Effect of chlorpropamide on phenformin-induced mortality in the dog

TOXICOLOGY

AND

APPLIED

Effect

PHARMACOLOGY

18, 253-262

of Chlorpropamide Mortality

(1971)

on Phenformin-Induced in the Dog1

G. SAGRITALO, G. CORTE,AND J. D. MCCOLL’ Research

Laboratories, Montreal, Receiced

Frank Quebec, September

W. Homer Canada

Limited.

17. 1969

Effect of Chlorpropamideon Phenformin-InducedMortality in the Dog. SAGRITALO, G., CORTE, G., and MCCOLL, J. D. (1971). Toxicol.

Appl.

253-262. During a chronic toxicity study of chlorpropamidein combination with phenformin in the beagle,it wasunexpectedly observed that the combination produced a lower mortality than anticipated from the effect of the individual drugsalone. Subsequentexperiments undertaken to elucidatethe mechanismof this antagonisticeffect demonstratedthat chlorpropamideuncoupledoxidation from phosphorylation in rat liver and rabbit kidney mitochondrial preparations. The action was similar to, but weaker than, that of 2,4-dinitrophenol (DNP). The inhibition of respiration produced by phenformin was reversedby chlorpropamide. In the beagleit wasfurther shownthat chlorpropamideand DNP reversedthe phenformin-induced increasein lactic and pyruvic acids in peripheral blood. It was concluded that the uncoupling action of chlorpropamidewasresponsiblefor the observedantagonism.

Pharmacol.

18,

It has been establishedthat chlorpropamide and phenformin are effective oral agents for the treatment of maturity-onset diabetes (Katz and Bissel, 1965; Singer and Hurwitz, 1967).The combined administration of a sulfonylurea and a hypoglycemic biguanide is advantageous in the treatment of diabetic patients unsuccessfully controlled by a sulfonylurea compound alone, or those who are “secondary failures” (Beaser, 1958; Clarke and Duncan, 196.5;Fineberg, 1968). During a routine chronic toxicity study in the dog with chlorpropamide and phenformin alone and in combination, it was observed that the mortality resulting from a given dose of phenformin did not occur when chlorpropamide was administered concurrently. Routine laboratory and histological studiesdid not satisfactorily account for this difference in mortality. The present investigation was undertaken to determine the mechanismof this antagonistic action of chlorpropamide to the toxic effects of phenformin in the dog. METHODS Adult male and female beagles(Horner strain, IO-14 kg) were used. The following drugs were administered po (5 days/wk) alone and in combination and for different ’ Presented in part 1968AnnualMeeting,CanadianAssociationfor Research in Toxicology,Montreal,Canada. 2 Presentaddress:MeadJohnsonResearchCenter,Evansville,Indiana47721. 253

254

SAGRITALO,

CORTE,

AND

MCCOLL

periods as described in the Results section: chlorpropamide, 100 mg/kg; phenformin, 40 and 50 mg/kg; 2,4-dinitrophenol (DNP), 2 and 4 mg/kg. Blood glucose concentrations were determined on serum using the Technicon Autoanalyzer method (Technicon Autoanalyzer Manual method N-2a). Blood lactic acid changes were measured by the enzymatic procedures of Hummel (1949) and Olson (1962) using lactic acid dehydrogenase. Pyruvic acid was assayed by the method of Bueding and Wartis (1940). Phenformin blood concentrations were measured by a modification of the procedure described by Freedman et al. (1961). A study of the action of the various compounds on rat liver and rabbit kidney mitochondria was carried out manometrically employing the usual Warburg techniques (Scholefield, 1956). Mitochondria were prepared by differential centrifugation following homogenization of the tissue in a Virtis “45” homogenizer. All incubations were carried out at 37°C. Oxygen uptake was measured at 5-min intervals over a 20-min period. Following this, I ml of 15 % trichloroacetic acid was added to each Warburg flask and the total phosphorus was determined by the method of Fiske and SubbaRow (1925). From the oxygen consumption and phosphorus content, the P/O ratio was calculated. The substrate employed was fumarate-stimulated pyruvate (6.7 x 10m4 and 6.7 x 10m3 M, respectively) with hexokinase and glucose (hexokinase trap to remove high energy phosphate bonds formed from the citric acid cycle) (Cross et al., 1949). All determinations were done in duplicate. With the rat kidney preparation, a pyruvate “blank” was included, and in all experiments appropriate controls were used. RESULTS Table 1 summarizes the mortality observed in the initial toxicity study and in subsequent experiments with chlorpropamide and phenformin, alone and in combination, at the doses shown. Death with phenformin occurred with 2-4 weeks of repeated administration. One animal in the chlorpropamide group died after 8 weeks treatment from acute tracheitis. TABLE MORTALITY

1

IN Dons AFTER REPEATED ORAL ADMINISTRATION OF CHLORPROPAMIDE AND PHENFORMIN

Treatment

Mortality

Control Chlorpropamide, 100 mg/kg Phenformin, 50 mg/kg Phenformin, 50 mg/kg + chlorpropamide, 100 mg/kg a Death

due to infection,

0 13 % (l/8) 64 % (9/14) 11% (l/9)

not to drug.

The concurrent administration of chlorpropamide (50 mg/kg) significantly (P ==I0.02) reduced mortality 11%.

(100 mg/kg) with phenformin due to phenformin from 64 to

CHLORPROPAMIDE

PHENFORMIN

755 _ -_

MORTALITY

The effect of these drugs on blood glucose when administered alone or in combination is shown in Fig. 1. Blood samples were taken 24 hr after daily po administration of the compounds for an I l-day period. Phenformin, as previously reported (Ungar rt al., 1957), produced no hypoglycemia in the dog. Chlorpropamide was potent, and a maximal hypoglycemic effect was observed after 24 hr. Concurrent administration of chlorpropamide and phenformin required approximately 3 days to become maximally effective; however. when the effect reached a plateau, it was of the sameorder as that of chlorpropamide alone. Phenformin and DNP given together produced slight and PHENFORMIN CHLORPROPAMIDE COMBINATION

- 60 1 1

I 1

I 5

I 3 DURATION

OF

TREATMENT

I 8

I 11

(DAYS)

FIG. 1. Effect of daily oral administration of drugs on blood glucose concentrations in dogs (chlorpropamide, 100 mg/kg; phenformin, 50 mg/kg; combination 100 mg/kg and 50 mg/kg, respectively; 5-8 dogs per group). Ordinate: percent change in blood glucose relative to pretreatment values. Abscissa: duration of treatment in days.

variable results. It did not seemlikely that alteration of hypoglycemia was the causeof the antagonistic action. Investigations were undertaken to determine whether modification of the activity of liver microsomal enzyme systemswas responsible for the decreasedtoxicity. To evaluate the gross effect in ciao, blood concentrations after a single dose of phenformin (50 mg/kg) were determined in the dog before and after 30 days of repeated daily doses of chlorpropamide (100 mg/kg/day). There was no significant alteration in phenformin blood concentrations after this treatment period as compared with the initial values. indicating that the system had not been stimulated (Burns et a/.. 1965).

256

SAGRITALO,

CORTE,

AND

MCCOLL

CHLORPROPAMIDE

PHENFORMIN

757

MORTALITY

Table 2 summarizes the in vitro effects of these drugs on oxygen and phosphorus uptake of rat liver and rabbit kidney mitochondrial preparations. In rabbit kidney chlorpropamide reduced phosphorus and oxygen uptake; this effect was proportional to the concentration. At the lower dose of chlorpropamide (I .*I 10m3 M) phosphate was affected to a greater degree than oxygen uptake; at the higher concentrations both parameters were equally reduced. As a consequence the P/O ratio was markedly reduced, indicating that chlorpropamide uncouples oxidation from phosphorylation. In rat liver mitochondria3 a similar reduction of phosphorus oxygen uptake and P:O ratio with chlorpropamide was seen. Again at the concentration employed the inhibition of phosphorylation was greater than that of oxygen uptake. With phenformin. as previously reported by Ungar et al. (1959), inhibition of oxidation closely paralleled

,A

Chlorpropnm~de

Phen’orml”

FIG. 2. Effect of chlorpropamide by rat liver mitochondria.

+

and phenformin alone and in combination

on total oxygen uptake

that ofphosphorylation. Phenformin at the sameconcentration as chlorpropamide was approximately twice as effective in reducing the P/O ratio. It was interesting to note that, when tested in combination in this preparation. the effects of chlorpropamide and phenformin on inhibiting the P/O ratio were not additive and that only the phosphorylation had been further inhibited. The results of a characteristic experiment with chlorpropamide and phenformin on oxygen uptake in rat liver mitochondria can be seenin Fig. 2. Oxygen uptake over a 20-min period was reduced by chlorpropamide (1 x 10e3M) and to a greater extent by the same concentration of phenformin. When the two compounds were tested in combination (1 x 1O-3 M each), the inhibitory effect on respiration was not additive but was lessthan that produced by phenformin alone. These results are interpreted to indicate that the inhibition of respiration produced by phenformin was released by the uncoupling activity of chlorpropamide as demonstrated in the previous experiments (Table 2). The similarity of these findings with chlorpropamide to those reported by Krueger 3 Most of the experiments were carried out with rat liver mitochondria was necessary; this simplified the procedure.

because no pyruvate blank

PYRUVK

40‘1 41

2I

pretreatment

(Day 0) value.

0.21 zk 0.03

0.12 I!z 0.01*

0.22 f 0.05b

0.02b 0.03b 0.11 0.05

0.09 i 0.02

i + k f

0.23 * 0.04

0.21 0.37 0.23 0.11

Day 3 & 0.03” i 0.04b & 0.12 It 0.04

0.25 zk 0.01

0.19 zk 0.02b

0.22 It 0.09

0.23 0.42 0.24 0.09

Day 5

Serum pyruvic acid” (meq/l)

”5

0.13 * 0.03

0.17 + 0.03

0.21 zt 0.01*

i% 0” F

8

0.24 & 0.05

6 0.20 It 0.04

r2 %

m

0.27 i 0.03b 0.55 ic 0.04b

~.

DOGS AFTER REPEATED ORAL DRUG TREATMENT

0.19 ck 0.06

ml 40

3z 0.02 i 0.04 rt 0.13 f 0.03

Day 0

IN

5o-L 1

-

CONCENTRATIONS

0.09 0.17 0.27 0.07

ACID

3

40 50 100 2

Dose (m&z)

OF SERUM

a Mean *SD; 4 animals per treatment group. b Statistically significant (P < 0.05) from individual

DNP

Chlorpropamide Phenformin DNP Phenformin

Phenformin Phenformin Chlorpropamide DNP Phenformin

Treatment

ALTERATIONS

TABLE

~__-.~

LACTIC

40-t 2J 401 41

2

50 100

40

Dose @w/k) ~

OF SERUM

’ Mean +SD; 4 animals per treatment group. b Statistically significant (P --: 0.05) from individual

Phenformin Chlorpropamide Phenformin DNP Phenformin DNP

Treatment __~Phenformin Phenformin Chlorpropamide DNP

ALTERATION

+ * zt *

0.2 0.1 0.6 0.3

REPEATED

ORAL

2.3 i

2.1 !L 0.3

I .7 I 0.2

(Day 0) level.

2.8

0.2

t 0.4

1.1 3zo.3

2.9 i 0.2’ 3.1 5 0.9 0.9 IL 0.2 0.9 -L 0.4

2.1 lim0.6

0.9 t 0.4

~-t 0.4 zt 0.2b i 0.2 f 0.3

Day 5 .~ _.

.--

1.8eO.2

1.2 -;0.3

i 0.4

i 0.1

i 0.2* 37 0.5

Day 11 3.4 2.1 0.7 0.9

DRUGTREATMENT

Serum lactic acid” (meq/l)

AFTER

Day 3 2.8 2.4 0.7 0.9

IN DOGS

4

I .9 -10.4

1.7 t 0.3

1.8 1.3 1.5 1.5

Day 0

CONCENTRATIONS

pretreatment

ACID

TABLE

/;

.’

c

z ? 5

zi

2 52

.J

260

SAGRITALO,

CORTE,

AND

MCCOLL

et al. (1960) with DNP in releasing the inhibition of oxygen uptake by phenformin suggested certain experiments to be conducted in the dog. According to these workers, phenformin produces an increase in the concentration of lactic and pyruvic acids supposedly by producing a partial anaerobic state, and as a result glucose uptake is increased together with the production of lactic acid. At the same time unused pyruvic acid accumulates. Accordingly, experiments were undertaken to determine the effect of these compounds on lactic and pyruvic acid concentrations in blood. Tables 3 and 4 summarize the results obtained following administration of phenformin, chlorpropamide, and DNP at the doses and combinations shown for an 1l-day period. Phenformin alone produced an increase in both lactic and pyruvic acids while chlorpropamide treatment resulted in a slight decrease. DNP produced adecreaseinlactic acid and a small increase in pyruvic acid. The concurrent administration of chlorpropamide with phenformin resulted in a marked reduction in the concentrations of lactic and pyruvic acids as compared with those following phenformin administration. When DNP was administered with phenformin, the blood concentrations of lactic and pyruvic acids were also reduced from those seen with phenformin alone. DISCUSSION The in vitro data presented demonstrate that chlorpropamide at the concentrations employed is capable of uncoupling oxidation from phosphorylation in mitochondrial preparations. This is shown by the reduction of P/O ratio to a degree proportional to the concentration and by the nonadditive reduction in oxygen and phosphate uptake by the chlorpropamide-phenformin combination. The lesser and nonadditive effect of the combined drugs on phenformin produced inhibition of oxygen uptake in rat liver mitochondria may be considered further evidence of uncoupling activity. The results are consistent with the data of Hollunger (1955) and Krueger et al. (1960), who demonstrated that an uncoupling agent (DNP) will release the inhibition of oxygen uptake induced by guanidine derivatives (including phenformin) in rat liver and rabbit kidney mitochondria. The present results are in excellent agreement with those of Krueger et al. (1960) except that chlorpropamide was substituted for DNP as the uncoupling agent. Krueger et al. (1960) concluded that their results were consistent with the concept of Hollunger (1955) that phenformin produces its in vitro metabolic effects by a primary interference with the transfer of respiratory chain high energy bonds to ADP and secondarily blocking electron transfer. This results in a decreased oxygen uptake and a partial anaerobic state. The increase in lactic and pyruvic acids can be explained on this basis. The mode of action of phenformin as an antidiabetic agent is still debatable. The recent observations of Searle and Cavalieri (1968) that phenformin enhances glucose oxidation to CO* is still not inconsistent with the hypothesis that it exerts its stimulatory effect on glucose uptake by the inhibition of aerobic glycolysis. The observation that chlorpropamide has uncoupling activity has not previously been reported. It is very doubtful if this has any relationship to its hypoglycemic activity in man, as the concentrations employed in these experiments are 4-10 times greater than clinically effective serum chlorpropamide concentrations (Sheldon et al., 1965). In any event it is approximately 300 times less potent in vitro than DNP as an uncoupling agent. On the basis of the ability of chlorpropamide to antagonize the phenformin-induced

CHLORPROPAMIDE

PHENFORMIN

MORTALITY

261

increasein lactic and pyruvic acids in the dog, it seemsreasonable to assumethat this is in fact due to the uncoupling activity. The similar effect of DNP in theseexperiments is further evidence for this. Finally, it must be kept in mind that these observations may apply only to the dog. Ungar et al. (1957) originally reported that in the dog phenformin was not hypoglycemic but produced a “complex effect.” Houssay and Penhos (1958) noted that an acute toxic effect preceded the hypoglycemic response. Ashkar et a/. (1958) also observed that, when large doseswere given iv in the dog, the toxicity which resulted was mainly due to hypotension and to action on the CNS. Penhos and Blaquier (1958) observed that epincphrine, glucose, and hydrocortisone protected rats but not dogs from the mortality produced by the dosesof phenformin used. They also reported no hypoglycemia in depancreatized dogs. Chenier et a/. (1968), in studying the acute toxic effect of phenformin and chlorpropamide in combination, did not observe any antagonistic effect of chiorpropamide in rats or mice. It may. therefore, be that these effects are speciesspecific. ACKNOWLEDGMENTS Thanks are due to Mr. Salvatore Galloro, Mr. Luigi Scaglione, and Mrs. Sian Robinson their skillful technical assistance in various aspects of the study.

for

REFERENCES ASHKAR, E., BURRIER, C. N., and RAMOS, M. C. D. P. (1958).Farmacologiade la fenetildiguanida. Rev, Sot. Argent. Biol. 34, 11-20. BEASER, S. B. (1958). Therapy of diabetes mellitus with combinations of drugs given orally. N. Engl. J. Med. 259, 1207-1210. BUEDING. E., and WARTIS, H. (1940). The stabilization and determination of pyruvic acid in the blood. J. Biol. Chem. 133, 585-591. BURNS, J. J., CUCINELL, S. A., KOSTER, R., and CONNEY, A. H. (1965). Application of drug metabolism to drug toxicity studies. Anu. N. Y. Acad. SC;. 123, 273-286. CHENIER. L. P., MAXWELL, J., and IRVING, H. (1968). Migration de la toxicit aiguC et du coCfficient de regression avec des combinaisons variCes de chlorpropamide et de phenformin. Personal communication: to be published. CLARKE. B. F., and DUNCAN, L. J. P. (1965). Combined metformin-chlorpropamide therapy in 108 sulfonylurea-failures. Lancet 2, 1248-1251. CROSS, R. J., TAGGART, J. V., Covo, G. A., and GREW D. E. (1949). Studies on the cyclophorase system. VI. The coupling of oxidation and phosphorylation. J. Biol. Chrm. 177. 655-678. FINEBERG, I. K. (1968). Combinations of oral hypoglycemic drugs in obese insulin-resistant diabetics. Geriatrics 23, 137-146. FISKE, C. H., and SUBBAROW, Y. (1925). The calorimetric determination of phosphorus. J. Biol. Chem., 66, 375-400. FREEDMAN, L., BLITZ, M., GUNSBERG, E., and ZAI~, S. (1961). Determination of phenformin in biologic fluid and tissues. J. Lab. C&l. Med. 58, 662-666. HOLLUNGER, G. (1955). Guanidines and oxidative phosphorylation. Actu Phcrmacol. Tuxicol. 11. Suppl., l-84. HOUSSAY, B. A., and PENHOS, J. C. (,1958). Action hipoglucemiante de la fenetildiguanida. Rec.. Sot. Avgent. Biol. 34, 53-63. HUMMEL, J. P. (19491. The flurometric determination of malic acid. J. Biol.Ch~~. 180, 12% 1228. KATZ, H. M., and BISSEL, G. (,1965). Blood sugar lowering effects of chlorpropamide and to]butamide. Diabetes 14, 650-657.

262

SAGRITALO,

CORTE,

AND

MCCOLL

F. A., SKILLMAN, T. G., HAMURI, G. J., GRUBBS, R. C., and DANFORTH, N. (1960). The mechanism of action of hypoglycemic guanidine derivatives. Diabetes 9, 170-173. OLSON, G. F. (1962). Optimal conditions for the enzymatic determination of L-lactic acid. Clin. Chem. 8, l-10. PENHOS, J. C., and BLAQUIER, J. A. (1958).Toxicidad dela fenetildiguanida.Rev. Sot. Argent. KRUEGER,

Biol. 34, 21-28.

P. G. (1956).Studieson fatty acid oxidation. 5. The effect of decanoicacid on oxidative phosphorylation. Can. J. Biochem. Physiol. 34, 1227-1232. SEARLE, G. L., and CAVALIERI, R. R. (1968).Glucosekineticsbefore and after phenformin in the human subject.Ann. N. Y. Acad. Sci. 148, 734-742. SHELDON, J., ANDERSON, J., and STONER, L. (1965).Serumconcentrationsand urinary excretion of oral sulfonylurea compounds.Diabetes 14, 362-367. SINGER, D. L., and HURWITZ, D. (1967).Long-term experience with sulfonylureasand placebo. SCHOLEFIELD,

N. Engl. J. Med. 277,45&456.

G., FREEDMAN, L., and SHAPIRO, S. L. (1957).Pharmacologicalstudiesof a new oral hypoglycemicdrug. Proc. Sot. Exp. Biol. Med. 95, 190-192. UNGAR, G., PSYCHOYOS, S., and HALL, H. A. (1959).Action of phenethylbiguanide,a hypoglycemic agent, on tricarboxylic acid cycle. Metabolism 9, 36-51. UNGAR,