db) to p-fluorophenethylbiguanide

Pharmacological Research Communications, Voi. 9, .No. l,~ 1977

RESPONSE OF "DIABETIC" MICE

39

(db/db)

TO p-FLUOROPHENETHYLBIGUANIDE

Terence T. Yen, Mark M. Greenberg, Jean A. Allan and June M. Act0n The Lilly Research Laboratories, and Company,

Indianapolis,

Eli Lilly

Indiana

46206

Received5 April 1976

SUMMARY The compound p-fluorophenethylbiguanide glucose and plasma insulin concentrations mice, C57BL/KsJ-db/db.

lowered the blood

of genetic diabetic

The compound also depressed the respira-

tion of the db/db mice and caused the accumulation of plasma lactic acid.

Since these mice did not respond to the sulfonyl-

urea type of hypoglycemic compounds, differentiating

they are thus useful in those

the two types of hypoglycemic agents:

that stimulate insulin secretion and those that do not. INTRODUCTION The mutation diabetes

(db) was discovered in an inbred

mouse strain C57BL/KsJ at Jackson Laboratory and Coleman, by obesity,

1966).

Mice homozygous

(Hummel, Dickie

for db are characterized

hyperglycemia and transitory hyperinsulinemia

(Coleman and Hummel, 1967).

Many studies have been conducted

on db/db mice in the past nine years

(Staats, 1975).

However,

Pharmacological Research,Communications, Vol. 9, No. I j, 1977

40

very little is known about the response of the db/db mice to various pharmacological

agents,

especially the hypoglycemic I

drugs.

This report describes

biguanide

(p-F-PEBG)

the effects of p-fluorophenethyl-

in db/db mice and.suggests

would be useful in discovering

that these mice

new hyp0glycemic

agents that do

not depend upon the stimulation of insulin secretion as the primary mode of action. We chose to use p-F-PEBG

instead of phenethylbiguanide

(PEBG) because the latter is first p-hydroxylated jugated to glucuronic p-Hydroxy-PEBG rats

acid in rats

(Murphy and Wick,

is not a very active pharmacological

(Cook, Blair, Gilfillan and Lardy,

(Beckmann,

and then con-

1973)

1968). agent in

and in mice

1966).

MATERIALS AND METHODS Male C57BL/KsJ-db/db mice were supplied by the Jackson Laboratory,

Bar Harbor, Maine.

Chow and water ad libitum. at about 25°C.

They were fed Purina Laboratory

Ambient temperature was maintained

Lighting was controlled by an automatic timer

to provide light from 6 a.m. to 6 p.m. For blood glucose determinations, tail with heparinized capillary tubes

blood was taken from the (Trident).

filtrates of blood were assayed with Worthington's using dextrose

(Mallinckrodt)

insulin

Glucostat,

as. the standard.

Plasma insulin concentrations the method of Wright et al

Deproteinized

were determined according

(1968) using single-component

(Lilly) as the standard.

12sI-insulin

(porcine)

porcine was

prepared by Dr. M. Bhatti and Mr. T. E. Cooper of our laboratories.

Guinea pig anti-porcine

from Miles Laboratories.

to

insulin serum was purchased

Pharmacological Research Colnmunications, Vol. 9, No. I, 1977

41

Respiration was measured in an apparatus described by McMahon et al

(1969).

Since the animal chamber was designed to

accommodate a rat, three db/db mice were placed in the chamber to occupy the space and their respiration was monitored at the same time.

Uniformly-labeled

l~C-glucose

(2.79 mCi/mmole,

New

L

England Nuclear) Nuclear)

or 1~C-fructose

was administered, i.p.,

hours after p-F-PEBG

(50 mg/kg,

(4.65 mCi/mmole,

New England

at 1 ~Ci/100 g body weight 3 i.p.) was given.

Measurement

of respiration was started as soon as the *~C-tracer was injected, and lasted for at least an hour. Plasma lactic acid concentration was determined on perchloric acid filtrates with Calbiochem Rapid Lactat~ Reagents. lactate

(Calbiochem)

Lithium

was used as the standard.

The sources of various hypoglycemic compounds used in this study were:

p-F-PEBG hydrochloride

acetohexamide

(Lilly);

tolbutamide

Glicodiazine and Glisoxepide Glipizide O'Connell,

(Dr. N. Belcher,

(Dr. J. Mills, (Dr. J. S. Ward,

(Dr. K. Zellerhoff,

Pfizer);

Lilly); Lilly);

Bayer);

and Glyburide

(Dr. P. W.

Upjohn).

In all experiments unless otherwise specified, 2% Emulphor EL-620

(General Aniline and Film)

a mixture of

and saline was

used as a vehicle to dissolve or suspend the above hypoglycemic compounds.

Emulphor EL-620 is a polyoxyethylated vegetable oil.

RESULTS

Blood ~lucose.

The effect of p-F-PEBG on the blood glucose

levels of db/db mice over a period of 7 hours is shown in Figure i.

Given 50 mg/kg,

i.p. of p-F-PEBG,

the blood glucose concen-

tration of db___/dbmice decreased at 1 hour after dosing although

42

Pharmacological Research Communications, !/ol. 9, No, 1, 1977

this decrease

was not significant.

glucose

became

level

ficant t h r o u g h

significant

the 6th hour.

The reduction

at 2 hours

It returned

of blood

and remained

to pre-dosing

signilevel

I00

80 4/t

oE

S

2"

mo

60

o~m 4 0 II

I

0

-

1

1

2 Hours

LEGEND TO FIG.

3 after

4

5

I

II

6

7

injection

i.

The time course of the h y p o g l y c e m i c activity of p-fluorophenethylbiguanide (p-F-PEBG, 50 mg/kg, i.p.) in C 5 7 B L / K s J db/db mice (solid line) as compared to that of vehicle (2% ~u[phor-saline, dotted line). Both curves were compiled from data gathered from the same 4 groups of db/db mice, w i t h 8 m i c e in each group. Each group of m i c e was sampled before injection of p-F-PEBG. They were then sampled at 1 and 4 hours (group i); or 2 and 5 hours (group 2); or 3 and 6 hours (group 3); or 7 hours after injection. On a separate day, 2 weeks prior to the p-F-PEBG experiments, these same mice were injected with vehicle, 2% emulphor-saline, and bled correspondingly. The data points on both curves at 1 and 4 hours after injection, for example, w e r e all from group i. The "S" indicates that the v a l u e is s i g n i f i c a n t l y d i f f e r e n t from the "before injection" blood glucose level of that day at p<0.025. The change of b l o o d g l u c o s e level at 4 and 5 hours after v e h i c l e p r e s u m a b l y reflects a diurnal rhythm. M~ce were 2-1/2 to 3 months of age at the time of control experlments and 3 to 3-1/2 months old at the time of p-F-PEBG experiments. Blood glucose c o n c e n t r a t i o n s were d e t e r m i n e d on d e p r o t e i n i z e d filtrates of 25 ~i of h e p a r i n i z e d . w h o l e blood with W o r t h i n g t o n ' s glucostat.

Pharmacological Research Communications, .Vol. 9, No. 1, 1977 on the 7th hour. vehicle

43

The db/db mice given the same amount of

(0.i m l / 2 O g

body weight,

i.p. of 2% Emulphor-saline)

showed a slight reduction in blood glucose level at 4 and 5 hours.

These decreases were not due to the injection o f vehicle

but due to a diurnal rhythm

(data unpublished).

p-F-PEBG was also active orally at 60 mg/kg suggesting that the comPound w a s w e l l doses,

absorbed.

(Table l) Atsimilar

i.p. or p.o., p-F-PEBG lowered the blood glucose level

of C57BL/KsJ-normal mice fed ad lib.

(data not shown).

compound was not active in db/db mice at 25 mg/kg,

Table i.

Hours after p-F-PEBG

The

i.p. or p.o.

Oral activity of p-fluorophenethylbiguanide (p-F-PEBG) on blood glucose of db/db mice I Blood glucose (mean +- s.e. in mg %)

% of 0 time (mean +- s.e.)

p(vs 0 time) 2 ..

|,

0

302.6 + 23.4

3

234.7 +- 26.9

75.0 +- 4.5

<0.0005

5

219.3 -+ 26.4

70.2 +- 4.7

<0.0005

IThe data presented in this table were compiled from three independent experiments using a total of 23 db/db mice with ages ranging from 3-1/2 months to 5-1/2 months. Each mouse was bled from the tail before p-F-PEBG and 3 and 5 hours after p-F-PEBG (60 mg/kg, p.o.). The compound was dissolved in 2% emulphorsaline. Blood glucose concentrations were determined on deproteinized filtrates from 25 ul of heparinized whole blood with Worthington's Glucostat. ZBased on Student's paired t-test.

In contrast to p-F-PEBG,

the following sulfonylureas did

not decrease the blood glucose level of d b / d b mice fed ad lib. at doses that were active in fasted normal mice: and tolbutamide at 150 mg/kg,

acetohexamide

p.o., and Glicodiazine,

Glisoxepide and Glyburide at 50 mg/kg, p.o.

Glipizide,

Pharmacological Research Communications, Vol. 9, No. 1, 1977

44

Plasma insulin.

At 25 mg/kg,

i.p. or 50 mg/kg,

i.p.,

p-F-PEBG decreased the plasma insulin concentration of d b/db mice which were hyperinsulinemic

to begin with

(Table 2).

This could

result from the direct suppression of insulin secretion from pancreatic islets by PEBG as shown by Schatz et al Alternatively,

(1972).

the lowering of plasma insulin level may indicate

a return of insulin sensitivity triggered by the reduction in blood glucose level.

Table 2.

Plasma insulin level of db/db mice after p - f l u o r o p h e n e t h y l b i g u a n i ~ (p-F-PEBG) I

Dose

(mg/kg,

i.p.)

0 3

N

0 TiRe 3 Hours (mean + s.e. in ~units/ml)

pZ

8

781.2 ± 103.2

615.0 ± 55.2

>0.05

25

8

988.8 ± 122.7

278.4 ± 61.6

<0.005

5O

16

788.6 + 138.8

58.8 ± 38.2

<0.0005

IEach mouse was bled before p-F-PEBG and 3 hours after p-F-PEBG. Blood was collected in heparinized capillary tubes. Ten ~i of plasma were assayed according to the procedure of Wright et al (1968). Mice were 2-1/2 to 3 months old at the time of the experiments, p-F-PEBG was dissolved in water or saline. ZStudent's paired t-test was used to compare the levels at 0 time and 3 hours. 3Water

(0.1 mi/20 g, i.p.) was injected.

Respiration in vivo. m

.

The recovery of l~CO 2 from three

db/db mice during the first hour immediately after uniformly labeled Z~C-glucose or 1~C-fructose was administered was much less in the p-F-PEBG the tracer)

(50 mg/kg,

i.p., given 3 hours prior to

experiment than in the vehicle experiment.

Using

l~C-glucose as a tracer, vehicle-treated mice expired 18.5% of the radioactivity

injected during that hour whereas the same

Pharmacological Research Communications, Vol. 9, No. 1, !977

45

three mice treated w i t h p-F-PEBG expired only 6.6% of the radiop

activity

given.,

i

For * ~ C - f r u c t o s e ,

the'vehicle-treated

mice

expired 18.6% of t h e radioactivity given whereas t h e same mice treated with p-F-PEBG expired only 8.8% of the radioactivity injected.

However, for both 19C-glucose and l~C-frL~ctose, the

specific activitY of the CO z expired (I~CO2/C02) did ~.ot change because of the p-F-PEBG treatment.

This indicated a depression

of respiration in general instead of a specific depression of the Oxidation of glucose or fructose.

D e p r e s s i o n of oxidation

of various substrates in different tissues by PEBG in vitro has been demonstrated and suggested by several groups of workers as a result of PEBG inhibition of oxidative, phosphorylation (S~ling et al, 1967, pereira, Jangaard and Pinson, Pereira and Pinson, 1968, Elkeles,

1972).

1967, Jangaard,

Our in vivo data

support this hypothesis. Plasma lactic acid.

The characteristic accumulation of

lactic acid in plasma caused by PEBG

(Assan, 1975) can also be

demonstrated with p-F-PEBG in both young and mature db/db mice (Table 3) .

DISCUSSION

The data reported here demonstrate unequivocally the effect of p-F-PEBG on db/db mice.

Administration of p-F-PEBG to d b/db mice

effectively lowers the blood glucose and plasma insulin concentrations, depresses respiration and produces a rise in plasma lactic acid level.

These pharmacological effects are characteristic for

biguanide s .

Similar effects have been observed in humans with

PEBG.

Our data on db/db mice thus support the n o t i o n that bigua-

n/des exert their hypoglycemic effects through alteration of

Pharmacological Research Communications, Vol. 9, No. 1, 1977

46 T a b l e 3. m,

,

Plasma lactic acid level of db/db mice after p-fluorophenethylbiguanide (p-F-PEBG)*

•

,

Age

,

Control2

(months) .

_ _

,,

3-1/2 - 4-1/2 ,

ilii

(mean ~

i-1/2 _ _

,

m

,,

_

,

,

p F PEBG2

p3

s . e . in mg %)

, • ,

39.4 +_ 4.1 31.9 -+ 3.9

,=

(N=6) I 54.6 + (N=I2) . m

I

3.8

83.5 + i0.9

(N=6)

<0.025

(N=I2)

<0.005

IMice were bled by decapitation or from the tail 3 hours after dosing. Blood was collected in heparinized tubes. A perchloric acid filtrate was prepared from 20 ~I or 50 ~1 plasma. Lactic acid concentration was determined with Calbiochem Rapid Lactate Reagents using lithium lactate as the standard. 2p-F-PEBG was dissolved in 2% Emulphor-water and administered at 75 mg/kg, i.p. (15 mg/ml). Mice serving as controls were dosed with the same volume of vehicle (0.i ml/20 g body weight). 3Probability was determined by Student's t-test comparing p-F-PEBGtreated db/db mice with vehicle-treated db/d b mice.

cellular oxidative processes,

possibly increasing anaerobic glyco-

lysis by inhibiting aerobic metabolism.

None of the six sulfonylureas tested affected the blood glucose level of db/db mice. tant and sulfonylureas least acutely,

Since db/db mice are insulin resis-

are known to promote insulin secretion,

the lack of response to sulfonylureas

at

is not a sur-

prise. The fact that db/db mice respond to biguanides and do not respond to sulfonylureas

prompts us to propose that these mice be

used in searching for new hypoglycemic agents.

Agents discovered

in such a system would have peripheral mechanisms and would not enhance llpogenesis should be therapeutically

as insulin does.

like biguanides Such agents

useful for both juvenile type and

maturity-onset diabetes. It should also be possible,

using db/db mice,

to find hypo-

glycemic agents llke.biguanides but devoid of their lactic acid-

producing activity.

Such agents would offer a great improvement

Pharmacological Research Communications, Vol. 9, No. 1, 1977 in diabetes

47

therapy by possibly acting by mechanisms distinct and

different from those of PEBG and p-F-PEBG. Studying effects of various hypoglycemic

agents such as

p-F-PEBG in db/db mice, on the other hand, will also lead to a better understanding

of the etiology of diabetes

an understanding which may be applicable

in these mice,

to certain types of human

diabetes.

ACKNOWLEDGEMENTS The authors are grateful to Dr. N. Belcher (Pfizer), Dr', J. Mills (Lilly), Dr. P. W. O'Connel (Upjohn), Dr. J. S. Ward (Lilly) and Dr. K. Zellerhoff (Bayer) for their generous supply of various hypoglycemic compounds used in this study. The authors are also indebted to Dr. R. J. Hosley (Lilly) for his assistance in obtaining some of these compounds.

REFERENCES m

Assan, R., Heuclin, C., Girard, J. R., LeMaire, F., and Attali, J.R.: Diabetes 24, 791 (1975) Beckmann, R. : Diabetol-6gia 3, 368 (1966) Cook, D. E., Blair, J. B., Gilfillan, C., and Lardy, H. A.: Biochem. Pharmacol. 22, 2121 (1973) Coleman, D. L. and Hummel, K. P.: Diabetologia 3, 238 (1967) Elkeles, R. S.: Horm. Metab. Res. 4, 178 (1972)n

•

Hummel, K. P., Dickie, M. M., and Coleman, D. L. : Sci. 153, 1127 (1966) Jangaard, N. O., Pereira, J. N., and Pinson, R.: Diabetes 17,

96 (1968) McMahon, R. E., Mills, J., Culp, H. W., Gibson, W. R., Miller, W. M., and Marshall, F. J.: J. Med. Chem. 12, 207 (1969) Murphy, P. J. and Wick, A. N.: J. Pharm. Sci. 57 , 1125 (1968) Pereira, J. N., Jangaard, N. O., and Pinson, E. R.: Diabetes 16, 869 (1967) Schatz, H., Katsilambros, N., Nierle, C., and Pfeiffer, E. E.: Diabetologia 8, 402 (1972)

ling, H. D., ZahTten, R., B~ttcher, M., and Willms, B.: Diabetologza 4, 377 (1967) Staats, J.: Diabe~ologia II, 325 (1975) Wright, P H., Makulu, D. R., Malaisse, W. J., Roberts, N. M., and Yu, P. L.: Diabetes 17, 537 (1968)