Systemic toxicity of polyinosinic acid: Polycytidylic acid in rodents and dogs

Systemic Toxicity Polycytidylic Acid

of Polyinosinic Acid: in Rodents and Dogs

BARRIE M. PHILLIPS,RALPH E. HARTNAGEL,PAUL J. KRAUS, RUY PEREZ TAMAYO, EMMA H. FONSECA, AND ROBERT L. KOWALSKI Pharmacology-Toxicology Laboratory, Therapeutics Research Division, Miles Laboratories, Inc., Elkhart, Indiana 46514, and Institute de Investigaciones Biomedicas, Universidad National Autonoma de Mexico, Mexico, D.F. Received August 22,1969

SystemicToxicity of Polyinosinic Acid: Polycytidylic Acid in Rodents andDogs. PHILLIPS,BARRIE M., HARTNAGEL, RALPH E., KRAUS,PAULJ., PEREZ TAMAYO, RUY, FONSECA, EMMA H., and KOWALSKI, ROBERT L. (1971). Toxicol. Appl. Pharmacol. 18, 220-230. The double-stranded polymer of polyinosinic acid: polycytidylic acid (poly I :C), an interferon inducer, was well tolerated by mice, rats, and guinea pigs following the acute iv or ip administration of 20 mg/kg doses.The acute LD50 of Poly I: C wascalculatedto be 75 and 150mg/kg in the mouseand 185and 365 mg/kg in the rat following iv and ip administration, respectively.Poly I :C waslethal to mongrel dogsfollowing acute iv dosesas low as 2.5 mg/kg; toxicity was characterized by depressionof spontaneousmotor activity, incoordination, vomiting, and flaccid prostration. Subchronic (42 days) administrationof poly I :C at daily dosesof 2.0 mg/kg ip to rats or 0.2 mg/kg iv to beagledogs was not accompaniedby signsof toxicity. However, severe toxic manifestationswere elicited in dogs upon the subchronic administrationof poly I: C at a doseof 2.0 mg/kg daily iv, and 3 of 6 dogs died during the course of the experiment. Toxicity was characterized by decreasedspontaneousactivity, incoordination, vomiting, anorexia, weight loss, hematologic changesreflecting decreasedhematopoiesis,increased alkaline phosphataseand transaminaseactivities, thymus degeneration, destructionof bonemarrow, dilatation of hepaticsinusoidalcapillariesin the centrolobular areas,necrosisof liver cells, collapseof hepatic structures, and a generalizedarteritis. It was concluded that useful systemictherapeutic application of poly I :C will require that the effective dosesbe quite small,but that, sincedaily dosesof 2.0 mg/kg in the rat and 0.2 mg/kg in the dog werewell tolerated in subchronicexperiments,cautiousexploration of low systemicdosesin man may be justified. Poly I: C is an interferon inducer (Field et al., 1967)which hasbeenshown to promote recovery from keratoconjunctivitis produced in the rabbit eyeby herpessimplex (Park and Baron, 1968)andtoincrease thelifespanofmiceinoculatedip withlethaldosesofl-1210 leukemic ascitescells (Zeleznick and Bhuyan, 1968).Poly I: C at singledaily iv dosesof 1.Omg for 6 days or at daily topical (ocular) dosesof up to 0.6 mg for 21 days did not elicit grosstoxicity in the rabbit (Park and Baron, 1968), but daily ip administration of 220

SYSTEMIC TOXICITY OF POLY I : C

221

the polymer at 20 mg/kg to mice inoculated with L-1210 leukemic ascites cells resulted in early deaths (Zeleznick and Bhuyan, 1968). The present communication describes the toxicity observed following acute or subchronic ip or iv administration of poly I : C to mice, rats, guinea pigs, or dogs. Immature animals were employed in some of these studies since it was of interest to evaluate the potential risk of toxicity following parenteral administration of poly I : C to the young. METHODS Compound Batch number ll-3-211 of poly 1:C was employed. Solutions of the polymer were prepared, using sterile technique, in pyrogen-free physiological saline, at concentrations of 0.02-l 5 %. The solutions were dispensed into multiple-dose vials, each containing enough material for 1 day of injections, which were kept frozen until shortly before use. Acute Toxicity All animals employed in acute studies were allowed a commercial dry food2 and water adlibitum and, with the exception of mice, were caged individually; mice were caged in groups of 5 of the same sex. All animals were observed for 14 days following polymer administration. LD50 values were calculated according to Litchfield and Wilcoxon (1949). Mouse. Groups of 5 male and 5 female Carworth CF-1 mice (21-30 g) received poly I: C, as a 1.5 % solution, iv at a dose of 75, 150,225,300, or 375 mg/kg or ip at a dose of 150, 300, 450, 600, or 750 mg/kg. Rut. Groups of 5 male and 5 female Charles River COBS-CD rats (8&l 28 g) received poly I: C, as a 1.5 % solution, iv at a dose of 75,150,225,300, or 375 mg/kg or ip at a dose of 150,300,450,600, or 750 mg/kg. Dog. Thirty immature mongrel dogs (1.6-10.6 kg) were randomly assigned to 5 groups (3 males and 3 females each) which received 2.5,5, 10, 15, or 20 mg poly I : C/kg iv as a 1.Oy/, solution. Subcizronic Toxicity Rat. Sixteen male and 16 female immature Charles River COBS-CD rats (males 102-l 12 g, females 7&103 g) were randomly assigned to 2 groups of 8 males and 8 females each. An additional 5 males and 5 females from the same shipment of rats were sacrificed in order to evaluate pretreatment histology. The rats were caged individually and received 2.0 mg poly I: C/kg, as a 0.04 % solution, or 0.5 ml/100 g sterile pyrogen-free physiological saline once daily ip for 42 days. This single dose level was employed since preliminary experiments indicated it would have no effect and since it was some 500-1000 times the human dose anticipated at the time. Water and a commercial dry food2 were available ad libitum. Body weights and food consumption values were obtained on day zero and twice each week during the study. Each rat was observed daily for behavioral changes and other pharmacologic responses. 1 Research Products Division, Process Industries Group, Miles Laboratories, Inc., Elkhart, Indiana. z Wayne Lab Blox, Wayne Guinea Pig Diet, Wayne Dog Food; Allied Mills, Inc., Chicago, Illinois,

222

PHILLIPS

ET

AL.

On days 20 and 41, 3 males and 3 females from the control and treated groups were transferred to individual metabolism cages for collection of urine. Twenty-four hours later these rats were anesthetized with chloroform and blood samples were obtained by open chest cardiac puncture. Clinical laboratory determinations (hemoglobin, hematocrit, total erythrocyte count, total and differential leukocyte counts, platelet estimation, erythrocyte appearance, serum total protein, serum and urinary sodium, potassium, and chloride, SGPT, serum glucose, serum urea nitrogen, urine volume, pH, and specific gravity, urinary protein, glucose, occult blood, ketones, bile, and microscopic solids) were made on the blood and urine samples obtained from these animals. All determinations were made using standard methods. The exsanguinated animals were then perfused with 0.9 % aqueous sodium chloride and subsequently with 10% buffered neutral formalin. A complete gross pathologic examination was performed. The liver, spleen, heart, brain, kidneys, thyroids, adrenals, reproductive organs, pituitary, and thymus were removed and weighed. Appropriate samples of these tissues plus lung, aorta, pancreas, salivary gland, esophagus, stomach, duodenum, jejunum, ileum, colon, urinary bladder, eye, parathyroid, bone and marrow, lymph node, and muscle were further fixed in formalin (with the exception of thymus, spleen, and lymph node, which were further fixed in Zenker solution). The fixed tissues were embedded in paraffin, cut at 7 p (14-p sections were cut from frozen sections of liver, kidney, and adrenal), stained with hematoxylin and eosin, Oil Red 0 (frozen sections), or periodic acid-Schiff (additional sections of kidney only), and subjected to histopathologic examination. On day 42, all surviving animals were killed with chloroform and subjected to gross and microscopic pathological examinations as described above. Dog. Nine male and 9 female purebred immature (9-12 weeks old) beagle dogs3 (males 2.0-3.6 kg, females 2.0-2.9 kg) were assigned at random to 3 groups of 3 males and 3 females each. The animals had been dewormed and immunized against distemper and infectious hepatitis prior to purchase. The dogs were caged individually and received 0.2 or 2.0 mg poly I:C/kg, as 0.02 or 0.2% solutions, or 1.0 ml/kg sterile pyrogen-free physiological saline iv once daily for 42 days. Water was available ad libitum, and 150 g of commercial canned dog food4 was offered to each dog twice daily. Food consumption was determined daily and averaged over weekly intervals, and body weights were recorded weekly. Each dog was observed daily for behavioral changes and other pharmacologic responses. Samples of blood and 24-hr urine collections were obtained from each dog on 3 pretreatment occasions and at the end of weeks 1, 2, 4, and 6 for determination of various clinical laboratory parameters (as described previously for the rat, plus prothrombin time, alkaline phosphatase, carbon dioxide combining power, and SGOT). Similar determinations were made on a sample obtained from a dog in moribund condition. At termination, a complete physical examination was performed on each dog, the dogs were killed with an iv overdose of barbiturate, and autopsied and sampled (including the gallbladder) as described previously for the rat. 3 Laboratory Research Enterprises, Kalamazoo, Michigan. 4 Fromm Blue Label Dog Food, Federal Foods, Inc., Thiensville, Wisconsin.

SYSTEMIC TOXICITY OF POLY I:C

223

RESULTS Acute Toxicity The acute iv LD50 of poly I: C was calculated to be 75 (19-48)5 and 185 (128-234) mg/kg in the mouse and rat, respectively, while the acute LD50 following ip administration of the polymer proved to be 150(96-236) and 365 (3 15-423) mg/kg in the mouse and rat, respectively. Toxicity was characterized by depressionof spontaneousactivity, flaccid prostration, and diarrhea. While generally occurring promptly at higher doses, death was frequently delayed one to several days at lower doses. In a preliminary experiment, an acute ip dose of 20 mg/kg poly I: C (as a 0.8 9; solution) was well tolerated by 5 male and 5 female random-bred guinea pigs (222-348 g). Depressedspontaneous activity was observed within 4 hr of polymer administration, but all animals had resumed normal activity within 24 hr. Although a valid LD50 could not be calculated, poly I :C proved to be lethal at considerably lower doses in dogs. Toxicity was characterized by depression of spontaneous activity, incoordination, vomiting, and flaccid prostration. Observations made in the course of this experiment are summarized in Table 1. TABLE 1 ACUTE

EFFECTS

OF

I : C ADMINISTERED

POLY

INTRAVENOUSLY

TO IMMATURE MONGREL Dons

Mortality Dose”

Male

2.5

l/3

5.0

l/3

213 Depressedspontaneousactivity within 10-20 min, and flaccid prostration within 2-5 hr; 1 male and 1 femalevomited within 3 hr. One femaledied at 3 hr, the other 2 deathsoccurred within 22 hr. Survivors were normal at 22 or 45 hr.

10.0

213

l/3

Depressedspontaneousactivity within 10 min. One male convulsed at 2 hr, death followed at 3 hr. Two other malesand 2 femaleswere prostrate and flaccid within 2 hr; death followed in a male and female within 3 hr. One female was normal in 3 hr, the remaining survivors recovered in 45 hr.

15.0

213

3/3

Depressedspontaneousactivity within 20min, followed by flaccid prostration and death in 5 animals within 2-4 hr. The single survivor vomited at 20 min and remaineddepressedat 22 and 45 hr, but recoveredwithin 72 hr

20.0

313

313 Ataxia within l-5 min, followed by flaccid prostration and death within 2-15 hr

a Poly

I:C, mg/kg

5 95 ‘A confidence

Female Observations 3/3 Depressedspontaneousactivity and vomiting within 20-120min. Flaccid prostration in 1femalein 2 hr, followed by deathin 3hr. Two other femalesand 1maledied within 15 hr; survivors were normal at 72 or 96 hr.

body limits.

weight,

as a 1.0%

solution.

224

PHILLIPS

ET AL.

Subchronic Toxicity Rat. Daily ip doses of 2.0 mg/kg poly I: C were well tolerated in the rat. There were no deaths, and no unusual responses were observed in either male or female rats. Poly I: C-treated female animals gained significantly less weight than controls, but food consumption was not influenced in either sex. With the exception of a reduction in hematocrit and serum sodium observed in female rats on day 21, poly I : C administration did not influence hematologic or blood or urine chemical values in either sex. The finding of an apparent increase in serum glucose in females on day 21 appears to be the result of an unusually low value in controls. No poly I: C-related gross pathologic changes were observed in any of the rats receiving ip injections of the polymer, but organ weight and/or weight ratio changes were observed in the adrenals and spleen of poly I: C treated rats. Adrenal weight and spleen to body weight ratio were increased in female rats sacrificed on day 21, adrenal weight and adrenal to body weight ratio were increased in both males and females sacrificed at termination, and the spleen to body weight ratio was increased in females sacrificed at termination. However, microscopic examination of the various tissues did not reveal any poly I:C-related pathologic changes, and the significance of the organ weight changes is not clear. The influence of poly I: C treatment on several of these parameters is summarized in Table 2. Without exception, the values for those parameters not listed were no different from control values. Dog. Poly I: C was well tolerated by dogs at the 0.2 mg/kg dose level. No consistent treatment-related responses were observed, although vomiting was seen occasionally. Poly I : C was not well tolerated at the 2.0 mg/kg dose level. One male and 1 female dog were sacrificed in extremis on days 27 and 36, respectively, and a second female dog was found dead on day 42. Observations in the 3 dogs which were sacrificed or which died were frequent vomiting, depressed spontaneous activity, and incoordination. Anorexia and weight loss were observed early in the study. Similar responses were observed in surviving dogs at this dose level, but the reactions were neither as frequent nor as severe. In addition to these typical responses, the female dog that died on day 42 appeared, from day 7 until death, to have difficulty remaining erect, and the head was held in a cocked position. Meaningful alterations in clinical laboratory values were observed only at the high dose level. Hematologic changes, which were definitely more marked in females, reflected decreased hematopoiesis. Changes in blood chemical values were large increases in alkaline phosphatase, SGOT, and SGPT activities, slight elevations in serum urea nitrogen, and marginal changes in serum electrolytes. The influence of poly I: C treatment on these parameters is summarized in Table 3. No gross pathologic change was observed at the low dose level, although the adrenal to body weight ratio was elevated significantly. Degeneration of the thymus was observed in all 6 dogs at the high dose level. The pathologic change, interlobular fatty infiltration, was most severe in the 3 animals which died or were sacrificed in extremis. Pulmonary congestion was also observed in 5 of the 6 dogs receiving 2.0 mg/kg poly I: C. Kidney and adrenal weights and organ to body weight ratios were elevated significantly, and thymus weight and weight ratio were depressed significantly. Similarly, microscopic changes were seen only in dogs given the high dose level;

OF POLY

y P -: 0.05.

Body weight (g) Initial Day 21 Terminal Gain Food consumption (g/100 g B.W.) Day 4 Day 21 Day 38 Hematocrit (g/100 ml) Day 21 Day 42 Serum sodium (meq/l) Day 21 Day 42 Serum glucose (mgi100 ml) Day 21 Day 42 Organ weights (g/100 g B.W.) Adrenal Day 21 Day 42 Spleen Day 21 Day 42

Parameter

INFLUENCE

I:C

0.374 0.285

3

156.33 136.33

3 3

0.0241 0.0183

147.33 148.33

3 3

3 5

52.50 57.17

3 3

Males

2.52 3.22

mt +

2

0.494 0.307

i T

0.056 0.056

0.0281 0.0253

140.67 142.33

146.33 145.33

53.17 51.83

12.99 8.69 8.10

108.2 246.4 326.6 218.4 0.62 0.46 1.17

0.58 2.89

z-c -+

0.073 0.024

; 0.003 .I~ 0.002”

+ 20.30 ~7 10.50

.r Ii;

-k 2.02 z!z 2.52

t + +

zk 1.06 + 9.95 * 17.09 -c- 16.54

0.410 0.211

0.0457 0.0354

114.33 142.33

148.67 141.33

56.17 55.50

11.77 10.05 10.15

78.6 175.0 206.6 128.0

0.58 1.16

0.58 5.77

0.77 0.42 0.75

6.11 5.83 5.65 4.98

.: 0.025

0.029

0.002 -I

.I:

0.002

~!

-6 19.50 . 14.15

i i

-c l

Ik i*

k zik t zk

Control

0.519 0.279

0.0502 0.0518

150.67 148.00

A A-

0.018" 0.016"

t 0.001” + 0.006“

I 6.11” - 37.24

1.73

It

139.00

2.93" 4.92

0.90 1.26 1.45

11_ 2.08"

t t-

+ $ l

4.82 7.65 6.79 4.55"

144.67

46.67 55.50

13.71 12.62 11.78

xt

zk

104.4

f +

89.0 169.0 193.4

Females

LABORATORYPARAMETERS

Mean value 1 SE

Poly I : C

I 0.001 k 0.001

20.60 30.89

5.29 4.37

+ zt

t I

0.67 0.54 0.73

k k t

I13.37 +13.00

k 1.69 + 10.45

Control 105.8 252.2 316.8 211.0 12.85 9.60 7.05

5

TABLE WEIGHT, FOOD CONSUMPTION, CLINICAL AND ORGAN WEIGHTS IN RATS

5 5

8

N

ON BODY

h) . .

i;

z kI

%

4

226

PHILLIPS ETAL.

TABLE 3 MAJOR ABNORMAL CLINICALLABORATORYFINDINGSIN

DOGS RECEIVING 2.0 MG/KG PolyI:C

Dog No.

Alterations observedin clinical laboratory parameters

215 M

Thirteen days prior to death, alkaline phosphataseactivity wasincreased4-fold to 106K.A. units, SGOT and SGPT activities were increased15 and 25 times, to 625 and 790 units, respectively, serumsodiumand potassiumwere decreased slightly, and serumurea nitrogen was increasedabout 70%, to 23.4mg/lOOml SGPT activity elevated, as much as 6-fold, on 3 of 4 samplingoccasions,to a maximum of 190units. Serum chloride and sodiumwere reducedslightly on 2 occasions,and serumpotassiumwas low, asmuch as 20% on all 4 occasions. Serum urea nitrogen was elevated on all 4 occasions,to a maximum of 19.8 mg/lOOml. Total leukocyteswerereduced,to a maximumof 15%, on 2 occasions and the hematocrit waslow by a maximum of 9 %, on 2 occasions Alkaline phosphatase,SCOT, and SGPT activities wereelevatedon the last 3 of 4 samplingoccasions,to maxima of 96.5 K.A. units, 118units, and 220 units, respectively.Serumurea nitrogen elevatedon all 4 occasions,to a maximumof 24.6 mg/lOOml. The hematocrit was reduced,by a maximum of 12x, on the last 2 occasions Alkaline phosphatase and SGPTactivities wereincreasedon the last3 of 4 sampling occasions,to maxima of 92 K.A. units and 340 units, respectively. Serum glucosewasreducedon all 4 occasions,to a low on 1 occasionof 68 mg/lOOml. Serumurea nitrogen waselevatedon all 4 occasions,maximum of 26.4mg/lOO ml. Hemoglobin and erythrocyte counts were reducedon the last 2 occasions, by a maximum of 15%. Hematocrit wasreduced,by a maximum of 20x, on the last 3 occasions Terminal total protein wasreduced 11%. Alkaline phosphataseand SGPT activities wereelevatedon the last 3 of 4 samplingoccasions,to maximaof 104K.A. units and 1440units, respectively; SGOT activity waselevated13-foldon day 14. Serumchloride and sodiumwere elevatedslightly on the last 2 occasions,and the last 2 glucosevalueswerereducedslightly. Serumureanitrogen waselevated on 3 occasions,to a maximum of 25.8mg/lOOml. Hemoglobinand hematocrit were reduced,to maxima of 50 and 45%, on the last 2 occasions,and the last erythrocyte count wasdown 14% Alkaline phosphataseand SGPT activities increasedon the last 2 of 3 sampling occasions,to maxima of 96.5 K.A. units, and 500 units respectively; SGOT activity was increasedto 1180units on day 14. Serum sodium was increased slightly on the last 2 occasions.Serumurea nitrogen waselevatedon all 3 occasions, to a maximum of 32 mg/lOOml on day 7, but eventually dropping to 25.0 mg/lOOml. Hemoglobin, hematocrit, and total erythrocyte counts were reduced on the last occasion,by ll-17%. On 2 occasionsthe total leukocyte count wasreduced, by no more than 20%

220 M

226 M

213 F

223 F

224F

3 major types of lesionswere observed. Destruction of bone marrow, with replacement by loose fibrous tissue and hemorrhagic material of recent origin, was seenin the two dogs sacrificed in extremis. Hepatic changeswere dilatation of sinusoidal capillaries in the centrolobular areas, necrosis of liver cells, and collapse of the structures, and were seenin 4 dogs. Generalized, acute, focal arteritis, involving different organs, was seen in 5 of the 6 dogs. With respect to the latter lesion, the arteries involved were of different sizes, and the type of lesion was different according to size; small arteries were completely replaced by fibrinoid material, at times with obliteration of the lumen, while

SYSTEMIC

TOXICITY

OF POLY

I :C

127

FIG. 1. Acute vasculitis in the kidney, showing focal involvement of the upper half of the wall. There is infiltration by fibrinoid material in the subendothelial and adventitial layers, as well as many polymorphonuclear leukocytes, PAS stain. x100.

228

PHILLIPS ET AL.

FIG. 2. Higher power view of the wall of another blood vessel with acute inflammation, to illustrate infiltration by fibrinoid material and polymorphonuclear leukocytes in the adventitia. PAS stain. x 150.

SYSTEMIC

TOXICITY

OF POLY

I:C

229

230

PHILLIPS

ET AL

larger arteries showed involvement of the outer muscular layer and adventitia, also with scanty inflammatory infiltrate. The kidney was always involved, while other organs (salivary glands, stomach, duodenum, gall bladder, urinary bladder, epididymus, thyroid, heart, and adrenal) were less frequently affected. Photomicrographs of representative vascular changes are shown in Figs. 1 and 2. The influence of poly I: C on body weight, food consumption, and organ weights in dogs is summarized in Table 4. DISCUSSION

At the doses employed in these experiments, clinically observable pharmacologic responses to poly I: C were seen primarily in the dog (except for depressed spontaneous activity, flaccid prostration, and diarrhea seen in rodents following the administration of larger acute doses of the polymer). Gross effects in dogs in both the acute and subchronic experiments (only at 2.0 mg/kg in the latter case) were characterized by decreased spontaneous activity, incoordination, vomiting, flaccid prostration, anorexia, and weight loss. With the exception of changes in spleen and adrenal weights (which were not accompanied by microscopic evidence of pathologic change) in the rat, meaningful toxicity in the subchronic experiments was seen only in dogs receiving 2.0 mg poly I:C/kg. Three major types of pathologic changes were observed; bone marrow destruction, hepatic changes (dilatation of sinusoidal capillaries in the centrolobular areas, necrosis of liver cells, and collapse of the structures), and arteritis involving both small and larger arteries in numerous organs. Bone marrow destruction was observed in only 2 dogs, both of which were sacrificed in extremis prior to termination of the study. However, 1 of the 2 dogs did not manifest any hematologic disturbances, while 2 female dogs which did not exhibit bone marrow changes evidenced significant reductions in hemoglobin, erythrocyte count, and hematocrit. Hepatic changes were always accompanied by marked elevations in transaminase activities, and in fact transaminase activities were elevated in all 6 dogs. These findings indicate that poly I:C elicited significant toxicity in the dog. Thus, while poly J: C has been shown to be free of gross toxicity when applied topically to the eye at therapeutic dose levels (Park and Baron, 1968), there would appear to be a very real risk of serious toxicity when the compound is administered systemically; useful systemic therapeutic application will require that the effective doses be quite small. On the other hand, the finding that 2.0 mg/kg in the rat and 0.2 mg/kg in the dog represent dose levels which were well tolerated for 42 days would seem to indicate that, ultimately, cautious exploration of low systemic doses in man may be justified. REFERENCES G. P., and HILLEMAN, M. R. (1967). Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes. Proc. Nat. Acad. Sci. U. S. 58, 1004-1010. LITCHFIELD, J. T., and WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96, 99-113. PARK, J. H., and BARON, S. (1968). Herpetic keratoconjunctivitis: Therapy with synthetic double-stranded RNA. Science 162, 811-813.

FIELD, A, K., TYTELL, A. A., LAMPSON,

ZELEZNICK, L. D., ~~~BHUYAN, stranded

polyribonucleotides.

(1968). Treatment ofleukemic(l-12iO)micewithdoubleProc. Sot. Exp. Biol. Med. 130, 126-128.

B. K.