffusion is the dominant mechanism which ex-
214
pl ai ns t he o b s e r v e d t r a n s p o r t o f p e p t i d e s a c r os s
BBB.
It has b e e n i n d i c a t e d t ha t t he p r e s e n t t r e a t me n t is
i mp o r t a n t f or u n d e r s t a n d i n g t he bi ol ogi c a l a nd ne u-
r o p h a r ma c o l o g i c a l e f f e c t s of pe pt i de s . We h a v e b e e n
a bl e t o d e r i v e a g e n e r a l e x p r e s s i o n f or t he e x p e c t e d
n o r ma l c a pi l l a r y c o n c e n t r a t i o n ~: o f a p e p t i d e
( Eq n . 19) i n t e r ms o f K t by i n v o k i n g a ma t h e ma t i c a l
c o n d i t i o n a p p r o p r i a t e t o o p t i mu m e f f i c i e nc y o f a nat -
ur al s ys t e m.
Fo r t h e p e p t i d e s i n v e s t i g a t e d we h a v e d e d u c e d an
i mp o r t a n t r e s ul t t ha t e = K t ( Eq n . 20). I n a d d i t i o n ,
we h a v e di s c us s e d br i e f l y t he n o n - s p e c i f i c a nd s pe c i f -
ic bi ndi ngs o f p e p t i d e s in t h e c e n t r a l n e r v o u s s ys t e m
in t he di s c us s i on s e c t i on whi c h l e a d us t o c o n c l u d e
t hat , in o r d e r t o h a v e mo r e s i gni f i c a nt a nd l o n g e r
p h a r ma c o l o g i c a l e f f e c t s c o mp a r e d t o t r ansi en~ c[-
f ect s of a c u t e a d mi n i s t r a t i o n , o n e nt ust i nf us e ~he
p e p t i d e s c o n t i n u o u s l y .
Th e p r e s e n t t h e o r e t i c a l mo d e l c o u l d ve r y wel l b e
e x t e n d e d wi t h o u t f u r t h e r mo d i f i c a t i o n s t o o b t a i n ex-
t r a c t i ons o r br a i n u p t a k e s f or s u b s t a n c e s s uch as am~-
no aci ds , h e x o s e s , ur e a , a mi n e s a nd o t h e r mo l e c u l e s
ha vi ng s i mi l a r t r a n s p o r t p r o p e r t i e s .
ACKNOWLEDGEMENTS
Thi s wo r k has b e e n s u p p o r t e d in pa r t by t he Re -
s e a r c h Bo a r d of t he Un i v e r s i t y o f I l l i noi s at Ch i c a g o
u n d e r Bi o me d i c a l Re s e a r c h Pr o j e c t s . Th e a u t h o r s
a r e t h a n k f u l t o Pr o f e s s o r R. Gr e e n b e r g f or v a l u a b l e
di s cus s i ons .
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