Absorption-promoting adjuvants: enhancing action on rectal absorption

advanced

drug delivery reviews Advanced Drug Delivery

Toshiaki of

28 (1997)

2055228

adjuvants: enhancing action on rectal absorption

Absorption-promoting Drparttnent

Review

Nishihata”,

Pharntuc~uticcrl Received 2X

Chemistry.

J. Howard Rytting The Univursi~

of

Kmscrs,

Kunsas,

USA

February 1996; accepted 28 June 1997

Abstract Studies of absorption-promoting adjuvants used for the modification of the barrier function of the rectal membrane are interesting for biochemical research as well as providing a basis for the development of new formulations of poorly absorbed drugs such as some moderately large water soluble drugs and peptides. Compounds such as chelating agents and sulthydryl depleters increase the rectal absorption of drugs through the paracellular route and transcellular route, respectively, by modifying the barrier function of each route. Salicylate, its derivatives and fatty acids also increase rectal drug absorption through both routes. Lectin increases rectal absorption of drugs by inducing microvillous infusion. With respect to lormulation development, ampicillin suppositories incorporating sodium caprylate as an absorption promoting adjuvant were confirmed clinically to be an effective formulation, and are a marketed product in Japan. In human trials, administration of 0 1997 Elsevier Science insulin in a combination with salicylate inhibited postprandial hyperglycemia in diabetic patients. B.V. Keyvordsc

antagonist;

Rectal absorption; Absorption-promoting adjuvant; Enamine Chelating agent; Sulfhydryl depleter; Lectin; Insulin

derivatives;

Salicylate;

Fatty acid; Calmodulin

contents I. Introduction ............................................................................................................................................................................ 2. Adjuvants for promoting rectal absorption ................................................................................................................................. 3. Enamine derivatives as ahsorption-promoting adjuvants .............................................................................................................

206 207 209

3. I. Chemistry of amino acid enamines of ethylacetoacetate ...................................................................................................... 3.2. Enhancement action of amino acid cnaminea of ethylacetoacetate ........................................................................................ 3.3. Enhancement mechanism of enamines ............................................................................................................................... 3.4. Use of enamine derivatives for medication or research as absorption-promotmg adluvants for rectal dehvery ......................... 4. Snhcylate and its derivatives .................................................................................................................................................... 4. I Adjrwent action of salicylate and its analogues.. .................................................................................................................

209 209 212 212 212 212

4.2. Enhancement mechanism of salicylate and SMSA .............................................................................................................. 214 4.3. Increased lymphatic uptake by coadministration of salicylate .............................................................................................. 217 4.4. Safety zthpect .................................................................................................................................................................. 218 4.5. Usefulnw of salicylate as an adjuvant for promoting rectal insulin delivery.. ....................................................................... 5. Fatty acids .............................................................................................................................................................................. 5 I Ahsorption promoting action of fatty acids and a proposed mechanism ................................................................................

218

5.2. Transport route enhanced by fatty acids ............................................................................................................................. 6. Others ...................................................................................................................................................................................

219

“Corresponding

author, Santen Pharmaceutical

Higashiyodogawa-ku.

Osaka 533, Japan. Tel.:

Ol69-409X/97/$32.00 P/I

SOlhY-409X(97)00073-2

0

Co. Ltd., Quality Control and Quality Assurance Division, 9.19,

+ XI 6 32174.56: fax:

+ XI 6 3217093.

1997 Elsevier Science B.V. All right< reserved.

218 218 220

Shimoshlnjo 3-chome,

0. I. Calmodulin

antagomht\

6.2. Strong chelating

6.1. Sulthydryl 6.4. Lectin 7. Formulation

220

agent

221

depleter _.....................

__....._...............,.,........ development

.._........................

223 223

of Insulm wppoGtorie\

8. Human trials

,..

X. I. Ampicillin

X.1. Insulin

221

suppository

221

[.55-.5X] ..,...

22‘l 22.5

wppositoq

References ,.. .,..

.._......

.,....

1. Introduction Although rectal dosage forms are not common because of cultural and psychological biases, there are several advantages to administration by the rectal route. For example, because the absorption site is near the administration site. 1. rapid absorption with a rapid increase in plasma drug levels can be achieved, 2. formulations can be readily prepared to provide desired release characteristics, and 3. methods to maintain high concentrations of the drug and additives at the absorption site are possible. These advantages for rectal dosing require devices or formulations with specific features to give the desired drug delivery system. Peptides and many other hydrophilic drugs are primarily developed as parenteral formulations. because of poor bioavailability after oral dosing. Pharmaceutical houses have generally attempted to modify the molecule of interest in order to achieve oral absorption. Although this approach has often been fruitful. there remains a host of drug entities that must be injected for reliable therapy. Two problems are associated with the oral absorption of these kinds of drugs. Firstly, most peptide drugs and some antibiotics are subject to chemical breakdown in either the stomach or the enzymatic milieu of the small intestine. If the target drugs are degraded before absorption can occur, then oral dosage forms are not usually feasible. Secondly, most peptide drugs and some antibiotics are simply absorbed too slowly to provide useful plasma levels for medication after oral administration. The nature of the mucosal barrier of the GI tract is such that many foreign moleculex - especially those that are large and water-soluble - are not well absorbed. Thus. in

226

order to devise an oral dosage form for such therapeutic agents, one must protect the drug from enzymatic degradation (in some cases) and simultaneously overcome the impermeable nature of the mucosal barrier. Some researchers have circumvented the problem of enzymatic degradation by concentrating on absorption sites that are free of digestive enzymes. The more popular of these administration routes have been the nasal and rectal mucosa. Both of these potentially drug-absorbing areas lack large concentrations of digestive enzymes but, nevertheless, maintain a selective barrier to the absorption of many drugs. The second problem, that of increasing the permeability of the target mucosa, has been approached by identifying permeation enhancers or absorption adjuvants. Traditional examples of these types of adjuvants are surface active agents such as synthetic or semi-synthetic surfactants [l-3 1 and bile salts. Rectal absorption of a drug is dependent on several drug characteristics such as partition coefticient and molecular size as has also been observed for absorption from the small intestine. Typical factors that have been identified for poor absorption from the intestinal (including rectum) route of administration are listed below: I 2. 3. 4.

small partition coefticient. large molecular size, charge, and high capability of hydrogen

bond formation.

To improve intestinal/rectal absorption absorbed drugs, three primary methods utilized:

of poorly are often

I. chemical modification - primarily used to increase the partition coefficient and decrease hy-

T. Nishihata,

.I. Hoard

Rwting

I Advanced

drogen bond formation to improve affinity to the membrane. This technique is also used to increase the solubility of very poorly aqueous soluble drugs to improve dissolution; formulation modification primarily used for poorly aqueous soluble drugs to improve the dissolution step. The development of an insulin suppository involved a combined technique of formulation modification and modification of the barrier system: modification of the barrier function of the rectal mucosal membrane using absorption-promoting adjuvants. As an example of chemical modification [4,5], there are marketed products of antibiotics which are modified to increase their partition coefficients. For some peptides, a technique for reducing the hydrogen bonding capability of the molecule has also been investigated. For formulation modification, approaches to maintain a high concentration of the drug on the intestinal epithelial surface by using a lipid vehicle for less aqueous soluble drugs have been suggested. In this chapter, the use of absorption-promoting adjuvants to modify the barrier system of the rectal mucosa with respect to poorly absorbable drugs having hydrophilic characteristics or large molecular weight is discussed.

2. Adjuvants

for promoting

rectal absorption

Transport from the rectal epithelium primarily involves two transport routes, the transcellular route and the paracellular route 161. An uptake mechanism which depends on lipophilicity is involved in a typical transcellular transport route. Active transport for amino acids, carrier-mediated transport for betalactam antibiotics and dipeptides [7,8], pinocytosis [9] and microvilli fusion [lo] are also involved in the transcellular transport system. The paracellular transport mechanism implies that drugs diffuse through a space between epithelial cells. To estimate the enhancing action of absorptionpromoting adjuvants on transport routes, a simple study [ 111 to measure the dependence of the transport rate on molecular weight was conducted by simultaneous administration of cefmetazole (CMZ,

Drug

Deliwr:y

Reviews 28 (1997)

205-228

207

M,: 47 1) and [AUS’.‘]-eel calcitonin (ECT, M,V: 3363). It was reported that strong chelating compounds such as EDTA increased intestinal mucosal permeability by depleting calcium ions from the area of the tight junctions, thereby opening the normally tight junctions as demonstrated by electron microscopy [ 121. Because strong chelating agents allowed transmucosal transport of even macromolecular compounds, the action of several different adjuvants were compared to that of strong chelating agents. It was demonstrated that permeation of substances through cellular junctions, loosened by strong chelating agents, seems to be dependent on the diffusion coefficient of the drug which is determined primarily by its molecular size and/or shape [ 121. Furthermore, the transport ratio of each substance via a paracellular route should be constant when molecular size is less than that of albumin (M,+: -75 000). The absorption of the compounds studied was expressed as the absorption clearance determined from plasma concentrations (A,,, for the clearance of ECT and A,,,,, for the clearance of CMZ in Fig. 1). As shown in Fig. IB’, the ratio of A,,,,, against A,,, (A,,,,,/A,c,) obtained after treatment with EDTA is constant even with changing concentrations of EDTA. This result indicated that the enhanced absorption of CMZ and ECT is dependent on their diffusion coefficients through loosened cellular junctions as described earlier. A constant ratio was also obtained when the drugs were treated with phenothiazine as a calmodulin inhibitor (Fig. IC). Diethyl maleate (DEM) enhanced only CMZ absorption (Fig. 1F) and the ratio of A,,,/A,,, increased in response to an increase in the concentration of DEM in a microenema; i.e. DEM seems to increase the transcellular route for relatively small compounds but does not loosen the cellular junction. When treated with salicylate (Fig. IE) or diethyl ethylenemalonate (DEEMM) (Fig. ID). only CMZ absorption was enhanced at low concentrations of the adjuvants. However, the absorption ratio of Arm,,/ Ato obtained at high concentration of salicylate or DEEMM became similar to that found with treatment by EDTA. Table 1 summarizes the improved transport route by each mechanism. Table 2 summarizes the classifications of absorption-promoting adjuvants which have been reported for enhancing rectal absorption of poorly absorbed drugs such as antibiotics and peptide drugs.

(C)

(8)

_

7r

60

l

.

0 z , c .-I I P N -

1 /’ 3

40

20

,” x H

/

0

E

0 15 ,

30

0 15

30

0.05

0.1 7.5 12.5

25

50 100

50 0.025 0.05

0.1 7.5 12.5

25

50 100

50 0.025

Concn. of adjuvant Fig.

I. Effect

of vxy~ng

and eel calcitonin Table

400 1.25

2.5 3.75

2.5

3.75

in microenema, mu

of each adjutant 111the m~rornema on the ahsxption clearance of cefmeta4e (A<,,,,. 0 and 0) (A--F) and on the rat10 ot .Aj,,,, /AL<, [A’- F’) determmrcl hy the AUC method for 13-Omin represented in

n)

represent

coadministrution.

and F and F’. DEM.

200

400 1.25

the concentration

(A,~,, r3 and

I. Open symbols

cysteamine

200

the result

without

qaanune

coadnlinistratlon

;md closed

symbols

reprerent

the result with

5 mM

A and A’, IN adjuwnt;

Each calur

K and B’. EDTA: C and C’. TEP: D and D’. DEEMM: E and E’, sodium sdicylnte; represents the mrenfS.D.. II .a (1. (a) [’ ,‘. 0.05 vs. no adjuwnt: (b) P < 0.05 vs. no cysteamine (Student’s

t-te\t1.

Table

I

ClasGtication Action I.

of absorption-protllotinp

adjuvanth

te\trd

in

1I I I Transport

style Enhancing

rectal absorption

of both low and high molecule aeieht

compounds

route

Adjuvants

Paracellulal

Strong chelating

route

agents, phenothiazinea

Transcellula~-

Diethyl-maleate

in a constant

Iratio of ahwrption 1. 3.

Only

enhancing

rectal abso!-ption

of low molecular

weight compounds

route

Absorption

changes ft-om Z

Combination

Salicylate.

routes

fatty

to

I

atylr

hy an increaw

concentwtion

in adjuvnnt

acids

DEEMM.

209

Table 2 Classifcatlon

of agents enhancing

rectal absorption

of drugs References

Classification Enamine Sulicylate

(3)

Calmodulin

f-1) (S) (6)

Phosphate derivative5 Strong chelating

(71

Fatty acid

(8)

Agents inducing

(9)

Lectinh

3. Enamine adjuvants

[14-18.62-651 [I 1.19-21.23-26.29-31.53.53.59.65-67.70~

derivatives

(I) (2)

and its derivatives

[ I I .49.6 I]

inhibitors and xetoacetate

169,721

derivatives

[ I I ,65,66.70]

agents

[I~??,701

Surfactants

derivatives

[29.36-4X47,48.5 non-protein

thiol

as absorption-promoting

There have been many investigations of the action of amino acid enamines (phenylalanine and phenylglycine) of beta-diketones (ethylacetoacetate) for enhancing rectal absorption as shown in Table 2. Since enamine derivatives of amino acids with betadiketones such as ethylacetoacetate can be easily prepared and the lipophilicity increases by the preparation of enamine derivatives, there are a couple of reports of the use of enamines for prodrug development of beta-lactam antibiotics [ 131. Because of the rapid absorption of enamine and the chelating capability of ethylacetoacetate and enamine derivatives, research to investigate the feasibility of enamine derivatives of amino acids as absorption-promoting adjuvants has started [14].

.?. 1. Chemistry of amino ucid enamines rth?lac,Ptocrcetcrtc

[ I I .46.52.60,65.68.7 [toI

loss

qf

It has been reported that the formation of enamines in aqueous solution is very difficult because the dehydration step for the formation of the enamine is the rate determining step in the aqueous phase. However, the amino acid enamine of ethylacetoacetate is easily formed even in aqueous solution under alkaline conditions as shown in Fig. 2 [ 151, and the formation constant (K) of the enamine derivatives of amino acids can be calculated according to the equation described in Fig. 2 [26]. As shown in Table 3, K values for enamine formation of each amino acid with ethylacetoacetate in aqueous solution increase with an increase in the pH of the solution. Enamine formation of each amino acid with

ethylacetoacetate is also related amino acid (Fig. 3) [ 161. 3.2. Enhcmcement

action

I ,SS-S8

]

I]

to the pK,, of the

qf amino acid emzninrs

of eth_vlacetoucetrlte Table 2 lists reports of the absorption-promoting action of enamine derivatives. The enhancing action of enamine derivatives on rat rectal absorption of CMZ (a hydrophilic antibiotic) is dependent on enamine formation capability as shown in Fig. 3. The action of the enamines appeared only for short periods, due to rapid absorption of the enamines and degradation at the site of action (hydrolysis in aqueous phase) [ 151. With respect to the adjuvant action of enamine derivatives for rectal absorption, there are many poorly absorbed drugs that have been tested such as beta-lactam antibiotics, peptides including insulin and ECT, and other high molecular weight compounds including inulin. As shown in Fig. 4 [17,18], an increase in serum insulin after rectal administration of an insulin suppository containing an enamine occurred rapidly, but the disappearance of serum insulin also occurred rapidly in depancreatized dogs. The disappearance of insulin after dosing with an insulin suppository is similar to that found following i.v. administration of insulin. When an enamine suppository without insulin was administered at 20 min after dosing with an insulin suppository containing enamine, an increase in serum insulin concentration was observed after the second administration of the enamine suppository. This result indicates that the time period during which enamines enhance rectal insulin absorption is very short.

R-NH-GCH-R

S

R-NH-C-CH,-R

kH,

step I’

ZH,

R-NH-C=CH-R’

+

H+

R-NH+=GCH,-R

-;t K

?H,

step 1

bH,

IrH’I

Fl

OH R-NH+=C-CH,-R’

+

H,O

zy-

CH,

R-NH,&CH,R’

step 2

t

[D’H”:,

UH+l OH

0-

R-NH,+&CH,-R

R-NH,*&Hz-R’

S K,

kH,

+

H+

step 3

i3Hs P’l

PH+I OR-NH,+i-CH,-R’

= K,

CH,

R-NH,

1

WI

u-1 -

H’

step 4

CH,-C=O kH,R’

P=l R-NH:

+

R-NH,+

-r

u-l

U-H+1

[El-cnamioe. [L)=pha~ythniac. K..-JE~W,,J~l~. IU=IY+ILH’I.

(hi)-cahyi

VW.

c. 2. Equilibrium Fiw

hetwern

enamine

K’-MWI.

K-K,K,KJ~~,-I~[H~~V(II-IMU

lor(K’/L)-PK-PH.

rt;-<r’bW*l. and its hydrolysis

products.

Table 3 Intrinsic

formation

constant”.

acids with ethylacetoacetate Amino

K. of enamines

of variouh

K

acid (pH) 73

8.5

9.0

Mean 15.15

43.6

46.30

45.36

45.32

Leu

40.29

42.84

43.32

42.52

42.24

Ala

42.31

38.8

32.52

39.6 I

40.x3

SS.96

51.89

53.12

Phe

I

8.0

GlY

Initial

54.27

I

SO.36

concentrations

of

amino

acid

and ethylacetoacetate

enamine were 0.01 M and the test solution “The

intrinsic

following

PH 8 h-‘1

amino

in aqueous solution

formation

equation

constant

(described

was determined in

the

ot

was incubated at 37°C. text)

by usmg the

log(KIK,>,,\

I

)=

PK,, ~ PH.

E

z Upon examining serum glucose levels after administering insulin suppositories containing enamines in depancreatized dogs, effective decreases in serum glucose levels were observed even at an insulin potency of 1.5 unit/kg. This strong absorption promoting action of enamine toward insulin

” Fig.

Obo* ”

”

a’ a

”

. 10.0 9.5 PJL of amino moiety of amino acid 3. Relationship

enhancement

with

of

the

extent

the PK,~ (from

of

cefmetazole

the Merck

index)

absorption (0)

of the

amino group of the amino acid and with the K,,,\ (A) of enamine formation Ala.

obtained

from Fig. 2. I. Phe: 2, Tyr: 3, Leu; 4, Gly: 5.

T. Nishiharu,

J. Hmwrd

Rwirzg

I Adwad

Drug

Delirvry

Rrviews

28 (1997)

20.%228

21 I

60C

666

,

0

I

1.0

2.0

Fig. 4. Upper: serum insulin (A) and glucose (B) concentration in depancreatized dogs as a function of time after administration of the insulin suppository at an insulin dose as follows: 5 U/kg (O), 2 U/kg (0) 1.S U/kg (A) and I U/kg (A). Experiments were carried out by cross-over using four depancreatized dogs. Each value represents the mean?S.D. Lower: serum insulin (A) and glucose (B) concentration m depancreatized dogs as a function of time after administration of the insulin suppository followed by the administration of the enamine suppository. Dose of insulin was as follows; 5 U/kg (O), 2 U/kg (0). I.5 U/kg (A) and I U/kg (A). Experiments were carried out by cross-over using four depancreatized dogs used in Fig. I The enamine suppository was administered 20 min after the administration of the insulin suppository. Each value represents the mean+S.D. (a) P
rectal absorption moting adjuvants

led to research in more detail.

of absorption-pro-

A detailed study of the mechanism for the adjuvant action of enamines has not yet been reported. Because enamines can enhance the rectal absorption of relatively large molecular weight compounds such as insulin and even a stronger action of enamine was observed for relatively small drugs. enamines belong to the same category with salicylate in Table I.

A primary concern of enamine derivatives for use in formulation clevelopment is the stability of the enamines themselves. In many cases. they undergo rapid hydrolysis at neutral or lower pH values and in suppository bases containing moisture [ IS]. Acute toxicity data in rats did not show any toxicity up to 1500 mg/kg and furthermore, rapid hydrolysis of enamines may reduce the probability of toxic effects. However, further drug safety studies will be required to use enamines as absorption-promoting adjutants. Since the environmental condition of the rectum can be easily controlled by the formulation, enamine derivatives may be more useful in prodrug development rather than as absorption-promoting adjuvants.

4. Salicylate and its derivatives There are many reports of the utility of salicylate and its derivatives as adjuvants in promoting rectal absorption of hydrophilic antibiotics and polypeptides, as shown in Table 2. The history of salicylic acid as a medication is more than one century and aspirin was initially developed as a prodrug of salicylate. Although aspirin itself has pharmacological action, the primary action after dosing aspirin, must be by salicylate as a metabolite because of the rapid degradation of aspirin to salicylate in the intestinal tract before absorption. It is also known that salicylate/aspirin may prevent heart attacks and the mechanism involved may be partly due to the

action of salicylate the cytosol [ 191.

in decreasing

free calcium

ion in

The enhancing action of salicylate is dependent on the concentration of salicylate at the site of action as shown in Fig. I; i.e. salicylate not only promotes rectal absorption of low molecular weight compounds. but salicylate also increases the rectal absorption of high molecular weight drugs [IO]. The enhancing action of salicylate against high molecular weight compounds has been demonstrated with insulin and dextran. Analogues of salicylate such as S-methoxysalicylate (SMSA) have also been demonstrated to act as rectal absorption-promoting adjuvant\. The uptake of trypan blue into cells has been used to monitor cell viability. Salicylate at a concentration of 1% in a microenema enhanced the permeability of rat rectal epithelial cells of the rectal lumen to trypan blue at pH 7.0, which closely approximates the natural pH of the rectal lumen. The salicylate-effect is reversible and occurs without substantial structural damage to the epithelial cells, as shown in Table 4 [ 201. Reversibility of the effect was clearly shown by exposing the rectal epithelium to salicylate, rinsing out the calicylate after IS min. and then demonstrating that the epithelium was impermeable to trypan blue. This finding indicates that compounds of low molecular weight enhanced by salicylate includes trypan blue. As shown in Table 5, SMSA increases the rectal absorption of antibiotics signiticantly in dogs when dosed as a suppository 121 1. Rectal bioavailability of antibiotic\ depends both on the concentration of the adjuvant and on the dosage form. A lipophilic suppository base seems to provide a satisfactory vehicle for the delivery of several antibiotic drugs resulting in good rectal bioavailability. The use of a lipophilic suppository base can easily maintain a high concentration of the adjuvant in the rectal compartment segment in comparison to an aqueous microenema, because salicylate easily dissolves when dosed as the sodium salt. As shown in Table 6, the enhancing action of SMSA against four antibiotics is stronger than that of salicylate. The stronger action of SMSA in comparison to salicylate

T. Nishiharu.

Table 4 Effects of sodium salicylate Treatment

J. Howrrrd

on surface

integrity

group

1

4. 5. 6.

I Advanced

Drug

Deliver?

and trypan blue permeability

2.16?1.42 I .46 i I .40

Control Trypan blue Salicylate Trypan blue + salicylate Phlortzm t trypan blue i salicylate Salicylate + rinse + trvnan blue

7.hl~3.01 45.02-+ 16.92

Revirwc\ 2X (1997)

in rectal mucosa

213

ZM-228

as determined

by light microscopy

pH 7.0

pH 4.8 7r Disrupted surface

I. 2.

Ryrtirlg

E

+++

pH 9.0

L

‘3%Disrupted surface

E

L

Disrupted surface

_

0.9 I 20.75 0.33t0.27

~ _

_

2.45 t I .09 4.OS~2.Sl

~

1+1

_

_

I .03-CO.38

~ +

+++

+++

19.3X? 13.92

1.76?1.13 22.68+4.09

+

1:

L

t+ (+)

t

t + -

+

+ +

_

L-1 +++

26.0224.19

+

2.41 20.97

Not done

l.66tl.36

+ +

t+

t+) +

2.61 -to.sx

t+

_

+ +I

(+)

Not done

+)

The “/r disrupted surface refers to the length of measured surface which was discontinuous as a percentage of the total distance measured. E and L refer to the epithelium and underlying lamina propria, and the column below the letters indicates the degree to which these regions showed trypan blue stain, The coding is as follows: + + + , intensely stained; + + , moderately stained; + , faintly stained; ~, unstained. The parentheses indicate that the tissue from one of the five rats showed the indicated difference in staining from the other four samples. The brackets indicate that the tissue from two of the five rats showed the indicated difference in staining. In all other cases the tissue from the tivse rats were stained in the same manner.

Table S Bioavailability administration

of four

Dose of drug tmg)

antibiotics

after

Dose of adjuvant

rectal

administration

from

(Witepsol

tmg)

0

G

Cefoxitin

300

compared

to intravenous

(%)

Cefmetazole

Gentamicin

0.3

I .4

IS0 300 200 300 I so

as base)

2

0

200

H-IS

Bioavailability” Penicillin

No adjuvant I50 300 S-Methoxysalicylate 7s 7s I.50 IS0 Salicylate I SO I SO I SO

suppository

49 74 80

was also reported for the enhancing action of small intestinal absorption of insulin in rats [22]. Because melting of a triglyceride base occurred rapidly, the release and dissolution of both the adjuvant and the antibiotics also occurred rapidly in the rectal cavity. Since the administration site is also the absorption site for rectal administration, enhancing effects appeared rapidly with T,,, at 30 min after rectal dosing in dogs. Based on this rapid increase in plasma antibiotic concentration, rectal dosage forms

31 54 44 Sl

37 48 49 49

30 36

2s 38 41

I8 30 44

II 2s 24

of these drugs may offer distinct advantages over a parenteral route. Both salicylate and SMSA also increase the rectal absorption of insulin, heparin, gastrin and pentagastrin administered in microenemas to rats [23,24]. It has also been reported that salicylate enhanced rectal absorption of dextran with molecular weights of more than 100 000 in rats [25]. Regarding rectal insulin delivery in healthy human trials, the insulin suppository containing sodium salicylate as an ad-

Table 6 Comparative bioavailability administration

of sodium penicillin

G. hod~um cefoxitin.

sodium cefmetazole and gentamlcm sulfate following

in four dogs (cross-over study) usin g sodium S-methoxysdicylate

Dose of drug (1112)

Dose of adjuvant (mp)

AK”

min

(pg.

Pemcillin

G

ml

rectal

as adjuvant

’ ) (comparative

bioavailabilityh ‘)

Cefoxitin

Cefnvswle

Gentamicin

I3SX~XO

IllXiXl

IS772

Intracenous injection 0

SO

Rectal suppository wtth Witepsol I SO

0

300

0

357ii.i Ii-

140

I5 as base 3Xx2

x2+17

1323 Yh-2-I

7S

75

240 + I I ( Ii

17_li76(X)

303_tSY( 13)

7S

I SO

4621-X3(24)

x2xi

I33(20)

61.5?68(25)

713542(lOY)

7.5

300

-136ih2(32)

IIOS~

12x27)

801 i65(33)

X48%162( 130)

I so

200

Yl2t-71(24)

IX045 lSY(22)

IS0

300

x9.3-t lO3(2-1,

20’)s t I hY( 2s

0.02 M phosphate buffer solution (pH = 7.4) or O.Y’;/ alme

I

16s3txO0~34)“

)

16.58i206(34)

mwoenema Il3il2

SX-tY

300

0

52% IO

7s

I so

11Y?l7(lI)

7s

300

2’)6?3’)(23,

I50

Iso

31215%131

330t3x(Y)

300

SXhL65(23,

656_tXY( 1X)

I so 4% felatin

71214 168?2O(Y)

IXY-t30(7)

201-t25(11)

333i.W

16)

micl-ocncm;i 6X

0

300 7.5

I so

7.5

200

7s

300

75

-100

Ii

1w+27

301 i2X( 1x1’ 1Xlt71(13) 1Y7~632Y)

6.5” IX

X3? IY 3o?tso(

IS)

-l3S_tX7(21)

3572 149(22)

382-i-61(23)

521247(Q)

1232.?53(36)

IS0

300

S63:Yl(21)

I so

300

715273(22)

_ 13xxt

645 i SO( I6 140(701

1

YXSi-7Y(24)

“ALIC = AUC 0 I:<>,I>,,,’uncertainties expresed as standard errors of the mean. “Numbers in parenthew = [AUC]p(Dose)a/[AUCla(Dose~p where a z adJu\ant absent. p = ndju\ant prewnt.

‘P *: 0.001

compared with Irectal Irdmmistrntion

“Experimental

in the absence of ad,ju\unt. Student’\ r-test.

number ~ 2.

‘Gentamicin. ‘ixperimental

number

X. two set\ of foul- dog\.

juvant seems to be a potential dosage form for clinical trials in comparison with an insulin suppository containing enamine as an adjuvant, since single administration of an insulin suppository containing sodium salicylate to healthy human subjects resulted in high serum IRI levels sufficient to lower serum glucose concentration signilicantly for a longer time than enamine-containing suppositories (Fig. 5) 1261. It was noted that serum insulin profiles as a function of time after administration of the insulin suppository containing sodium salicylate are close to those found after an oral glucose tolerance test in healthy adult Japanese. These findings indicate that the insulin suppository containing sodium salicylate is effective in diabetic patients, who have basic serum IRI levels ( IO-20 FUlml) under fasting conditions but have a pancreatic disorder with respect to insulin secretion

in response to high serum after a meal.

glucose

concentrations

In isolated rat intestinal epithelial cells. protein thiol loss and/or an increase in the cytosolic Cal’ concentration seems related to accelerated cell aggregation (Fig. 6) 1191. Since calmodulin antagonists inhibited the acceleration of cell aggregation, such aggregation may occur through a calmodulin-dependent mechanism. Because adsorption of aresenazo III to isolated epithelial cells was enhanced by 2,4dinitrophenol or DEM, and decreased when cell aggregation was inhibited by a calmodulin antagonist or salicylate, it is theorized that cell aggregation is

T. Nishihnfn,

_I. Hmwrd

(A)

Ryrring I Advnnwd

Dray

Delivery

Reviews 28 (1997)

205-2228

215

(B)

a

lotlb (A')

(B’ 1

a,a

7!

!3

2

I

0

0.5

1.0

1.5

2.0

Tine,

h

Fig. 5. Serum glucose concentration (A and B) and IRI levels (A’ and B’) in human subjects after administration of the insulin suppository containing either enamine (A and A’) or sodium salicylate (B and B’). A and A’: a cross-over study was carried out with the following three subjects; 24 yearsold and 60 kg,33 years old and 65 kg, and 36 years old and 67 kg. The code of insulin suppository was as follows: m, for Code-A2

(dose of insulin in human; 2 U/kg);

0,

for Code-E2

(2 U/kg);

and 0,

for additional administration of Code-E0

(containing

enamine alone) at 20 min after administration of Code-E2 (2 U/kg). B and B’: a cross-over study was carried out with the following three subjects; 23 years old and 54 kg, 26 years old and 58 kg, and 36 years old and 76 kg. The code of the insulin suppository was as follows; 0, for Code-S I

(I

U/kg);

0, for Code-S I .5 (I .5 U/kg);

and &

for Code-S2 (2 U/kg).

The dotted line in B’ represents the serum IRI levels

after an oral glucose tolerance test at a dose of SO g glucose in healthy human subjects

(

[73]). Each value represents the meantS.D.

(n = 3). (a) P i 0.05 vs. the value at zero time (Student’s f-test): (b) P < 0. I vs. the value at zero time.

I

I

I

I

0.6

I

0

4

8

12

16

0 Time

Fig. 6. Effect of various agents on the aggregation of isolatd

4

I

I

8

12

I 16

(min)

rat small mte\tmal epithelial cells. Aggregation was determined by a decrease

in absorbance of the wspenhion at 540 nm as a function of time. Experiments were performed at 37°C and in Ca’ +- and Mg”-free Krebs-Henseleit experiments. (A)

buffer. Cell suspensions of

0.

I.5

ml (2 mg of cell protem/ml)

were wed and the initial absorbance was 0.9% I .02 In all

no additive: 0, no additive in Kreba-Henheleit buffer: a.

I% bovine serum albumin: A, 500 mg/ml concanavalin A:

0, 25 mM arsemwo III. (B) a, SO mM W-7; OA SO mM W-7 chlorpromazme: huspensiona contained SO mM DNP): 0,

dithiothreitol. (E, F) (cell suspension contained 5 mM DEM): sahcylate: 0,

n,

SO mM sdicylate; 0. S mM dithiothreltol. (C, D) (all

no other additive; 0. SO mM W-7: A. SO mM chlorpromazine;

n,

50 mM salicylate: 0. 5 mM

0. no other additive: 0. SO mM W-7; A, SO mM chlorpromazine;

n,

SO mM

S mM dithiothreitol

induced by the appearance of specific components on the cell surface to which aresenazo III bind (Fig. 7) [ 191. Salicylate may inhibit cell aggregation by two possible mechanisms; i.e. salicylate may modify the cell surface function responsible for binding aresenazo III and/or inactivation of calmoduiin by decreasing the Ca’+ concentration in the cytosol [ 191. The action of salicylate in inhibiting cell aggregation may relate to the adjuvant action of salicylate in enhancing the paracellular transport route at high salicylate concentrations. The action of salicylate at high concentrations increases the paracellular route of rectal absorption. The intestinal absorption of salicylate itself is depen-

dent on the concentration; i.e. salicylate is transported primarily through epithelial cells at low concentrations, but salicylate is transported via the intercellular route through tight junctions at high concentrations which are reached at therapeutic doses 127,281. The enhancing action of salicylate at low concentration has not yet been discussed. As described above. salicylate at high concentrations acts directly on the cell surface Ca’+ or decreases cytosolic Ca’+ to induce inhibition of calmodulin action. Adsorption of salicylate on the plasma membrane has been reported by Kajii 1291 and Nishihata 1.701. Another series of experiments with beagle dogs

Arsenozo

III recovered 60

40

I

used the transmucosal electric potential as an indicator of increased ionic permeability. In this case, salicylate and SMSA were compared to lauryl sulfate with respect to both the extent of transmucosal potential reduction and duration of the effect. Lauryl sulfate caused a slow deterioration of the potential difference which was sustained for many hours. Salicylate and SMSA, however, caused an immediate and extensive reduction of the transmucosal potential difference which recovered much more rapidly. In fact, if the salicylate was washed out of the rectal compartment at a time when the effect was maximal, recovery of the original potential difference was achieved within 15 min.

in supernatont (‘1.) 80

I

I

I

I

I

I

I

I

I

w additive 50pM

SA

50pM

W-7

5opM 5mM

CPZ DTT

~OJJM DNP + none +50mM

SA

+~OJJM

W-7

t5OpMCPZ + 5 mM DTT 5mM + none +50

DEM

mM SA

+50 j&l w-7 +5OpM CPZ +5mM

DTT

I

I

The rectal absorption of pepleomycin in rats was increased significantly by co-administration with sodium SMSA. diclofenac and enamines. SMSA also increased lymphatic uptake of pepleomycin after rectal administration while diclofenac and enamine did not (Table 7) [31]. The enhanced lymphatic transport of pepleomycin when injected into rectal connective tissue with SMSA was also observed. The mechanism behind the enhancing action of SMSA on the lymphatic uptake of pepleomycin may be due to a suppressing action of SMSA on the vascular permeability to pepleomycin. Diclofenac increased the vascular permeability to pepleomycin as did bradykinin.

Fig. 7. Effect of various agents on the adsorption of arsenazo 111to isolated epithelial cells. Adsorption of arsenaao III was determined by measurement of the percent recovery of arsenazo III

in the

supernatant after I5 min (initial concentration of arsenazo III was 25 mM). Cells ( 12.5 mg

of cell protein/ml)

were suspended in the

medium containing various agents and the suspension was centrifuged to obtain the supematant. Concentration

of arsenazo 111

was measured by (absorbance at 575 nm)-(absorbance in the presence of 20 mM promazine: mean?S.D.

DTT.

EDTA.

dithiothreitol.

(II = 4-8).

animals. (a) P
at 685 nm)

SA. salicylate: CPZ, Each

value

chlor-

represents

the

‘II’ refers to batches of cells from different vs. values for cells with no additive: (b)

P < 0.05 vs. values for cells with no additive: (c) P < 0.01 vs. SO mM

DNP; (d) P CO.1

vs. values for cell with no additive; (e)

P < 0.01 v \. 5 inM DEM:

and (f) P < 0. I vs. 5 mM DEM.

Table I Bioavailability”

of pepleomycin sulfate (PEPS)

after rectal administration and accumulative amounts of PEPS in thoracic lymphatic duct

during 3 h after administration Administration

route

Ad,juv,ant

[AUC] O-3 h

[(B) x 1001

,ug h/ml

([AUCl,<~I IAW, ~1

Accumulative

(dose, mg/kg)

amount, c(S (B)

Dose X (A)]

30.481-5.23

I .o

15.021-2.46

0.15 0. IS

I.v. administration

(7~)

Rectal administration Diclofenac

(20)

30. I I 2.5.34

0.988

14.84i2.16

Sodium S-methoxysalicylate

(40)

10.73-t.1.s3

0.352

35.96i4.62

Enamine

(40)

0.117

3.96+

Dose of PEPS was IO mg/kg “Bioavailability

= [AUC],_,

3.572

I .03

I .02

I .04

0.34

for all experiments.

O-3 h X (Dose),

,

/[AUC],

\ . O-3 h X (Dose)r_,

curve of PEPS during 3 h after rectal and i.v. administration. respectively.

where [AUC],L,L and [AUC],

j

represent the area under the

4.4.

S@ty

aspect

There are a variety of experiments conducted to assess the cytological effects of salicylate and SMSA on the rectal mucosa of rats, including acute and chronic studies with 30 days of consecutive dosing. At the light microscopic level. adjuvant-treated tissue was not distinguishable from sham-treated and controls, and at the electron microscopic level, the only obvious effect of salicylate treatment at neutral pH, was a thinning of the glycocalyx coat on the epithelial cell membrane 1201. These experiments, when take together with the observation that aspirin suppositories have been successfully utilized for years in some countries. indicate that salicylate is probably not harmful when administered rectally as the sodium salt.

Historically, salicylate was widely used as an oral hypoglycemic agent as part of the treatment of diabetes mellitus during the latter half of the 19th century 132,333. In addition to the use of salicylate as an anti-inflammatory agent, it has an action in inhibiting gluconeogenesis in the liver 134.351. This was also found with SMSA. Salicylate and SMSA are found to inhibit metabolism from lactate to pyruvate in TCA cycles. Thus, coadministration of salicylate or SMSA with insulin in a suppository may induce a synergistic therapeutic effect as well as an absorption promoting effect.

5. Fatty acids Fatty acids are very common excipients used in formulation development of many types of dosage

Effect

of fatty XI&

Sodium

on rat rectal absorption

salt of fatty acid

of ampicillin

forms including oral and rectal formulations. There are many reports about the enhancing action of fatty acids on rectal and small intestinal absorption. As shown in Table 8, medium chain length fatty acids showed the most effective action as absorption-promoting adjuvants for ampicillin and hydrophilic antibiotics for rectal delivery 136).

It has been reported that the enhancing action of fatty acids is dependent on the partition coefficient [ 361: i.e. caprate. caprylate and lauryate with log partition coefficients of 3-6 against n-octanol have strong absorption promoting effects. The optimal partition coefficient was calculated at log P = 4.2. This action of fatty acids to enhance antibiotic absorption from the rectal compartment was also observed in the small intestine of rats (Fig. 8) 1371. This apparent correlation with partition coefficient indicates that the uptake of fatty acids into rectal tissue must be a key factor in their potency as absorption-promoting adjuvants as shown in Fig. 9. In the rat experiment using caprylate and its derivatives as absorption-promoting adjuvants, it has been suggested that the importance of uptake of the adjuvant into rectal mucosal tissue was supported by pharmacokinetic analysis [ 38,391. With respect to long chain fatty acids such as oleic acid, dosing of oleic acid itself does not cause significant enhancements for the rectal absorption of hydrophilic antibiotics, but when dosed in a mixed micelle formulation prepared with sodium taurocholate, significant absorption promoting action appeared I40 1. Muranishi et al. have proposed that even long chain fatty acids can promote rectal absorption whenever the fatty acids are taken up into epithelial cells 1411.

(ABPC)

ABPC

(‘,,,,,\(pCLg/nll)

AUC (pg

Hexnnoatr

3.7’0.8

4.O~O.X

Octanonte

12.51-0.9

IO. I -t I .o

DrcanoLLtr

19.31-3.1

IX

Dodecanoate

I I.Xi2.l

I I .ot

i

h/ml)

Bioavailability 20.1 50.x

I .c)

61.8

I.?

55.3

Myristate

s.0to.h

3.520.6

17.6

PaImitate

I .7?0.7

7.0+0.4

IO.1

2.3-to.1

7. I kO.7

IO.6

Control

(no xljuvant)

T. Nishihatu,

Carbon

J. Howard

Rytting

I Advmced

number

Fig. 8. Area under the curve (AK) of plasma cefoxitin concentration for I80 mitt following administration of sodium cefoxitin, IS mg kg ‘. into rat duodenum with fatty acids in the form of triglyceride (O), free acid (a) and sodium salt (A) at a dose of 250 mg kg- ‘. Administration of cefoxitin in preincubated medium of triglyceride with (0) or without (0) lipase in bile is also shown. The hatched area and dotted area represent the AUC of cefoxitin after administration of cefoxitin alone into rat duodenum and after cefoxitin intravenous administration, respectively. Each value represents the meantS.D. (11> 4).

x4

3

1000-

3 I

P f

8oo-

3

9 .E

P t t

Drug

D&very

Ret?ewbs 28 (1997)

219

20.5-228

reports of Kajii et al. [42], Tomita et al. and Sawada et al. [43-451 using a fluorescence polarization method. The perturbation by fatty acids occurs in both the lipid fraction and protein fraction. Nishihata et al. 1461 have proposed that protein thiols and non-protein thiols play an important role for the permeability of plasma membranes and Murakami et al. 1471 have reported that the adjuvant action of fatty acids was suppressed by pretreatment with N-ethylmaleimide, a sulfhydryl modifier; i.e. one of the action sites of fatty acids must be the protein fraction of the plasma membrane. As described later, sodium caprylate is used as an absorption-promoting adjuvant in ampicillin suppositories, a marketed product in Japan for children. 5.2. Trmsport

route enhanced

by f&y

acids

There have been several reports regarding transport routes enhanced by fatty acids. As shown in Fig. 10, a good relationship was observed between the enhanced transport of several hydrophilic compounds by caprylate, and the molecular weight of the hydrophilic compounds [38]; i.e. absorption promoting action of sodium caprylate appears to involve increasing the pore size of membrane. Nishimura et al. [36] have reported that the enhanced transport of antibiotics by caprylate is correlated better with the cellulose membrane permeability of antibiotics rather than with molecular weight. However, this correlation was obtained only in tested compounds with a

600 -

400 -

.E 4

mo-

log P of fatty add Fig. 9. Accumulation of fatty acid in rat rectal tissue in the in vitro rectal sac method at 37°C. Sodium salt of fatty acid was loaded in the rat rectal sac and the rectal tissue was removed at 37°C.

They also have demonstrated using an ESR spectrum method that fatty acids perturb the membrane structural integrity by being incorporated into the plasma membrane [40]. This proposal is also supported by

Fig. IO. Relationship between the permeability index, Pa and reciprocal value of square root of molecular weight of compounds (O), PABA; (A). PSP; (M), trypan blue: (7). FD-4; (+), FD-10s. Each point for Pa represents the meaniS.E.

small molecular weight range. A more detailed report is available by Sawada et al. in a study using an Ussing-type chamber to investigate the effect ot caprate and caprylate on the paracellular permeation of water-soluble non-electrolytes (inulin. PEG 900, mannitol. erythritol, glycerol. thiourea and urea) across the isolated rat colonic epithelium 1451. The permeation clearance (P,,, in Fig. I I ) for inulin ( I ?IS i in molecular radius) to erythritol (3.2 A) increased linearly with an increase in their free diffusion coefficient (D,, in Fig. 8) showing the existence of a paracellular shunt route unrestricted by any molecular size. Glycerol (2.9 A,, thiourea (2.6 A) and urea (2.3 A) had higher clearance5 than the expected linear value in Fig. I I. showing the existence of a restricted pamcellular route or tl-anscellulal

-1 2

4

6

6

IX+ (cm*/=)

10

12 xl@

route 1451. Caprylate can increase the paracellular route transport in the colonic segment but not in the jejunal segment 1481.

6. Others Absorption-promoting adjuvants listed in this section may be more useful for research purposes rathe than for the development of formulations for medication.

Phenothiazines are known to act as calmodulin antagonist tranquilizers. An adjuvant action of pheand N-(6-aminohexyl)-5-chloroI nothiazine naphthalensulfonamide (W-7) in enhancing rectal absorption of hydrophilic antibiotics occurred at concentrations from I to 100 mM, in which they also act as calmodulin inhibitors. Maximum action of prometazine and perfenazine was observed at concentrations of S-SO mM (Fig. 12) 1491 and their mechanism of action seems to be different from that of other adjuvants such as strong chelating agents and surfactants which generally act more strongly with an increase in their concentration. Calmodulin antagonists increase the paracellular route of transport 1I II. As also reported, the adjuvant action of EDTA is inhibited by the presence of ouabain, because diffusion of solutes through the paracellular route r-equires a driving force attained by high osmotic pressures in the intercellular space between epithelial cells. Since it has been reported that phenothiazines inhibit Na -K ATPase [SO]. suppression of the ad.juvant action of prometazine and perfenazine at concentrations above 100 mM may be due to their own Na -K ATPase inhibition at high concentration. Because W-7 is only a calmodulin antagonist. no suppression on the adjuvant action was observed at high concentration. On the other hand, trifluoperazine and chlorpromazine which have halogen moieties in their structures XI as surfactants at high concentration. Thus, these phenothiaaines have two different mechanisms for the adjuvant action: i.e. act as calmodulin antagonists at low concentration and act as surfactanta at high concentration.

T. Nishihutu,

J. Howwrd

Rytting

I Advunced

DY!? Deliver?,

Reviews 28 (1997)

205-228

221

9

8r

U

5

10

20

50

100

Concn. ofphcnothiazines in mucosal medium Q.4 Fig. 12. Effect of trifluoperazine (A), perphenazine (B), profenamine (C) and propericlazine (D) on the clearance rate of cefmetazole (CLLm, 0) and Insulin (CL),, 0) through rat colonic sac. The clearance rates shown in this figure were determined by the mean value from 0.5 to 2 h after recirculation. The ratios of CLL,,I,, against CL,,, were shown with a. Number of colonic sacs used for each study was more than four. Each value represents the mean2S.D.

6.2.

Strong

chrluting

(a) P < 0.05 vs. no additive. (b) P < 0.1 vs. no additive. (c) P < 0.05 vs. maximum value.

agent

Strong chelating agents such as EDTA and EGTA have been employed for the study of tight junctions between intestinal epithelial cells. Treatment of the intestinal epithelium by strong chelating agents resulted in loosened tight junctions [6], which induce an increase in diffusion of solutes through tight junctions. Further, strong chelating agents inhibited the aggregation of isolated intestinal epithelial cells by removing or masking Ca’+ on the surface of the isolated intestinal epithelial cells [ 191. EDTA increases the transport of both antipyrine (relatively small molecular weight) and phenol red (relatively larger molecular weight). The transport enhancing action of EDTA was inhibited by ouabain which is an Na+,K+-ATPase inhibitor. Hayashi et al.

[5 l] predicted that the enhancing action of EDTA involves loosening tight junctions which can allow the diffusion of solutes even with large molecular weights and the diffusion of the solute can also involve a solvent drag effect for which sodium ions in the intercellular space act as the driving force. 6.3. Sulfhydryl

depleter

Living cell membranes show greater barrier selectivity to the transport of hydrophilic compounds such as trypan blue, while membranes of dead cells allow the free permeation of trypan blue. This finding raises the question of which common constituents of living cells and/or cell membranes are essential in regulating the permeability of a wide range of solutes. Nishihata et al. [46] found that there was a

222

DEM

Qzsg2gLL

I 1.5

1.o

Concn. ofnonprocein nrlfhydryis in cat lwtal tkuc, pnoug-cisuc

15

10

5

00

0.5

Concn. of cefmctazok in SCWSA side. &ml

Fig. 13. Effect of DEM

relationship between non-protein thiol content and membrane permeability in isolated rat intestinal cells (Fig. 13). DEM, a sulfhydryl depleter, provides high cell toxicity at a concentration of more than 5 mM in an isolated rat intestinal epithelial cell medium. DEM at concentrations up to 1 mM caused nonprotein thiol loss, but did not influence protein thiol levels in isolated cells 1521. Along with non-protein thiol loss. uptake of water soluble antibiotics increases into the isolated cells. When the isolated cells were treated with DEM at high concentrations of 5

mM for a short period of time (before apparent cell death). a significant loss of both non-protein and protein thiols occurred and cell membrane permeability against water soluble antibiotics decreased (Table 9). Protein thiol loss induced by high concentrations of DEM was inhibited by the co-presence of calmodulin inhibitors in the media of the isolated cells. However, calmodulin inhibitors did not provide for the recovery of the loss of non-protein thiol by DEM. Further, when protein thiol loss by a high concentration of DEM disappeared in the co-pres-

Table 9 Effect of DEM

on thio” and on cefmetalole

Cont. of

Non-protein thiol

EM (mM)

(

pmol/g

uptake” In the iwlated cell\ Protein thiol

protein)

(

pmolig

CefmetaLole uptake

protrm)

(nmol/g

protein)

Tlmc after incubation (min) S

20

0

14.222.1

I2.6?2.

0.025

I I A +- I .6

5 I

20

2

29.7 t I .4

31.9-+2.1

0.37-+-0.06

Y.7E2.1

‘7.4-t

30.2z2.7

0.40~0.09 O.Sh-t0.07’

I .Y

0. IO

9.22 I .Y”

7.X? I .-I”

i3.2+3.1

29. I + I .9

o.so

6.YL3.2”

6.1:

i2.6+2.Y

29.x-t4.1

0.62i-0.

I .o

6.2% 1.1”

5.X+-2.6”

i3.224.6

27.5 2 I .o

0.7OfO. 13”

s.0

3.3 IL I .7”

3.X-‘l.I”

2O.htJ.O”

1x.2t3.7”

0.22to.O~’

No htudy

IY.312.5”

No study

I .52?0.26’

3.2+0.7”

IO.0

Each value represents the mean5S.D.

1.7”

at 5 OI- 20 men after the \txt

“Cefmetazole uptake was measured at 2 min after addition of cefmetwolr for 5 min. Before the incubation. non-protein thiol WBD l3.Yi ‘Cells were preincubated with IO mM DEM “P i 0.01

vs.

no additive.

‘P < 0.0.5

vs.

no additive.

I ).

”

01 = 3-S).

“Thiols isolated intestinal epithelial cells were meawred

cefmetazole uptake (Table

I I <’

1.7 ~molig

(4 mM)

of incubation with DEM.

to cell

suspension which had been incubated with DEM

protein and protein thiol was 31.222.4

for 15 min before addition of cefmetwole

pmollg

protein.

in order to investigate the effect of cell death on

ence of a calmodulin inhibitor but non-protein thiol loss continued, the cell membrane permeability against water soluble antibiotics increased again [52]. Thus, non-protein thiol loss induced an increase in cell membrane permeability with respect to water soluble compounds which have relatively small molecular weights. Treatment of the isolated cells with calmodulin inhibitors alone, resulted in an increase in cell membrane permeability for water soluble compounds. These data also indicate that calmodulin inhibitors do not influence cell membrane permeability. 6.4. Lectin Lectin has been isolated from a wide variety of organisms including bacteria, plants and mammals. Plants provide the most common source of lectin. It has been reported that lectins stimulate the endocytosis process of the cells allowing for targeting of anticancer agents into some cells. With regards to the action of lectin on intestinal epithelium, it was discovered that Con A, one of the lectins, induces microvillous fusion and pinching-off, but does not induce endocytosis of mucosal epithelial cells of the rat colon. During this process, a significant uptake of trypan blue was observed, but this action of Con A disappeared rapidly after rinsing. Thus, the uptake of trypan blue was not due to the death of epithelial cells [lo]. 7. Formulation suppositories

development

of insulin

The enhancing action of salicylate and enamines appeared when they are present at the site of action (rectal segment); i.e. an enhancing effect is expected when they are located at an appropriate concentration at the rectal epithelium. Therefore, drugs which are enhanced in their absorption must be dissolved when the absorption-promoting adjuvants start their action. As shown in Fig. 4, additional administration of enamine alone 20 min after administration of an insulin suppository containing insulin and enamine caused a further absorption of insulin; i.e. remaining insulin in the rectal compartment after the disappearance of enamine was further absorbed by the action of additional enamine which was administered following the initial dose. These experimental data

indicate that enhanced dissolution of drugs increase the efficacy of the absorption promoting adjuvant, especially for drugs which dissolve slowly such as peptides. As an example for the effective development of an insulin suppository, a suppository which promotes the dissolution step of insulin should be developed. The increase in efficacy of an absorption-promoting adjuvant for rectal absorption of insulin can result in a decrease in the insulin dose, which should result in a safer formulation. In earlier trials of insulin suppositories, an acidic insulin solution was mixed with a triglyceride base to improve the dissolution rate of insulin after the suppository melts (formulation Code-l 1 and -12 in Table 10) 1531. Even in this formulation, the dissolution of insulin at 1 h was about 50%, as shown in Table 1 1. This incomplete dissolution of insulin might be due to recrystallization of insulin in the suppository. To accelerate insulin dissolution from such a suppository, a solid dispersion technique for insulin was employed; i.e. insulin powder was dissolved in a 0.05 M citric acid solution containing 0.001% polysorbate 80 and then sodium salicylate was added to the solution gradually to obtain complete dissolution. The solution was dried to obtain a solid dispersion of the insulin. The use of this solid dispersion of insulin accelerated insulin dissolution after melting as shown in Table 1 1 (Code 9). On the other hand, rapid dissolution of the absorption-promoting adjuvant causes a short enhancing period when the absorption-promoting adjuvants can be absorbed rapidly, e.g. salicylate and enamine. It has been reported that the incorporation of phospholipid into a triglyceride suppository base caused a sustained dissolution of diclofenac [ 541. Therefore, the use of such a technique has been employed for the development of an insulin suppository. In Table 10, the formulation composition of various insulin suppositories are listed (Code 1 to 6 with various insulin potency). From an optimally designed insulin suppository, dissolution of insulin occurred rapidly and dissolution of salicylate was maintained (Table 11). The use of such a suppository into normal dogs caused a significant decrease in serum glucose concentration even at 5 units per dog (less than 0.5 unit/kg), as shown in Fig. 14. In the formulation development of rectal suppositories containing an

Table IO Codes and constituents of suppwitoriea Base

Code Triglyceride

Sodium salicylate (mg)

(111~)

Insulin

Lecithin (mg)

I

630”

70

299.93

2

1

6.30”

70

209.X?

s

3

630”

70

299.63

IO

4

630”

70

29X.76

20

5

h30

70

3X.89

10

6

630

70

29x.

0

I5 x3.x’)

so 30

0

7

700

7’

700”

208.X9

30

x

S60”

II0

29X.XY

30

Y

390

x3.x9 300

30 20

I0

540”

210 ho

II

630”

70

300

so

IZ

700”

0

300

50

Code I-Y,

use of solid dispersion

(U)

form of insulin

with

wdlum

wlicylate

(amounts of hodium salicylate and insulin

is 300 my/g

wppo5itory). Code IO. use of solid dispersion form of Inwlin Code

II

and

12.USC of’ acidic

Mith manmtol (amount\ of mannitol and insulin

Insulin solution (volume of the inwhn

“Mixture of triglyceride of Pharmasol A- IO0 and Phxmaaol

wlution

added is SO PI/~

is 100 m&/g suppoGtory). suppository).

B- IO0 wtth :t ratio of 30:60.

“Pharmasol B-100.

Table

II

Diaholutlon of insulm and sdicylate from auppositorleh Cod+?

Insulin

Salicylate

0.5 h

I h

0.5 h

I I1

I

73.2-tS.Y

Y1.4kl.h

62.1-+1.X

9.3.hk 2.1

2

71.4i4.Y

Yl.2?S.Y

66.3k3.7

97.

3

68.62X.I

90.9 i I .6

61.3-tj.7

94.2t3.9

4

70.31-6.7

93.2-t7.0

6 I. I il.6

Y4.2l4.Y

5

67.9x5.1

92.??7.Y

6Y.l+_X.J

90214.6

6

68.257.

YO.624.4

70.11-1.5

91.1 13.1

7

16.714.5

2Z.li8.2

21.3iY.h

44.4 + 7. I

I

I i 5.2

7’

79.1+9.1

Yh. I i-t.7

x.3.9+-5.2

96.h-t2.0

8

74.3-th.Y

X6.723.X

64.3i7.2

X6. I -7.2

Y

1Y.Xth.l

70.31-3.x

39.2i7.s

72.4 I x. I

IO

hX.4-tX.3

89.7~5.9

72.6?5.1

Y6.YiJ.Y

II

17.2+7.4

S-l.h?6. I

X6.4-tS.2

04. I + 3.9

I2

16.7-t 7.2

50.2~7.1

YO.3i1.2

YS.6?X.

I

Values we ‘k.

absorption-promoting adjuvant, timing of the dissolution of the active drug and the adjuvant from the suppository is a key factor.

8. Human trials 8.1. Atnpicillin

.suppo.sitoty 155581

suppositories incorporating sodium Ampicillin caprylate were tested as a formula for children in

Japan. In a human kinetic study [%I, the maximum plasma concentration after rectal dosing of an ampicillin suppository at a dose of 125 mg was 4.5 pug/ml at 15 min, which was half the level found 15 nun after intravenous dosing of the same dose. The maximum concentration in plasma of ampicillin after rectal dosing of the ampicillin suppository was much higher than that found after oral dosing of ampicillin. The time required to reach the maximum concentration after rectal dosing of the ampicillin suppository was much shorter than that required after oral dosing of ampicillin. In clinical trials of ampicillin suppositories containing sodium caprylate as the adjuvant, rectal dosing of the suppository was carried out only for children who had difficulty in swallowing the oral tablet or capsule or who rejected intravenous injection. The patients were diagnosed with pneumonia, acute respiratory infection or tympanitis. The efficacy of the ampicillin suppository for more than 85% of the children was equal or greater than oral dosing or intravenous dosing. In the clinical trials, physicians selected the rectal ampicillin suppository as an alternative dosing form for oral dosing or intravenous dosing. Thus, an ampicillin suppository containing sodium caprylate as an absorption pro moting adjuvant is an effective alternative dosage form both for oral and intravenous administration in

22s

a

a

(A)

I 1

.Q

I

!

I

2.0

3.0

0

Time, Fig.

14.Plasma

I

I

1

1.0

2.0

3.0

h

glucose concentrations in dogs after the administration of each suppository. (A) 0, Code. I;

Code 2; A. Code 4: W, Code 6. Each value represents the meantS.D. 12.2 and 12.9 lip. (a) P
A.

0. I 1.6.

Code 3; LJ. Code 5. (B)

(II = 3, cross-over study). Body weights of the three dogs were

vs. zero time: (b) P
the children. Another advantage in the use of ampicillin suppositories obtained in the clinical trials, was that the suppository could be inserted even while the patient is sleeping.

Insulin treatment for diabetes leaves much room for improvement in its delivery to its site of action. A study, in which insulin delivered via the hepatic portal vein in depancreatized dogs after regular feeding, demonstrated less hyperinsulinaemia than that observed in a peripheral infusion study. This indicates the importance of insulin delivery via the portal vein in normalizing both blood glucose and insulin levels in the postprandial state. Oral or partly rectal insulin dosing could have an advantage by achieving portal insulin delivery in a convenient way. However, insulin is not normally absorbed from the intestine. The use of absorption-promoting adjuvants for rectal insulin delivery may be one possible approach to achieve this advantage. Practically, the rectal route should have advantages over the oral route because of insulin’s rapid degradation enzymatically in the small intestine. The following clinical trials were performed with insulin

suppositories containing an acidic insulin solution and lecithin (Supp-1 in Fig. 15) and with an insulin suppository described in the section on formulation development of insulin suppositories which contain a solid dispersion of insulin and lecithin (Supp-2) [S9]. The dosing of the insulin suppository in the form of Supp-1 at a potency of 100 units caused a normalization of serum glucose concentration in diabetic patients with a significant increase in serum insulin concentration (Fig. 15A) 1591. The dosing of insulin suppositories in the form of Supp-2 at a potency of 30 units resulted in a serum level of 32.7k6.9 punitlml at maximum. In a diabetic patient eating a regular meal for a tolerance test (Fig. 15B), the administration of an insulin suppository at a potency of 60 units in Supp2 and 150 units in Supp-1 was found to inhibit postprandial hyperglycemia. Insulin suppositories were found to control postprandial hyperglycemia in diabetics, and peripheral insulin profiles were similar to those in healthy subjects after eating. The observations suggest that an insulin suppository may control postprandial hyperglycemia in diabetic patients in a more natural manner than found with conventional parenteral insulin therapy.

226

phenol red and gentamycine. J. Pharm. Pharmacol. 30

(197X)

17%1x5.

121 M. Shlchiri. Y. Ynmalaki. Hakut,

R. Kawamori.

M.

Kikuchi,

N.

H. Abe, Increased intestinal absorption of insulin:

insulin suppository. J. Pharm. Pharmacol. 30

( 1978)

806.

I31

Y. Nishioka,

111

E.J. Ariens. Drug Deulpn. vol. I. Academic Press. New York

ISI

7‘

T. Kawamura.

Effect of surfactant on rectal

insulin absorption in rabbits, Yakuzaigaku

38 (1978)

67-7.5.

1971.

r 0

I

I

1 120

I

60

Higuchi.

Sy\rem.

V. Stella.

Prodrup

ACS-Symposium

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Ihl

Novel

as

Drug

DeliLet-y

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D.W. Powell. Barrier function of epithelia, Am. J. Physlol. 24

I c19X I ) G275-G28X.

I71 E. Naknahima, A. Thuji. H. Mizuno, T. Yamana. Kinetics and mrchanim

of in vitro uptake of amino-p-lactam

by rat hmall intestine and relation transporr system. Blochem.

0’ Fi p.

IS. (A)

II 0 15

135

195

Carrier-mediated

admin. (min)

Plusma ~rlsulin cil.

of Supp-I

IRI)

containing

value represents the mtxm-tS.D.

and pluco\e

(0)

con-

100 units of insulin. Each

;‘P c: 0.0.5 v\. the value befot-e

334S-

of insulin (dashed line). or Supp-2 containing 60 unitz, of insuhn (solid line). on plasma glucose concentration

of seven diabetic

patient!, (six male and one female) Lifter a regular Japanese meal (270 g of boiled rice) for the tolerance test. The suppository wab IS min before the meal (patien& ate at the IS min

point m the figure). Plasma glucose concentration< under fasted condition. which were measured 7 days before the administration were

between

140-180

mp/lOO

ml for

\ix

patients, and 310 mgl 100 ml for one patient marked *d:C.

( 1078)

262-267.

A on mouse

peritoneal macrophageh. I. Stimulation of endocytic activity and Inhibition of phago-lysosome formation. I40 (1974)

1101 T.

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P.R.

Burton.

L.J.

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fusion and pinching-off

Blochem. Bmphys. Rcs. Commun.

IIll

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1364-1386.

micro\iIIoll\

(H)

The effect of rectal administration of Supp- I containing I SO unit\

I

191 PJ. Edelson, Z.A. Cohn, Effects ofconcan~alin

kg and 42-64

administration; “-“p ‘: 0. I vs. the \aluc before administration.

of suppositories.

( 1984)

transport systems for amino penicillins in

rat small intestine, J. Pharmacobio-Dyn.

years old), who were fasted for I6 h before administration, after

administered

33

1x1 I‘. Kimura. H. lzndo, M. Yoshikawa. S. Muranihhl, H. Se/&i. 75

in five male diabetic patient\. (Sl-64

administration

Phxmacol.

.3352.

Time after

centration

antibiotics

tv the intact-peptide

T. Nishihnta. M.

Miyake,

Induction

ot

by concanilvalin

A,

I40

( 1986)

H. Takahata,

A.

766-772. Kamada,

The

cffcct of adjuvants an the colonic abaorption of cefmetaLole and eel calcitonin in rata, Int. J. Pharm. 33 (19X6)

1121 A.

Martined-Palomo.

I.

Me/a.

G.

Beaty.

M.

89-97. Cereijido.

Experimental modulation of occluding junctions m a cultured

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1131 T.

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lY86~lYY7.

1141 A. Kamada. T. Nishihata, S. Kim. M. Yamamoto, N. Yata,

There may also be some incidental advantages in the use of salicylate as an absorption-promoting adjuvant for insulin. It has been reported that salicylate is an effective agent to treat vascular disease which often occurs in the diabetic condition. This may be because it inhibits glycosylation of collagen and because it suppresses cyclooxygenase activity, also acting as an antiplatelet agent. Salicylate also inhibits gluconeogenesis from L-lactate and I.alanine.

Study of enamine derivatives of phenylglycine as adjuvants for the rectal absorption of insulin, Chem. Pharm. Bull. 29 (19x1)

2012-2019.

1151 T. Nishihata. J. Kate. K-H. Kim. M. Kobayaahi, I. Kitagawa. A. Kamada, Formation and hydrolysis of enamine in aqueous solution. Chem. Pharm. Bull. 32 (1984)

1161 R. Choh, H

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T. Nishlhata,

4545-4550.

A. Kamada,

Enamine

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Chem. Pharm. Bull. 33

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3027-3030.

1171 T. Nishihata. Y. Okamura. A. Knmada. T. Higuchi. T. Yagi. R.

Kawamori.

M.

Shichiri,

Enhanced

bioavailability

of

insulin after rectal administration with enatnine as adjuvant in depancreatired

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