25. Carstensen, J. T., Su, K. S. E .. Maddrell, P., Johnson, J. 8., and
`ewmark, H.
`., "Thermodynamic and kinetic aspects of parenteral
`bcnzodiazcpincs," Bull. Parent er. Drug Ass()('., 2S, 193 ( I 971 ).
`26. Wang, D. I. C., Scharer, J., and Humphrey, A. E., "Kine1ics of death
`of bacterial spores al elevated temperatures," Appl Microbio/., 12,
`451 (1964).
`27. Pflug, I. J., and Smith, G. M., "Survivor curves of bacterial spores
`
`heated in paren1cral solution," in Sport Research /976, Vol. II. E....
`Barker, A. N., cl al., Academic Press, Inc., London 1977.
`28. Rahn, 0 ., " Injury and dcalh of bacteria," Biodynamica M~
`3, (I 945).
`29. Cavalieri, L., and Rosenberg, 8. H .• " ludics on the muctu~
`nucleic acids, XI. The roles of heal and acid in deoxyribonucleic
`dena1Ura1ion," J Am. Chem. SI)('., 79, 5352 (1957).
`
`RES UM£: En la optimizaci{m de los ciclos en el autoclave para u11 prod11c10 de uso parenteral, la temper(cid:173)
`atura de esterilizaci611 y el tiempo de exposici6n puede11 ser reducidos al mismo tiempo al aume111ar la ac(cid:173)
`ci6n termica /eta/ sabre las esporas de bacterias, coma resultado de cambios e11 la formulaci6n. £11 co11Se(cid:173)
`cue11ica, se puede esperar u11 degradaci6n quimica me11or con 11110 alto seguridad de esterilizaci611. la acci6n
`/eta/ termica sabre el Bacillus stearothermophilus (sus esporas) Jue increment ado en forma efectiva con el
`uso de acido citrico (0. I M , pH 2),fosfato dis6dico (0. I M , pH JO), glico/ de propileno al 50% y glicol polie(cid:173)
`tilenico 300 tambie11 al 50%. Los parametros de la cinetica de la acci6n esporicidafueron ca/cu/ados usando
`el trata111ie1110 de la cinetica no isotermica.
`
`Hemolysis Study of Aqueous Polyethylene Glycol 400, Propylene Glycol and
`Ethanol Combinations In Vivo and In Vitro
`
`FARREL L. FORT'\ IRWIN A. HEYMAN, AND JAMES W. KESTERSON
`Dfrision of Drug Safety E,a/uation, Abbott Laboratories, orth Chicago, Illinois
`
`ABSTRACT: Propylene glycol:erhanol:water (5:/ :4) given i111rave11ously for 2 weeks to rats and dogs re(cid:173)
`sulted i11 hematuria and hematologic cha11ges consistent with il11ravascu/ar hemolysis. Several combinations
`of propylene glycol, polyethylene glycol 400, ethanol , saline a11d/or water were then studied for hemolytic
`activity both in vivo in rats after intravenous administratio11 and i11 vitro. Solutions containing propylene gly(cid:173)
`col:ethanol:saline (1:3:6), ethanol:sa/ine (3:7), and polyethylene glycol 400:etha11ol:water (3:2:5) were
`found to cause less hemolysis in vivo than the other solutions tested. Polyethylene glycol 400:ethanol:water
`(3:2:5) was chose11 for further evaluation and when given intravenously to dogs for I week or rats for 2
`weeks, this solution caused no adverse hematologic effects.
`
`Introduction
`onaqueous solvents such as propylene glycol (PG),
`polyethylene glycol (PEG), and ethanol are currently used
`as components of vehicles for several pharmaceutical
`preparations for parenteral use in man. One or more of these
`solvents may be found at concentrations as high as 50% in
`certain injectable formulations (I , 2). We used a solution
`of 50% PG, I 0% ethanol, and 40% water as a vehicle for
`intravenous (i.v.) repeat-dose toxicity studies in rats and
`dogs. During the course of these experiments we observed
`frank hematuria in animals treated only with vehicle, as well
`as in drug-treated animals. In order to find a less toxic ve(cid:173)
`hicle for the drug under development a series of vehicles
`containing nonaqueous solvents were subsequently exam-
`
`Received ovcmber 22. 1983. Acccp1cd for publica1ion January 7,
`1984.
`4 Au1hor 10 whom inquiries should be dircc1ed.
`
`82
`
`ined for hemolytic activity both in vivo after i.v. adminis- 1
`tration and in vitro. The vehicles chosen for evaluation were
`limited to those having solubility characteristics suitable
`for the particular drug under development, and, therefore,
`a systematic examination of all possible combinations of the
`solvents used was not undertaken. To our knowledge there
`have been no previous reports comparing the hemolytic
`activity of specific mixtures of PG, PEG 400 and/or ethanol
`in water and saline both in vitro and in vivo. Therefore, our
`results should be useful to others involved in the develop-I
`ment of parenteral formulations containing these non(cid:173)
`aqueous solvents.
`
`Methods
`Sprague-Dawley Crl:CD<e (SD)BR rats were obtained
`from Charles River Breeding Laboratories, Portage, Ml.
`The rats were about 5-8 weeks of age at the time of treat(cid:173)
`ment. They were housed individually in hanging metal.
`
`Journal of Parenteral Science and Technology
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`

`mesh-bottom cages equipped with feeders and automatic
`watercrs. Purebred beagle dogs were obtained from Mar(cid:173)
`;hall Research, North Rose, NY and were about 8- 13
`months old. They were housed individually in stainless steel
`cages equipped with feeders and automatic waterers. Food
`and tap water were available ad libitum except as noted
`below. Ambient temperature was maintained at 72 ± 5 °F.
`Animal rooms were on a 14-hr daily light cycle.
`PG, USP, was obtained from Dow Chemical Co. PEG
`400, Abbott Grade, was obta ined from Union Carbide.
`Ethanol, USP, was obtained from U.S. Industrial Chem(cid:173)
`ical.
`Urine was obtained by placing the animals in metabolism
`cages immediately after treatment and collecting urine over
`a period of 3- 4 hr. The urine was subjected to routine uri(cid:173)
`nalysis using a Clini-Te~ automated instrument and mi(cid:173)
`croscopic examination of sediment.
`Blood samples were obtained after an overnight fast and
`analyzed for erythrocyte count, hematocrit, hemoglobin,
`mean corpuscular volume, mean corpuscular hemoglobin,
`and mean corpuscular hemoglobin concentration. Blood
`samples were obtained from dogs via the jugular vein, while
`m rats cardiac blood samples were obtained at necropsy.
`In vitro hemolysis tests were conducted using blood ob(cid:173)
`tained from nonfasted, healthy, untreated beagle dogs. Fifty
`microliters of heparinized blood were added to 5-ml aliquots
`oftest solution, mixed thoroughly and incubated at room
`temperature for 20 min. After centrifugation the super(cid:173)
`natant was examined for color and clarity, absorbance of
`the supernatant was measured at 540 nm, and the presence
`or absence of a cell pellet was noted. Distilled water and
`physiological saline were used as positive and negative
`controls, respectively, during each test run. Absorbance of
`test supernatants relative to positive control supernatants,
`after correction for absorbance of negative control super(cid:173)
`natants, were used to calculate percent hemolysis. Absence
`
`of a cell pellet, reddish colored supernatant and calculated
`hemolysis of about 90% or more were taken as indications
`of complete hemolysis. The presence of a cell pellet together
`with a discolored supernata nt and/or a calculated hemolysis
`of about 20% or more were interpreted as partial hemolysis.
`Test samples were analyzed undiluted and as 3: I, I: I and
`I :3 (test solution:saline) dilutions. Comparisons of in vitro
`hemolytic activity were made by comparing the degree of
`hemolysis obtained with the diluted test solutions.
`In the initial repeat-dose studies, a solution of PG:etha(cid:173)
`nol:water (5: I :4) was administered i.v. to groups of 5 male
`and 5 female rats or to I male and I female dog daily for 2
`weeks. Normal saline was concurrently administered to
`equal numbers of rats or dogs as control groups. Rats re(cid:173)
`ceived 5 ml/kg/day at a rate of 0.3 ml/min, while dogs
`received 4 ml/ kg/day at 6 ml/ min. The subsequent re(cid:173)
`peat-dose studies were conducted in a similar manner except
`that the test solution was PEG 400:ethanol:water (3:2:5),
`and the duration of the dog study was only I week. Also, in
`these latter studies rats received 2 ml/ kg/ day at a rate of
`0.15 ml/ min, and dogs received 1.2 ml/ kg/ day at 3 ml/ min.
`At the conclusion of each of these four studies all the ani(cid:173)
`mals were subjected to necropsy and histopathologic ex(cid:173)
`amination.
`
`Results
`
`Frank red urine was observed after the first dose in both
`rats and dogs treated with PG:ethanol:water (5: I :4) in the
`initial repeat-dose studies. This was frequently observed in
`rats a nd occasionally seen in dogs throughout the 2-week
`treatment period. Some animals excreted red urine within
`I hr after dosing, whereas others had red urine within 3- 5
`hr after dosing. After 2 weeks of treatment both rats and
`dogs given this solution had decreases in hematocrit, he(cid:173)
`moglobin and erythrocyte count (Tables I and 11). Rats also
`had minimal to moderate anisocytosis and polychromasia.
`
`TA BLE 11. Hematology after 2 Weeks Intravenous Administration of Propylene Glycol, Ethanol, Water (5: I :4) to Dogs•
`Incidence-Serum
`Color
`
`RBC
`(106/ mm3)
`6.26, 7.33
`5.43, 6.92
`4.54, 5.45
`
`HCT
`(%)
`
`41 .8, 49.3
`36.1, 46.8
`31.3, 37.5
`
`Hgb
`(g/ dl)
`15.3, 18.2
`13.2, 17.3
`10.6, 13.1
`
`2/ 2-straw
`2/ 2-straw with tinge of red
`1/ 2-straw, 1/ 2-straw with
`tinge of red
`2/ 2-cherry red
`
`Saline
`
`Test Solution
`
`• n = 2.
`
`Pre-dose
`Post-dose
`Pre-dose
`
`Post-dose
`
`4.00, 4.86
`
`27.3, 33.8
`
`9.2, 11.7
`
`TABLE I. Hematology after 2 Weeks Intravenous Administration of Propylene Glycol, Ethanol, Water (5:1 :4) 10 Rats•
`
`Males
`Females
`Males
`Females
`
`6.48(0.54)
`6.81 (0.32)
`5.14(0.26)
`4.96(0.27) 6
`
`Saline
`
`Test Solution
`
`• n = 5, Mean (SD).
`h n = 4.
`
`Vot 38. No. 2 I March- April 1984
`
`HCT
`(%)
`
`40.0(4.2)
`40.6(2.3)
`33.0( 1.8)
`33.7( J.7)b
`
`Hgb
`(g/ dl)
`14.7(1.0)
`14.8(0.8)
`11 .7(0.7)
`11.9(0.6)6
`
`83
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`

`TABLE 111. Urinalysis after 2 Weeks Intravenous Administration of Propylene Glycol, Ethanol, Water (5: I :4) in Rats•
`Incidence-
`RBC
`(High po-..tt
`Field)
`
`Ketones <
`
`Bilirubin <
`
`Occult Blood '
`
`Incidence-Color
`
`Protein b
`(mg/ di)
`
`Saline
`
`Males
`Females
`Males
`Females
`
`24( 13)
`0(0)
`300(0)
`300(0)'
`
`0,0,0,1,1 d
`0,0,0,0,1
`0,1,1,1 ,1
`I, I.I , I,'
`
`0,0,0,0,1
`0,0,0,0,0
`l, l,1,1,2
`1.1,2,2'
`
`Test
`Solution
`0 fl• 5.
`b \1can (SD).
`' I nd1vidua I scores presented.
`'0 = none. I = small amount, 2 = moderate amount. 3 = large amount.
`'n = 4, urine from one animal could not be instrument assa}ed due to excessive red di.coloration.
`
`0,0,0,0,0
`0,0,0,0,0
`1,1.2,2,2
`1.2,2,2'
`
`2/5-straw, 3/ 5-ycllow
`3/5-straw, 2/ 5-yellow
`I / 5-amber, 4/5-bloody
`1/5-amber, 4/ 5-bloody
`
`5/5-nonc
`5/5-nonc
`5/5-nonc
`5/5-nonc
`
`TABLE IV. Urinalysis after a Single Intravenous Dose of Propylene Glycol, Ethanol. Water (5: I :4) in Rats•
`Incidence--
`RBC
`(High Po-..er
`Field)
`
`Ketones d
`
`Bilirubin d
`
`Occult
`Blood d
`
`Dose Volume b
`(ml/ kg)
`
`Protein <
`(mg/ di)
`
`2.0
`1.0
`0.5
`
`300(0)'
`300(0)'
`300(0)
`
`1 , 1 ,2•✓
`I, I, I '
`l, l,1,1
`
`2,2,3'
`1,1,2'
`1,1,2,2
`
`I, I. I'
`I, 1,2'
`1,1,2,2
`
`0,0,0.1
`0,0,1,1
`
`0. 125
`108(135)
`0.03
`15( 17)
`• n = 4. males and females combined.
`b lnrusion rate was 0.3 ml/ min.
`' Mean ( D).
`4 Individual scores presented.
`'11 = 3. urine from one animal could not be instrument assa)ed due to c,cessive red discoloration.
`IO= none. I = small amount, 2 = moderate amount, 3 = large amount.
`
`0,0,0,0
`0,0,0,0
`
`0,1,3,3
`0,0,0,0
`
`4/ 4-none
`4/ 4-none
`4/ 4-none
`
`4/ 4-none
`4/ 4-nonc
`
`Incidence--
`Color
`
`4/ 4-blood)
`4/ 4-blood>
`I/ 4-ambcr,
`3/ 4-blood}
`4/4-}ello-..
`4/ 4-yellow
`
`In Vitro and In Vivo Hemolysis Tests of Solutions Containing
`Solution Com~ ilion
`Propylene
`Glycol
`(% • / •)
`
`0.9%
`aline
`(% \ / •)
`
`Infusion
`Volume
`(ml/ kg)
`
`Ethanol
`(% • / •)
`
`Water
`(% • / •)
`
`Infusion
`Rate
`(ml/ min)
`
`Hematuria
`In Vho
`(Rats)
`
`Hemolysis
`In Vitro
`(Dog Blood)
`
`60
`50
`50
`40
`40
`30
`0 11 • 4. 2 males and 2 fem.ii~.
`• 11 = 12. 6 male, and 6 females.
`' All urine samples "ere di,colorcd (red or port" inc color) and/or positive for occult blood.
`d Complete hemolysis (,cc M etlwd.r) 11,ing undiluted ,olu11on,.
`'Given at greater volume and rate or infosion because of poorer ,olubihty of the drug under dc,clopmcnt in these ,olu11ons.
`I Compari,ons were ba,ed on rc,ults for diluted ,olution; (,cc Mrthods).
`
`TA BLE V.
`
`Solution
`umber
`Jb
`2
`3
`4
`5
`6
`7
`
`10
`10
`10
`10
`20
`
`40
`40
`
`50
`
`50
`
`100
`
`40
`
`50
`
`10
`5
`5
`5
`10'
`10'
`5
`
`0.6
`0.3
`0.3
`0.3
`0.6'
`0.6'
`0.3
`
`0
`Yes'
`Yes
`Yes
`Yes
`Yes
`Yes
`
`0
`Y esd
`Yes
`Yes
`Yes
`Yes (less than # 5)-1
`Yes
`
`Urinalyses were positive for occult blood, bilirubin, ketones,
`and protein, but no erythrocytes were fo und (Table 111 ).
`Splenomegaly and renal hemosiderin deposits were also
`ob erved in rats. These results were consisten t with intra(cid:173)
`vascular hemolysis ca used by i.v. administration of the test
`
`84
`
`solution. Blood samples were taken from dogs both before
`and 5 10 min after injection in order to veri fy this h}·
`pothesis. Although there were no differences in hematoloin
`parameters between pre- and post-dose sample relative to
`controls, the post-dose blood sa mples showed marked he-
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`

`TABLE V (cont.).
`
`In Vitro and In Vivo Hcrnolysis Tests of Solutions
`olution ComeQsition
`Propylene
`Glycol
`(% v/ v)
`
`Ethanol
`(% V / v)
`
`Water
`(% v/ v)
`
`0.9%
`aline
`(% v/ v)
`
`lnfu ion
`Volume
`(ml/ kg)
`
`onaqueous Solvents0
`
`Infusion
`Rate
`(ml/ min)
`
`Hematuria
`In Vilo
`(Rats)
`
`l-lemol) is
`In Vitro
`(Dog Blood)
`
`Solution
`\'umber
`
`20
`30
`30
`30
`30
`40
`40
`
`50
`
`60
`
`50
`
`50
`
`50
`
`60
`
`50
`
`5
`5
`5
`5
`5
`5
`5
`
`0.3
`0.3
`0.3
`0.3
`0.3
`0.3
`0.3
`
`Ycsb
`Yes
`Yes
`Yes
`0
`Yes
`Yes
`
`Yes<
`Ycsd
`Yes (less than # 9)d.,
`Yes
`Yes
`Yes
`Yes
`
`8
`30
`9
`20
`10
`20
`11
`10
`12
`10
`13
`10
`14
`10
`• 11 = -l. 2 mafos and 2 rcmalcs.
`• \II urine samples were discolored (red or port"ine color) and/ or positive for occult blood.
`Complete hcmolysis (sec \lerhods) using undiluted solutions.
`' I c,- than # 13 and # 14
`' Comparison, " ere based on results for diluted solutions (sec M er hods).
`
`TAB LE V(cont.).
`
`In Vitro and In Vivo Hemolysis Tests of Solutions Containing onaqueous Solvents0
`Solution ComeQSition
`PEG
`400b
`(% vf , )
`
`Ethanol
`(% vf , )
`
`Water
`(% v/ v)
`
`0.9%
`aline
`(% v/ v)
`
`Infusion
`Volume
`(ml/ kg)
`
`Infusion
`Rate
`(ml/ min)
`
`50
`50
`30
`20
`
`10
`10
`20
`30
`
`40
`
`50
`
`40
`
`50
`
`5
`5
`5
`5
`
`0.3
`0.3
`0.3
`0.3
`
`1-lcmaturia
`In Vilo
`(Rats)
`I /4 occult
`Yes<
`0
`3/ 4 occult
`
`Solution
`\'umber
`
`15
`16
`17
`18
`
`Hemolysis
`In Vitro
`(Dog Blood)
`
`Partial, RBC discolored
`Partial, RBC discolored
`Partial. RBC discolored
`Partial (less than # 19),d
`RBC discolored
`Partial. RBC discolored
`
`19
`10
`40
`• 11 = -l. 2 males and 2 rcmales.
`• Poly ct hylcnc glycol 400.
`' \II uri ne sample, " ere di;colorcd (red or port"inc color) and/ or positive ror occult blood.
`d Comparisons were ba,ed on results ror diluted ,olutions (,cc Mer hods).
`
`50
`
`5
`
`0.3
`
`Yes
`
`TA BLE V (cont.).
`
`In Vitro and In Vivo Hcrnolysis Tests of Solutions Containing onaqueous Solvents0
`olution ComeQ ition
`
`Solution
`',umber
`
`Ethanol
`(% v/ v)
`
`Waler
`(% v/ v)
`
`0.9%
`aline
`(% v/ v)
`
`Infusion
`Volume
`(ml/ kg)
`
`Infusion
`Rate
`(ml/ min)
`
`1-lematuria
`In Vilo
`(Rais)
`
`l-lemolysis
`In Vitro
`(Dog Blood)
`
`70
`
`60
`
`70
`
`60
`
`5
`5
`5
`5
`
`0.3
`0.3
`0.3
`0.3
`
`Yesb
`0
`Yes
`Yes
`
`Yes<
`Yes (less 1han # 20) d
`Yes
`Yes
`
`20
`30
`21
`30
`22
`40
`23
`40
`• 11 = 4. 2 males and 2 rcmalc,.
`• \II urine samples " ere d1>colorcd (red or port wine color) and/ or positive for occult blood.
`' (omplctc hcmolysi; (,cc M1•1hods) using undiluted solution,.
`1 Comp.iri,ons " ere b.1, cd on results for diluted ~oluuon, (,cc M nhods).
`
`molysis (Table 11) . Other treatment-related findings in(cid:173)
`cluded swelling (dogs) or necrosis (rats) at the infusion site,
`decreased body weight ( I 0%) and food consumption (25%)
`b; the end of the study in rats. a nd occasional decreased
`activity after dosing in rats.
`When the same formulation was given to rats at the same
`rate (0.3 ml/ min), bu t at decreasing infusion volumes, the
`amount of solution that could be infused without causing
`hcmaturia was only 0.03 ml/ kg (Table IV).
`
`A series of solutions were then tested both in vivo in rats
`and in vitro in dog blood for hemolysis potential (Table V).
`PG, PEG 400 a nd/ or ethanol were combined in various
`proportions with both \\ ater and norma l saline. PG com(cid:173)
`bined with ethanol and/ or water caused hematuria and
`complete in vitro hemolysis with all combinations examined.
`When combined with saline, all PG solutions gave similar
`results except the one containing PG:ethanol:saline ( I :3:6).
`This solution produced complete hemolysis in vitro but no
`
`Vol. 38, No. 2 / March- April t 984
`
`85
`
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`

`

`hematuria in vivo in rats. Upon dilution with saline, two
`other solutions of PG, ethanol, saline (2:3:5 and 4: I :5)
`caused less hemolysis in vitro than their counterparts con(cid:173)
`taining water instead of saline.
`The solutions containing PEG 400 all caused partial
`hemoly is in vitro and hematuria in rats except the one
`containing PEG 400:cthanol:water (3:2:5). This solution
`gave partial hemolysis in vitro but no hematuria. All of the
`PEG 400 solutions also cau ed a brown discoloration of the
`cell pellet and, in some cases, the supernatant in the in vitro
`assay.
`Forty percent ethanol caused hemolysis in vivo and in
`vitro when combined with either water or saline. However,
`30% ethanol in saline did not cause hematuria even though
`hemolysis was observed in vitro.
`Based on the above observations, subsequent repeat-dose
`studies were conducted in which PEG 400:ethanol:water
`(3:2:5) was administered i.v. to rats for 2 weeks and to dogs
`for I week as described in M e1hods. In rats, bruised and
`discolored tails (infusion site) were observed occasionally
`during the first week and frequently during the second week.
`There were no infusion site changes in dogs. There were no
`treatment-related effects on urinalysis, hematology, body
`weight gain, or food consumption in either rats or dogs.
`The other two solutions that did not produce hematuria
`in rats (i.e., PG:ethanol:saline [ I :3:6) and ethanol, saline
`[3:7)) were not studied further.
`
`Discussion
`
`In Vi1ro S1udies
`Aqueous PG at any concentration has been reported to
`cause lysis of erythrocytes in vitro, even though 2% PG in
`water is isosmotic (3). It has also been reported that no
`hcmolysis occurs in solutions containing up to about 30%
`PG in saline instead of water (3, 4). However, in our studies
`undiluted a line solutions containing as little as I 0% PG
`gave complete hemoly is in vitro. This was thought to be due
`to the additional innuence of ethanol in the solutions. For
`example, 40% ethanol in saline caused hematuria in rats in
`our study, but 30% ethanol in saline did not (Table V). Also,
`upon dilution with saline, solutions #9 and# 10 appeared
`to have less hemolytic action than solutions # 13 and # 14.
`These solutions all contained 50o/o water or saline; solutions
`#9 and # 10 contained 30o/o ethanol versus 40% ethanol
`in solutions # 13 and # 14. Polyethylene glycols of various
`molecular weights have also been reported to cause he(cid:173)
`molysis in vitro, however PEG 400 in water was reported
`to cause hemolysis only at concentrations less than about
`8- 11 % (5). The hemolysis with lower concentrations of
`PEG 400 could be prevented by formulating in saline in(cid:173)
`stead of water (5, 6), as was the case for PG. Although the
`PEG solutions we tested were chosen with these data in
`mind, we observed hemolysis in vitro with concentrations
`of PEG 400 ranging from I 0% to 50% in saline (Table V).
`Again, this was probably due to the presence of ethanol in
`all of the PEG solutions studied. For example, solutions
`# 18 and # 19 both contained 50o/o saline, but solution # 18
`gave less hemolysis in vitro and in vivo even though it con(cid:173)
`tained more PEG than solution # 19. Solution # 18 con-
`
`es
`
`tained 30% ethanol versus 40% in solution # I 9. On the
`basis of these comparisons, it is possible that lower con(cid:173)
`centrations of ethanol in our studies may have resulted in
`more favorable results with given concentrations of PG or
`PEG 400.
`
`In Vivo Studies
`Intravenous PG has been previously reported to cause
`adverse changes in blood and urine. In dogs, 40o/o PG in
`saline administered i.v. caused an increase in plasma he•
`moglobin which peaked within 1- 5 min after injection (7).
`Hematuria has been reported in rats (4) and calves (8) after
`i.v. administration of PG. On the other hand, i.v. adminis(cid:173)
`tration of PEG solutions has been reported to cause no ad(cid:173)
`verse hematologic effects in several studies in rats and d~
`(9- 11 ). Our results were consistent with these previous
`reports. Also, the injection site injury we observed in our
`repeat-dose studies was consistent with previous studies or
`i.v. PG (12, 13) and PEG (14- 16).
`Our results, taken together with previous reports, suggest
`that i.v. administration of PEG solutions may generally be
`expected to result in fewer adverse hematologic changes
`than solutions containing PG.
`
`Acknowledgments
`All test solutions were prepared by Dr. J. Collins. Uri·
`nalysis, hematology and in vitro hemolysis measurements
`were performed under the supervision of Mr. J. Langdon.
`ecropsies and histopathological examinations were con·
`ducted by Dr. P. Cusick, Dr. B. Buratto, Dr. S. Tekeli, and
`Dr. M. Pratt.
`
`6.
`
`References
`I. Spiegel, A. J .. and oscworthy, M. M., .. Use of nonaqucous solvent!
`in parenteral products," J. Pharm. Sci., 52,91 7 ( 1963).
`2. Wang, Y.-C. J .. and Kowal, R. R., .. Review of cxcipients and pH·s
`for parenteral products used in the United States," J. Parenteral Drug
`ASSIX'., 34, 452 ( I 980),
`3. Cadwallader, D. E .. "Behaviour of erythrocytes in various solvent
`systems. I. Water-glycerin and water-propylene glycol,'. J. Pharm
`Sci., 52, 11 75 (1963).
`4. Ruddick. J. A .... Toxicology, metabolism and biochemistry of 1.2·
`propancdiol,'' Toxicol. Appl. Pharmacal .. 21, 102 (1 972).
`5. Smith, B. L .. and Cadwallader, D. E .. " Behavior of erythroC)tCS in
`various solvent systems. Ill. Water polycthylne gl)cols,'' J. Pharm
`ci .. 56, 35 1 ( 1967).
`ish10, T .. I lirota, S .• Yamashita. J .. Kobayashi. K .. fo1ohash1. Y
`and Kato. Y., .. Erythrocyte changes in aqueous polethylene gl)col
`solutions containing sodium chloride,'' J. Pharn,. Sci .. 71. 9r
`( I 982).
`7. Gentry. P. A .. and Black. W. D .... Jnnuence of pcntobarbital sodium
`anesthesia on hematologic values in the dog," Am. J. Vet. Res .. 37.
`1349 ( 1976).
`8. Gross, D. R .. Ki11man. J. V .• and Adams. H. R .. "Cardiovascular
`effects of intravenous administration of propylene glycol and of
`o, ytetracyclinc in propylene glycol in calves: · Am. J. Vet. Res .. 40.
`783 ( 1979).
`9. Carpenter. C. P .. Woodside. M. D .. Kinkead. E. R .. King. J. M .. and
`ullivan. L. J .• " Response of dogs to repeated intravenous injecuon
`of pol)ethylene gl)col 4000 with notes on excretion and scnsiti1.ation:·
`Toxicol. Appl. Pharmacol .. 18, 35 (1971 ).
`JO. Pfordte. V. K .. "Polyathylenglykol. ein wenig toxisches losungsmutcl
`1ur intravenosen applikation wasserunloslicher substanzcn:· 7bl
`Plwrm., 110,449 ( 197 1).
`11 . Lee. C.-C .. and Anderson. R. C., .. Toxicologic studies on vancom)Cln
`and polyethylene glycol 200:· Toxicol. Appl. Pharmacol .. 4, 206
`( I 962).
`
`Journal of Parenteral Science and Technology
`
`Novo Nordisk Ex. 2032, P. 5
`Mylan Institutional v. Novo Nordisk
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`

`' Paµno. R . R .. Graham. C. W .. Galligan. \1 .. Conner. J T .. and l-.,111.
`R I ·• ""I liMop.itholog) of veins after intravenous lora1epam and RO
`11-~39 I:· Ca11 Anaesth. Soc. J .• 25, 50 ( 1978).
`1 Braun. 11
`.. and Cartland. G. F .. "The to~icity of prop)lcnc gl)col.""
`J ~m Pham, As.roc .. 25. 746 ( 1936).
`J Lockard. J . S. and Lev). R. H .. ··Pol)ethylcne gl)col 400; Solvenl
`
`and an1iconvul,ant."" Ufe ri. 23. 2499 ( 1978).
`15. Loda rd. J . .. Lev) . R. II .. Congdon. \\, C.. and Ducha rme. L. L..
`·•Efficac) and to, icll) of the ,ohcnt pol)cth)lenc gl)col 400 in
`monkc) model."" Ep,lepsia. 20. 77 ( 1979).
`16. Christenson. P J .• \1aclead. P F. Belt. \\'. R .. and Arce. L. ... ,n(cid:173)
`travenou, use of nitrofurantorn:· Curr. Thl'r. Res .. 2. 45 ( 1960).
`
`RE U M £ 1 : El tratamiento intravenoso usando glicol de propileno:etanol:ag ua (5:4:1) se aplic6 durante
`do semanas a rotas ya perros y dib por res ultado 11110 hemalllria y ca111bios he111a10l6gicos que correspon(cid:173)
`den a hem 6/isis intravascular. Varias co111bi11acio11es de glicol de propileno, polietilen glicol 400, eranol
`soluc i6n salina y/o agua fueron entonces usadas en al estudio de la actividad hemolitica, y a sea in vivo en
`rotas, despues de 1m s11111inistro i111rave11oso o in vitro. e e11co111r6 que las soluciones que co111ie11e11 glicol de
`propile110:etanol:sol11ci6n salina (J :3:6), etanol:soluci6n salina (3:7) y polietilen glicol 400:etanol:agua (3:
`2:5) causan menos hem6/isis in vivo que las otras soluciones ecaluadas. Se decidi6 continuar la ei·al11aci611
`de po lietilen glicol 400:etanol:agua (3:2:5) y cuando se la s11mi11istr6 enforma intracenosa a perros, durante
`I semana. o a rotas d11ra111e 2 semanas. esta sol11ci611110 caus6 efectos hematol6gicos adi•ersos.
`
`ERRATUM
`
`In the paper, "The Targeting of Drugs Parenterally
`Using Microspheres," by L. Ilium and S.S. Davis, J. Par(cid:173)
`ent. S ci. and Tech., 36, 242 ( 1982), the legends for Figures
`2 and 3 were inadvertently transposed.
`
`ct 38, No. 2 / March-April 1984
`
`87
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`Novo Nordisk Ex. 2032, P. 6
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