Response of dogs to repeated intravenous injection of polyethylene glycol 4000 with notes on excretion and sensitization

TOXICOLOGY

AND APPLIED PHARMACOLOGY

Response with

18, 3540(1971)

of Dogs to Repeated Intravenous of Polyethylene Glycol4OUO Notes on Excretion and Sensitization’

Injection

CHARLES P. CARPENTER, MURRAY D. WOODSIDE, EDWIN R. KINKEAD, JOHN M. KING, AND LLOYD J. SULLIVAN Carnegie-Mellon Pittsburgh, Received

University, Mellon Institute, Pennsylvania 15213 December

8, 1969

Response of Dogs to Repeated Intravenous Injection of Polyethylene Glycol 4000 with Notes on Excretion and Sensitization. CARPENTER, CHARLES P., WOODSIDE,MURRAY D., KINKEAD,EDWIN R.,KING,JOHN M., and SULLIVAN, LLOYD J. (1971). Toxicol. Appl. Pharmacol. 18, 3540. Repeated intravenous injections of sterile 10 y’, polyethylene glycol4000 in 0.85 % aqueous NaCl caused no detectable ill effects in groups of 9 beagles on dosages of 90, 30, 10, and 0 mg/kg/day. Two dogs from each level were killed after 43 daily injections in 2 months, 2 more after 99 injections in 6 months, and the remaining 5 after 178 injections in 12 months. There were no statistically significant differences between any of the injected groups and the control group that received the 0.85 o/0NaCl vehicle in an amount equivalent to the volume of the highest dosage, i.e., 9 ml. The criteria of effect included body and organ weights, hematology, biochemical tests, gross and histopathologic examination of cranial, thoracic, and abdominal organs. Two importantly related studies reveal that PEG 4000 is not a sensitizing agent in guinea pigs and that the major routes of excretion of 14C equivalents by rats after iv and po administration are via urine in the first instance and feces in the second. Its proposed use as a complexing agent in the preparation of a high purity antihemophilic factor for human intravenous use prompted these studies. These studies were undertaken to determine the effect of repeated intravenous administration of small amounts of polyethylene glycol4000 (CARBOWAX 4000)’ used as a complexing agent which might accompany the administration of a High Purity Antihemophilic Factor (Polson et al., 1964; Johnson et al., 1966; Howell-Karpatkin et al., 1967) for the treatment of hemophilia. The investigation of polyethylene glycol 4000 (PEG 4000) and a summary of the more important toxicologic findings emanating from this laboratory were reported by Shaffer and Critchfield (1947a, b) ; Shaffer et al. (1948a, b, 1950); Smyth et al. (1947, 1950, 1955); and Carpenter and Shaffer (1952). Its use by a major drug company in preparations for intramuscular administration is to be found in their literature.3 I This study was supported by Union Carbide Corporation to aid the American Red Cross in the development of a High Purity AntihemophilicFactor. 2 Registeredtrademarkof Union CarbideCorporation, who manufacturedand suppliedthe compound. 3 Upjohn Company, 7171 Portage Road, Kalamazoo, 35

Michigan 49001.

36

CARPENTER

ET AL.

Before the injection studies were undertaken, the absence of sensitizing potential was established by a modified Landsteiner test using male albino guinea pigs. Subsequently, the major pathways of excretion for the rat were established after administration by both the iv and po routes. The dosage regimen for dogs was designed to parallel the dosage requirements for the treatment of humans afflicted with hemophilia. Preliminary potency assays by the American National Red Cross Laboratory at the New York University Medical Center revealed that the amount of PEG 4000 that would be contained in the average human dose of the High Purity Antihemophilic Factor (AHF) would be equivalent to 10 mg/kg of body weight. Accordingly, dosages of 1, 3, and 9 times this level or 10, 30, and 90 mg/kg of PEG 4000/kg of body weight were selected to evaluate any possible effects and to establish a reasonable safety factor. Because the initial treatment of hemophilia may be quite intensive, it was decided to administer 5 doses each week for a 2-month interval to simulate such treatment. It was also postulated by the clinicians that extensive therapy would not be likely to exceed three injections per week, and the latter regimen was adopted for the subsequent intravenous injections. METHODS Beagles ranging from 7 months to 5 years of age were distributed randomly by sex and by age to provide closely matching groups of 9 dogs each. Five dogs from each level were randomly selected to receive the full complement of doses and to have all tests performed upon them at each of the serial sacrifice intervals. This left 4 dogs in each group available for earlier evaluation of gross and micropathology. It was decided that one of each sex at each level should be killed after 2 months and 6 months. This plan allowed for 5 dogs to receive injections for the entire duration of the study. Injections were made in the cephalic veins of the foreleg or the saphenous of the hindleg. To restrict the volume of the largest daily injection to about 10 ml per dog, the PEG 4000 was prepared in sterile solution at a 10% concentration. When this concentration is administered by means of a 23-gauge needle the speed of delivery is restricted to a rate at which any possibility of damage to the red blood cells is unlikely. Sterilization was achieved by passage of the 10% PEG 4000 solutions made in 0.85% NaCl through a sterile No. GS WPO47SO Millipore Filter.4 Prior to each use all glassware, needles and hypodermic syringes were sterilized at 15 lb pressure for 15 min in an autoclave. Injections were made under aseptic conditions to avert the possibility of infection. Each dog was examined carefully by the veterinary pathologist prior to dosing. The only abnormalities detected were several reducible hernias and one avascular optic disk. Records were made of these inherent deficiencies so there would be no confusion as to their causation. During the course of the study, appetite, body functions, and general behavior of each dog were observed daily when the dogs were fed or brought out for injection. They were weighed weekly for the first 8 weeks and monthly thereafter so that weight curves could be charted and individual doses adjusted. Any deviations from the control group were called to the attention of the veterinarian for follow-up. ’ Millipore

Corporation,

Bedford, Massachusetts 01730.

INTRAVENOUS

INJECTIONS

OF PEG

4000

37

Preliminary or baseline values on the subsequently described criteria of response were established on all dogs allocated to this study, prior to the first injection of PEG 4000, on blood drawn from the jugular vein. To follow any hematologic events that might transpire, hematocrits were run, leukocyte counts were made via the Coulter Counter, coagulation times were determined by the capillary tube method, and sedimentation of heparinized blood was recorded at intervals of 30 and 60 min. In addition, the following determinations were made on blood from the jugular vein : total protein and albumin (Natelson, 1963) serum alkaline phosphatase (Sigma Chemical Co., 1963) serum glutamic-oxaloacetic transaminase (SCOT), serum glutamicpyruvic transaminase (SGPT) (Sigma Chemical Co., 1964), blood urea nitrogen (BUN). and plasma prothrombin time (Warner Chilcott Lab., 1965). The dogs on the 90 mg/kg or highest dosage level were checked most frequently, for they would be the first to show evidence of any adverse response. Therefore, all of the hematologic and biochemical tests were done on this group and the controls after 2,4. and 6 weeks and after 2,6, and 12 months of dosing. All dogs on the 30 and 10 mg/kg levels were checked at the end of the 2nd, 6th and 12th month of dosing. Two animals per dosage level were killed, for tissue study and evaluation of effect upon internal organs, after the cessation of 2 months intensive dosing, 5 doses per week for 43 doses. The second sacrifice of 2 dogs was made after 4 more months on the reduced schedule of 3 doses per week or after a total of 95 doses in 6 months. The remaining 5 dogs per group were carried 12 months: during this time they received 178 injections of PEG 4000. At each of the serial sacrifices body weights were determined prior to killing, and kidneys, liver, and spleen were weighed after exsanguination. Representative tissues from the cranial, thoracic, and abdominal viscera were taken at autopsy for histopathologic evaluation after the usual hematoxylin-eosin stained sections had been prepared. When data from the full complement of 36 dogs were available, namely. for the preliminary and S-week biochemical and hematologic surveys, the chi square for homogeneity of variance and the F test for analysis of variance (Snedecor and Cochran. 1967) were used in the statistical calculations. Body weights were similarly evaluated. For the 2nd, 4th, and 6th week and the 2nd, 6th, and 12th month data, derived from 5 high level (90 mg/kg) and 5 control dogs, the F test and Fisher’s f test (Snedecor and Cochran, 1967) were utilized.

RESULTS

AND

CONCLUSIONS

Body Weight Analysis of variance and Bartlett’s test of homogeneity of variance were applied to the body weight data. No effect was found at any period of observation that differed in a statistically significant manner from the controls. This statement also applies to the depressionapparent in Fig. 1 during the final 5 months of the study. In a further check. the Wilcoxon rank sum test showed that the ranked weight changes of the groups of 5 dogs were not significantly different (Snedecor and Cochran, 1967). All of these animals were lean but in good physical condition and behaved normally throughout the study.

38

CARPENTER ETAL. 90 30 10 0

1

q/kg mg/kg mg/kg q/kg

-----._._,_.

-.-

0.9 E

0.8 -

-0.2 0

./‘)...““.

!‘?

3

I 6

I 9

I 12

I 15

I 18

I 21 Weeks

..... .

I 24

I 27 of

I 30

I 33

I 36

\ --I I 39

/I 42

I 45

1 48

!51

doses

FIG. 1. Mean body weight change (kg) for groups of 5 beagles receiving daily intravenous injections of polyethylene glycol4000 for 2 months (43 doses) followed by 3 injections per week for 10 months (178 doses total). Control group received physiological saline in volume comparable to highest dosage level.

Biochemical and Hematological Tests

Statistical evaluation of the parameters described above revealed no differences between the saline-injected control group and the three dosage groups. This applied to the determinations made preliminary to the study and for the six subsequent evaluations made during the year. As would be anticipated from the preceding paragraph, the 2 dogs from each of the 3 dosage levels that were killed after 43 or 95 injections at 2 or 6 months, respectively, were found to be unaffected by the treatment when hematologic and biochemical findings were compared to the saline-dosed controls. Gross and Micropathology

After 43 injections in 2 months the two dogs killed from each of the dosage levels were found to have neither gross nor microscopic lesions in any organ or tissue that were considered by the pathologist to result from the injections of the PEG 4000. Two dogs from each of the dosage groups likewise were killed after 95 doses so that histopathologic examination of cranial, thoracic, and abdominal viscera could be made. None showed any pathologic changes referable to the PEG 4000 administration. The finding of glomerular hyalinization in the kidney of the male dog on 10 mg/kg is considered by the pathologist to have been a sporadic lesion. The remaining 5 dogs in each of the 3 dosage groups were continued on the 3-day-perweek schedule for another 6 months to achieve a total of 178 doses during 12 months. There were no lesions, such as regional lymphadenopathy, Kupffer cell hyperplasia, or hypertrophy or pulmonary embolism, which might have been anticipated as sequelae to this rather formidable intravenous regimen. An average 10 kg beagle receiving 90 mg/kg/day for 178 days would have had to clear its blood of 160,200 mg of PEG 4000.

INTRAVENOUS

INJECTIONS

OF PEG

4000

39

This appears to have been acomplished without any evidence of tissue reaction. It is the opinion of our veterinary pathologist, who did the physical examinations and the autopsies and who studied the tissue sections of those dogs, that the few cases of testicular hypoplasia had no relationship to the administration of the PEG 4000. This condition occurs sporadically among untreated inbred, celibate laboratory beagles. Organ Weights No statistically significant differences from the saline-dosed controls were apparent when the data from both sexes that received the full complement of 178 injections were analyzed. Neither was there a difference in the response between the sexes on any given dosage level, nor was there any significant difference when all males were compared with all females. Route of Excretion of PEG 4000 A material balance or route of excretion study was done using 14C-labeled PEG 4000 in order to test the hypothesis, based on the results of our previously annotated repeated feeding studies, that such doses may be considered as a series of single doses because of their rapid elimination from the body. Study of the excretion pattern of 14C equivalents in rats revealed that this compound is so rapidly cleared after an iv dose of 10 mg (circa 70 mg/kg) that the major portion is excreted via the urine in 24 hours while po doses pass through the alimentary tract with but little absorption in a like interval. The mean cumulative, 7-day recovery after iv injection by tail vein was 8 I”/, . Of this, 617; appeared in the urine, none in the exhaled carbon dioxide, and 20% in the feces. Only one of the 10 rats retained any detectable radioactivity in the carcass, but the results on this animal were not considered reliable and were therefore excluded as only 42.9% of the 14C equivalents could be recovered. Peroral administration to rats resulted in a mean 7-day cumulative recovery of 86 “/;I. Only 4.1% appeared in the urine, none in the carbon dioxide, and the remainder was eliminated in the feces. Sensitization To ascertain that the parenteral use of PEG 4000 would not induce sensitization, a group of 20 male albino guinea pigs, weighing between 430 and 530 g were subjected to a modified Landsteiner intradermal sensitization test. After measuring and evaluating the initial response to a 0.05 ml intradermal dose, a series of 8 doses of 0.1 ml of a 0.1 y/, aqueous solution of PEG 4000 in 0.85% NaCl was given on alternate days, 3 per week. Following a 3-wk incubation period a challenge dose of 0.05 ml was given intradermally and the initial response on that animal was deducted from the final response as well as any similarly obtained response from the saline control. None of the guinea pigs was sensitized. Purification of the ethylene oxide starting material used in the current production of PEG 4000 has eliminated the sensitizing properties reported by Smyth et al. (1950) who utilized the identical intracutaneous test described above. Based upon these findings, it is concluded that polyethylene glycol4000, administered by repeated iv injection to dogs at 9 times the projected dosage level for treatment of

40

CARPENTER

ET AL.

human cases of hemophilia, caused no adverse physiological responses after 178 injections within a 1Zmonth interval. Adjunct studies indicate that the major portion of an intravenous dose is excreted via the urine within 24 hr while po doses pass through the alimentary tract with but minor absorption in a like interval. Furthermore, there was no indication of sensitization following a modified Landsteiner intradermal sensitization test using the albino guinea pig. In the light of this evidence, there seems to be no reason why PEG 4000 should not be used by the intravenous route in humans. REFERENCES C. P., and SHAFFER, C. B. (1952). A study of the polyethylene glycols as vehicles for intramuscular and subcutaneous injection. J. Amer. Pharm. Ass., Sci. Ed. 41, 27-29. HOWELL-KARPATKIN, M. B., NEWMAN, J., PIOMELLI, S., Moss, S.J., and JOHNSON, A. J. (1967). Managementof comprehensivedentalsurgeryin severehemophiliawith ahigh purity human antihemophilicfactor (AHF) concentrate. Clin. Res. 15 (2). JOHNSON, A. J., NEWMAN, J., HOWELL, M. B., and PUSZKIN, S. (1966).Two large-scaleproceduresfor purification of human antihemophilicfactor (AHF). Blood 28 (l), 1011. NATELSON, S. (1963).Microtechniques of Clinical Chemistry, 2nd ed., p. 346.Thomas,Springfield, Illinois. POLSON, A., POTGIETER, G. M., LARGIER, G. E., MEARS, G. E. F., and JOUBERT, F. J. (1964). The fractionation of protein mixturesby linear polymersof high molecularweight.Biochim. CARPENTER,

Biophys. Acta 82, 463-475.

C. B., and CRITCHFIELD, F. H. (1947a).The absorption and excretion of the solid polyethylene glycols (CARBOWAX Compounds).J. Amer. Pharm. Ass., Sci. Ed. 36, 152157. SHAFFER, C. B., and CRITCHFIELD, F. H. (1947b).Solid polyethylene glycols (CARBOWAX Compounds).Quantitative determinationin biologicalmaterials.Ind. Eng. Chem., Anal. Ed. SHAFFER,

19, 32-34.

C. B., CRITCHFIELD, F. H., and CARPENTER, C. P. (1948a).Renalexcretion andvolume distribution of somepolyethylene glycols in the dog. Amer. J. Physiol. 152, 93-99. SHAFFER, C. B., CRITCHFIELD, F. H., and CARPENTER, C. P. (1948b). The renal clearanceof somepolyethylene glycols in the dog. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 7, 111. SHAFFER, C. B., CRITCHFIELD, F. H., and NAIR, J. H., III (1950).The absorptionandexcretion of a liquid polyethylene glycol. J. Amer. Pharm. Ass., Sci. Ed. 39, 340-344. SIGMA CHEMICAL COMPANY (1963).SigmaTechnical Bulletin No. 104,Phosphatase,rev. ed., SigmaChemicalCompany, 3500DeKalb Street, St. Louis 18, Missouri. SIGMA CHEMICAL COMPANY (1964). SigmaTechnical Bulletin No. 505, Glutamic-Oxalacetic and Glutamic Pyruvic Transaminase,rev. ed., SigmaChemical Company, 3500DeKalb Street, St. Louis 18, Missouri. SMIYTH, H. F., JR., CARPENTER, C. P., and SHAFFER, C. B. (1947).The toxicity of high molecular weight polyethylene glycols; chronic oral and parenteral administration.J. Amer. Pharm. Ass., Sci. Ed. 36, 157-160. SMYTH, H. F., JR., CARPENTER, C. P., andWEIL, C. S.(1950).The toxicology of the polyethylene glycols. J. Amer. Pharm. Ass., Sci. Ed. 39, 349-354. SMYTH, H. F., JR., CARPENTER, C. P. ,and WEIL, C. S. (1955).The chronic oral toxicology of the polyethylene glycols. J. Amer. Pharm. Ass., Sci. Ed., 44, 27-30. SNEDECOR, G. W., and COCHRAN, W. G. (1967).Statistical Methods, 6th ed. The Iowa State CollegePress,Ames, Iowa. WARNER-CHILCOTT LABORATORY (1963). Urograph B, Folder 3OOG250, General Diagnostics Division, Warner-Chilcott Lab., Morris Plains,New Jersey. WARNER-CHILCOTI. LABORATORY (1965).Simplastic0, Folder 3516133,rev. ed.,GeneralDiagnosticsDivision, Warner-Chilcott Lab., Morris Plains,New Jersey, SHAFFER,