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Experiments on the toxic, sedative and muscle relaxant potency of various drug solvents in mice
Pharmac Ther Vol 5 pp 467-474 1979 Pergamon Press Ltd
Pnnted m Great Britain
EXPERIMENTS ON THE TOXIC, SEDATIVE MUSCLE RELAXANT POTENCY OF VARIOUS SOLVENTS IN MICE
AND DRUG
R BUDDEN, U G KUHL and J BAHLSEN Department Pharmacologtcal Research I, Kah-Chemze Pharma OmbH, Hannover, Hans-Bockler-Allee 20, Germany
1. S U M M A R Y The solvents dlethyleneglycol monoethylether (Transcutol®), N,N-dmthylacetamlde, dimethylsulfoxlde, glycenne, N-(/3-hydruxyethyl)-Iactamlde, polyethylene glycol 400 (Lutrol 9~), "l.2-propanediol, tetrahydro= furfurylalcohol d~ethyleneglycolether (Tetraglycol ®) and polyoxyethylene sorbttanemonooleate (Tween ® 80) were tested m mice for their tox~cRy, tketr CNS-profile, thew acnwty m the mchned screen and balance rod tests a f t e r , p admm~straUon as well as for their influence on the hexobarbltone sleeping t~me and their muscle relaxant properties on the rota-rod after p o admmlstrauon It ~s demonstrated that the compounds tested cause a wtde variety of pharmacodynamtc effects m concentraUons m which they are used as drug solvents m the laboratory As proved by the potenaatmn of hexobarbRone sleeping time, interactmns wtth the drugs to be tested cannot be excluded (Just m this test all solvents but three exhlbtted thetr h~ghest actavRy Only glycenne. N-(//-hydroxymethyl)-Iactamlde and polyethylene glycol 400 were most potent m the rota=rod test ) So, whenever a drug solvent is used, ~ts concentratmn must be specdied and lack of acuvlty m the respectmve test system has to be demostrated Based on thetr actlvRy m the most sensmve test, the drug solvents examined are recommended not to exceed the following concentrataons m drug solutmns for screening purpose dlethyleneglycol monoethylether 0 7%, N,N-ðylacetamlde 0 3%, dlmethylsulfoxlde 0 9%, glycenne 3.0%, N-(/3=hydroxyethyl)lactamtde 50%, polyethylene glycol 400 6 0%, 1,2=propanedml 20%, Tetraglycol ® 6 7% and Tween ® 80 6 8%
2 INTRODUCTION O n e o f t h e first p r o b l e m s
facing the experimentally
working pharmacologist
when
screening water-insoluble drugs is to transfer them into a hqmd water-mixable phase Therefore some common drug solvents were tested for their applicability m the pharmacological laboratory The solvents were screened for acute Lp. toxicity, influence on behavior as well as for their sedative and muscle relaxant properties m mice 3
MATERIALS
AND
METHODS
3.1 ANIMALS All tests except the hexobarbRone sleeping tune test were performed m male NMRI m l c e ( 1 8 - 2 2 g) F o r d e t e r m i n a t i o n o f h e x o b a r b l t o n e s l e e p i n g t i m e f e m a l e N M R I m l c e ( 1 8 - 2 2 g ) w e r e u s e d All a n i m a l s w e r e b r e d at t h e Z e n t r a l i n s t i t u t f u r V e r s u c h s -
tterzucht, Hannover. They were kept m air=conditioned rooms at 20-30°C tern= perature and 50-60% relative humidity in groups of 20 anunals per cage (Macrolon Type III). The animals were fed with a commercial diet ("altrominmR 1324", Altrogge) and tap water ad libitum. In case of oral drug admmtstratlon fasted animals were used.
3.2
DRUG SOLVENTS
The chemical and physical data and the origin of the solvents tested are listed m Table 1 467
78 13
92 10
133 15
DImelhylsulfoxlde
Glycerine
N-(O-hydroxyethyl)-Iactamlde
190 23
Tetrahydrofurfurylalcohol dlethyleneglycolether (Tetraglycoi®)
Polyoxyethylene sorbltanemonooleat (Tween® 80)
76 i0
i ,2-Propanedlol
400
115 18
N,N-dlethylacetamlde
Polyethylene glycol 400 /(Lutrol9®)
134 18
Dmthyleneglycoimouoethylether (Transcutol®)
Solvent
Molecular wetght
CsHjsO4
C~HsOz
HO(C2H,O).H
CsH.NO3
C3HsO3
C2H6OS
C6Ht3NO
C6HI,Oj
Molecular formula
OH
H
O
I
OH
I
OH
I
H2C C H--C H2--O--C Hz--CHz--O--CH2-- CH2 \/ I O OH
I
OH OH
H2C'-CH--CH~
HO--(C H2--C H 2---O--),- - H
N H--'CH2---CH2--'OH
I
OH
H2C--CH--CH2
H~C~ S--CH3
f/° CH3-- C H - - - C \
Hz(~-~Hz
/CH~--CH3
H3C--C--N \CH2---CH~
O II
HO--"C H z--C H 2"-'-O-'-CH~----C H 2--O--C H z---C H ~-
Structural formula
TABLE I Chemical, Physical and Ongm Data of the Tested Drug Solvents
I 079
1 086
I 036
I 126
I 144
! 221
1099
0 907
0990
(d 20°C)
Specdic gravtty Ongm
Pfannenschmldt
Merck
BASF
C H Boehrmger & Sohn, lng
Merck
Fluka
Merck
Gattefoss~, Lyon
DAB7 Atlas-Chemle /USP XVIi
chem pure
USP XVII
DAB 7
pharm qual
DAB 7
purlss
pro synth
pharm qual
Purity
o m z
Toxic. sedative and muscle relaxant potency of vartous drug solvents m m,ce
469
3.3 METHODS 3.3 1. Acute mtrapentoneal toxiczty T h e solvents w e r e diluted with w a t e r and administered In geometrically increasing d o s e s (dose f a c t o r 2) at a v o l u m e of 10ml/kg, exceptionally at 2 0 m l / k g in high d o s e s Six animals w e r e used per dose. The LDs0 values were d e t e r m i n e d by problt analysis.
3 3 2 CNS-profile test according to Campbell and Richter, reclined screen and balance rod tests The solvents were administered as described in Section 3.3.1 T h r e e animals were used per dose T h e p e r f o r m a n c e of the CNS-profile test and the classtficatlon of the solvents were m a d e according to the criteria of Campbell and Richter (1967) S u b s e q u e n t to the CNS-profile test the inchned screen test w a s p e r f o r m e d In the s a m e animals T h e animals w e r e set d o w n at the b o t t o m end of an inclined screen (a = 30 °) consisting of a wtre grating ( a p p r o x 40 × 30 c m side length, square m e s h e s of 2 m m side length) Animals not c h m b m g up the screen within 5 m m were regarded as reactors. T h e EDs0 value was d e t e r m i n e d by the m e t h o d of Behrens and K h r b e r (1935) S u b s e q u e n t to the inchned screen test the balance rod test was p e r f o r m e d In the s a m e a m m a l s (30-35 rain p.l.) T h e mice had to run on a horizontal metal bar 40 cm long and d i a m e t e r 8 ram. Animals that d r o p p e d off or did not run at all w e r e designated as reactors. The ED~0 value was calculated b y the Behrens and K a r b e r (1935) m e t h o d 3.3.3 Potentiation of hexobarbitone sleeping time T h e solvents were administered to fasted mice as a 6.25-100 per cent a q u e o u s solution at a v o l u m e of 10 ml/kg p.o Sixty*minutes later the animals w e r e given hexobarb~tone at a dose of 64 mglkg, intravenously. The sleeping antrnals were put on a w a r m plate (37°C) The time untd r e a p p e a r a n c e of the righting reflex was measured. By m e a n s of probit analysis the d o s e of the drug solvent w a s calculated wh,ch caused a 1 2 ( E D ~ ) or 1 "4 (EDs0b) potenUatlon of sleeping Ume in half of the a m m a l s as c o m p a r e d with the control group (aqua bidest. 10 mllkg) Six animals w e r e used per dose 3.3 4 Rota-rod test Mice w e r e t r a m e d to run on a rotating rod (dla 4 cm; 12 rev/mln) without d r o p p m g off for a 5 min period T h e drug solvents were administered orally at a v o l u m e of 10 ml/kg T h e time until dropping off (max 60 sec) w a s d e t e r m i n e d 40, 60, 90 and 135 m m p a. Five d o s e s (four a m m a l s per dose) w e r e tested
4 RESULTS 4.1
ACUTE INTRAPERITONEAL TOXICITY
T h e L D values, with hterature data and r e f e r e n c e s , are listed In Table 2 As part of the literature data were given in ml/kg they w e r e c o n v e n e d mto g/kg by m e a n s of their density 4.2
CNS-PROFILE TEST ACCORDING TO CAMPBELL AND RICHTER, INCLINED SCREEN AND BALANCE ROD TESTS
T h e r e s u l t s are s u m m a r i z e d in T a b l e 3 I n the C N S - p r o f i l e t e s t the f o l l o w m g p h e n o m e n a w e r e o b s e r v e d w i t h i n 30 m l n p l at JPT
~ i ~
~F
LDse (gJkg) I p
65 43
78 (7 54-8 06) 76 (7 22-7 98)
Tetrahydrofurfurylalcohol dlethyleneglycolether (Tetraglycol ®) Polyoxyethylene sorbltanemonooleat (Tween ® 80)
Polyethylene 81ycol 400 (Lutroi 9 ° ) 1,2-Propanedlol 83
10 I
13 8
16 3 (157-170) 13 2 (12 7-13 5) II 2 (11 0-11 5)
N -(/3-hydroxyethyl)-Iactamlde
73
98
14
I7
LDos (8]kg) t p
88 (8 49-9 04)
(12 6-13 3)
130
(1 58-1 67)
ib
(2 25-2 34)
23
(confidence hmtts)
Glycenne
Dtmethylsuifoxtde
I~ethyleneglycol monoethylether (Transcutol ®) N,N-dlethylacetamtde
Solvent
II 4 17 2 3 8(t v )
42 14.5 97
13 8 14 7 18.0 6.9 8.7 12 2 (s c ) 15 8 (s c )
--
--
LDso(gJklg), p (hteraturc)
Davis and Jenner (1959) Zaroshnskl el a/ (19"/I) Sptegelberg el al (1956)
Spector (1956) Beurtsch el ai (19/6) i_~mpc and Easlerday (1953)
Bartsch el a/ (1976) Farrant (1964) Caujolle et al (1967) Farrant (1964) Bartsch et a/ (1976) Latven and Mohtor (1939) Neumann and Vlehmann (1959)
--
Reference
TABLE 2 Acute lntrapentoneal Toxlc,ty o[ the Tested Drug Solvents m Mice (LDso, 95% Conj~dence Limits, LDos, and Literature Data) Number ol Animals per Dose n = 6
4~
u~
O
3 3 3 3
N -(0-hydroxyethyl)-Iactamtde
Polyethylene glycol 400 (Lutrol 9~)
Tetrahydrofurfurylalcohol dtethyleneglycolether (Tetraglycol ®) Polyoxyethylenesorbltanemonooleat (Tween®80) No classification, abduced hind legs ante mortem
3
Glycerine
3
SympatholyUc AntJconvulsant, weak analgeslc No classtflcatton, pdoerectmn and convulsions ante mortem No classlficatmn, ataxla, hypothermia, convulsions ante re®nero Antidepressant AntidepresSant, skeletal muscle relaxant NeurolepUc
3 3
1,2-Propanedaol
Neuroleptic
3
CNS-profile classdicatlon, symptoms
Dtethyleneglycol monoethylether (Transcutol ®) N,N -dlethylacetamlde DImethylsulfoxide
Solvent
No of animals per dose (n)
10 8
07
28 50
II 4
60
06 28
50
Mm sympt dose (g/kg) I p
• > 10 0
I4
99 77
> 50
> 50
> I ! 11 0
50
Inclined screen ED~o(g/kg) i p
> 10 0
I4
II 3 45
> 50
- 50
08 36
3I
Balance rod EDse(g/kg) I p
TAnLE 3 Results of CNSoprolile Test (Campbell and Richter. 1967), Inclined Screen Test and Balance Rod Test (Classl~cation, Symptoms, Mm Sympt Dose. ED~o(g/kg) for Inclined Screen and Balance Rod Tests Determined by Method of Behrens and K~rber (1935)) All Tests were Performed with Mtce
4~
.s
,if ft
e~
O
¢f
g~ f~
O
g_
e~
go
e~
R BUDDEN et al
472
the mmzmum symptomatic dose Dlethyleneglycol ethylether
mollo-
N , N -dtethylacetamtde
Dlmethylsulfoxlde Glycerine N -(~ -hydroxyethyl)-Iactamtde 1,2-Propanedml Tetraglycol® Tween ® 80
ptos~s, plloerectlon, hypothermta, mus-
cle relaxation, mhlbmon of Hatfner reflex, cyanosls increased paw temperature, mmsts, mhtbttmn of Hafiner reflex muscle relaxation pdoerect~on hypothermm, ataxm Straub tad phenomenon muscle relaxation exRus letahs
E x c e p t for polyethylene glycol 400, all preparatmns caused the ammals to react more sensitively m the balance rod test than m the lnchned screen test Yet the mimmum symptomatic doses of N,N-dtethylacetamtde, dimethylsulfoxtde, polyethylene glycol 400 and Tetraglycol ® m the CNS-profile test are even below the EDs0 values m the balance rod test 4 3 POTENTIATION OF HEXOBARBITONE SLEEPING TIME
The results are presented in Table 4 Except for N-(//-hydroxyethyl)-lactamlde and polyethylene glycol 400 all drug solvents caused at least a 1:2 potentiatmn of sleeping t~me in the dose range tested. It is noteworthy that m all solvents except polyethylene glycol 400 the minimum dose leading to a potentmtion of sleeping time is even below the mtmmum symptomatic dose In the CNS-profile test 4 4. ROTA-ROD TF.ST
The results are presented in Table 5. Dlethyleneglycol monoethylether, dtmethylsulfoxlde, and glycenne possessed the highest, N-(//-hydroxyethyl)-lactamtde the lowest potency of the tested solvents m producing premature drop off. It is remarkable that the lowest dose of diethyleneglycol jnonoethylether (0.3 g/kg) produced premature drop off by excRatmn, not by muscle relaxant effects. TAaLE 4 Potentmtion o f Hexobarbltone Sleeping Time in Mice by Drug Solvents (EDos..*EDso~,*EDosb,* ED~ob,* 95% Confidence Lzm~ts) Number of Ammals per Dose n = 6 All Numbers m g/kg p o
Solvent
ED05.
ED~ (confidence hmRs)
EDosb
ED~, (confidence hmRs)
l~ethyleneglycol monoethylether (Transcutol ®) N,N-chethylacetamtde
0 07
07 (0 43- ! 2 I)
0 42
24 (I 37-4 25)
0 03
0 12
DImethylsulfoxlde
009
Glycenne
078
06 (0 45--0 77) 25 (1 38-4 51) >50
N *(~-hydroxyethyl)-Iactamtde Polyethylene glycol 400 (Lutroi 9° )
62t 56t
02 (0 11-0 22) 08 (0 52-1 3) 36 (2 45-5 18) >114 >113
1,2-Propanedtol
2 03
Tetrahydrofurfurylalcohol dlethyleneglycolether (Tetraglycoi ®) Polyoxyethylene sorbltanemonooleat (Tween ® 80)
067 0 68
52 (4 26-6 42) 14 (1 11-1 71)
043
~i14 ~.113 > 104 15
22 (I 71-2 79)
*Index "'a" means i 2 and mdex "b" means 1 4 potenttatlon of sleepmg ttme tApprox values, no 1 2 potentlatton w a s seen m the htghest dose
29 (247-3 34) > 50
Toxic, sedative and muscle relaxant potency of various drug solvents m mice
473
T^aLe 5 Muscle Relaxant EMects o f Drug Solvents, Results From Rota-Rod Experiments m Mice Number o f Ammals per Dose n = 4
Mm dose producingdrop off (g/kg p o )
Solvent Dlethyleneglycolmonoethylether(Transcutol®) N , N -dIeth ylacetamIde
Uqmethyisulfoxlde Glycerine N-(fl-hydroxyethyl)-iactamlde Polyethyleneglycol400 (Lutrol 9®) 1.2-Propanediol Tetrahydrofurfurylalcohold~ethyleneglycolether (Tetraglycol®) Polyoxyethylenesorbltanemonooleat(Tween® 80)
03* 13"t
03 03 50 06 25 25 13
*Strong stimulation of ammals at lowest dose tLDtee at 50 g/kg p o
5 DISCUSSION Except for dlmethylsulfox,de and 1,2-propanedtol, knowledge about drug solvent effects in screening tests ~s rare Most reference data are on toxic effects The reference values of Lp. LD~0 are in agreement with our results. Only the LDs0 value of 4.2 g/kg for polyethylene glycol 400 reported by Spector (1956) and the value of 17.2 g/kg found by Zaroslmski e t a l (1971) for 1,2-propanediol differ notably from our results The latter LD~0 value also differs from the other reference values for 1,2-propanediol (Lampe and Easterday, 1953; Davis and Jenner, 1959) The LDs0 value for polyethylene glycol 400 reported by Spector (1956) is not congruent w,th the value found by Bartsch e t a l (1976) and our own results. No reference data were found concerning the effects of drug solvents m psycopharmacological screemng tests (e.g CNS-profile tests according to Campbell and P,achter (1967) or to Irvm (1962), mchned screen test, and balance rod test) As the effects on behavior predominantly occurred m subtoxic doses, we assume that they are toxic effects rather than specific actions Zaroslinski e t a l (1971) mvestigated the action of 1,2-propanediol on hexobarbttone sleeping tune. The authors found a significant potentiation of sleeping time by more than 4 0 ~ at 4.16g/kg 1,2-propaned,ol p.o Our E D ~ value for this compound (5.2 g/kg) is consistent with the above result. Potenuatton of hexobarbitone sleepmg tune m all solvents except one ,s effectuated m doses below the mmunum symptomatic dose m the CNS-profile test The relative potency of the drug solvents in the hexobarbltone sleepmg tune as compared with the other tests is underhned by the fact that the solvents were administered orally Except for glycenne, N-(fl-hydroxyethyl)-lactamlde and polyethylene glycol 400, thts test seems to be even more sensmve than the rota-rod test, as most ED05, values were below the mmtmum dose producing drop off. In contrast to our results with dunethylsulfoxtde, Gergely (1970) found a shortening of hexobarbltone sleepmg time m female and a potenUaUon only in male m~ce We have no apparent explanation for this dlscrepancy The conclusions from our results are summartzed m Table 6, where recommendations for the h,ghest concentrations of the tested drug solvents are g~ven Except for glycerine, N-(13-hydroxyethyl)-lactamide and polyethylene glycol 400, these data correspond to the ED05a values m the hexobarbitone sleeping time test For the above compounds the recommended value corresponds to the mmunum effecUve dose m the rota-rod test These data were calculated assuming a volume of 10 ml/kg ~s not exceeded m drug admm~stration m m~ce The results presented emphaslze that the drug solvents tested cannot be regarded as "inert" cosolvents in pharmacologic studies, whether the oral or lntrapentoneal route Is employed Possible rarefactions w~th the dissolved drugs must be kept m m i d .
R BUDDEN et al
474 TAet~
6 Recommendation for Tolerable Concentrations of Psychopharmacologtcal Screemng Tests
Drug
Solvents
m
Tolerable concentration m % (w/v)
Solvent Dtethyleneglycol monoethylether (Transcutol ®) N,N-dlethylacetamlde l~methylsulfoxlde Glycerme N -(~ -hydroxyethyl)-lactam,de Polyethylene glycol 400 (Lutroi 9®) 1,2-Propanedlol Tetrahydrofurfurylalcoholdaethyleneglycolether (Tetraglycoi©) Polyoxyethylene sorbRanemonooleat (Tween ® 80)
07 03 09 30 50 0 60 200 67 68
W h e n e v e r a d r u g s o l v e n t is u s e d , Rs c o n c e n t r a t i o n m u s t be s p e c i f i e d a n d l a c k o f a c t w l t y m t h e r e s p e c U v e t e s t s y s t e m h a s to be d e m o n s t r a t e d
REFERENCES BARTSCH, W , SPONER, G , DIEYMANN, K and FUCHS. G (1976) Acute toxicity of vartous solvents in the mouse and rat Arzneun-Forsch. (Drug Res ) 26, 1581-1583. BEUI~NS, B and K ~ I g R , G (1935) Wte stud Rmhenversuche for btolo~sche Auswertungen am zweckmaBtgsten anzuordnen "t Naunyn-$chmledeberg's Arch Pharmac 177, 379-388 CAMPaSLL, D E. S and 17dct-rr~ W (1967) An observattonai method esttmatmg toxlc:ty and drug actions m mice apphed to 68 reference drugs Acta Pharmac Toxicoi 25, 345-363 CAUJOL[.~ F, M. E , CAUJOLLI~, D H , CROS, S B and CALvL~r M-M J (1967) LimRs of toxic and teratogemc tolerance of dimethyl sulfoxtde Ann. N Y Acad Scz 141, 110-125 DAVIS. K J and JENNER,P M (1959) Toxtclty of three drug solvents Toxicol appl Pharmac 1,576-578 FARRAIcr, J (1964) Pharmacolotccal acUons and toxicity of dlmethyl sulphoxlde and other compounds which protect smooth muscle during freezmg and thawing J Pharm Pharmac 16, 472-483 GERGELY, J (1970) Injekc16k old6szeremek hatisa a hexobarbR~ti haUtstartamira Acta pharmaceut huns 40, 97-101 IRVlN, S (1962) Drug screemng and evaluaUve procedures Science, N Y 136, 123-128 LAMPE, K. F and EASTERDAY,O D (1953) A note on a contramdzcatlon to propylene glycol, as a solvent m toxicity stud,es J Pharmaceut S o 42, 445 L^TVeN, A R and MoLrroa, H (1939) Comparison of the toxic, hypnoUc and trntatmg propemes of eight orgamc solvents J Pharmac exp Ther 6S, 89-95 NEUMANN,H and Vlth~tlANN, P (1959) Stabtle wissrtige l..6sungen yon Antiblottka der Tetracychn-Gruppe mlt verhesserter Gewebsvertraghchkett A~nelm -Forsch 9. 711-713 SPECTOR, W S (1956) Quoted m SPIIgGt~Land NOSEWORTHY(1963). q v SPXEGEL, A J and NOSEWOttTHY, M M (1963) Use of nonaqueous solvents m parenteral products 2" Pharm Scl !2, 917-927 SPlELGELBERG, H , SCHL,gPFER, R , ZaiNDEN, G and STUD~a, A (1956) Em neues mjtzterbares LosungsmRtel (Glycofurol) Arznelm -Forsch 6, 75-77 ZAROSLINSKI, J F , BROWNIL R K and POSSLEY, L H (1971) Propylene glycol as a drug solvent m pharmacologic stu&es Toxwol appl Pharmac 19, 573--578
NOTE
ADDED
IN PROOF
W h i l e th,s r e p o r t w a s in p r o d u c t i o n , f u r t h e r i n v e s t i g a t i o n s w , t h the a b o v e d r u g s o l v e n t s w h e r e p e r f o r m e d ( e s p e c i a l l y c a r d i o v a s c u l a r ) , the r e s u l t s o f whzch w e r e p u b l i s h e d ,n A r z n e ~ m . - F o r s c h ( D r u g Res.) 28. 1571-1593 (1978)
Pnnted m Great Britain
EXPERIMENTS ON THE TOXIC, SEDATIVE MUSCLE RELAXANT POTENCY OF VARIOUS SOLVENTS IN MICE
AND DRUG
R BUDDEN, U G KUHL and J BAHLSEN Department Pharmacologtcal Research I, Kah-Chemze Pharma OmbH, Hannover, Hans-Bockler-Allee 20, Germany
1. S U M M A R Y The solvents dlethyleneglycol monoethylether (Transcutol®), N,N-dmthylacetamlde, dimethylsulfoxlde, glycenne, N-(/3-hydruxyethyl)-Iactamlde, polyethylene glycol 400 (Lutrol 9~), "l.2-propanediol, tetrahydro= furfurylalcohol d~ethyleneglycolether (Tetraglycol ®) and polyoxyethylene sorbttanemonooleate (Tween ® 80) were tested m mice for their tox~cRy, tketr CNS-profile, thew acnwty m the mchned screen and balance rod tests a f t e r , p admm~straUon as well as for their influence on the hexobarbltone sleeping t~me and their muscle relaxant properties on the rota-rod after p o admmlstrauon It ~s demonstrated that the compounds tested cause a wtde variety of pharmacodynamtc effects m concentraUons m which they are used as drug solvents m the laboratory As proved by the potenaatmn of hexobarbRone sleeping time, interactmns wtth the drugs to be tested cannot be excluded (Just m this test all solvents but three exhlbtted thetr h~ghest actavRy Only glycenne. N-(//-hydroxymethyl)-Iactamlde and polyethylene glycol 400 were most potent m the rota=rod test ) So, whenever a drug solvent is used, ~ts concentratmn must be specdied and lack of acuvlty m the respectmve test system has to be demostrated Based on thetr actlvRy m the most sensmve test, the drug solvents examined are recommended not to exceed the following concentrataons m drug solutmns for screening purpose dlethyleneglycol monoethylether 0 7%, N,N-ðylacetamlde 0 3%, dlmethylsulfoxlde 0 9%, glycenne 3.0%, N-(/3=hydroxyethyl)lactamtde 50%, polyethylene glycol 400 6 0%, 1,2=propanedml 20%, Tetraglycol ® 6 7% and Tween ® 80 6 8%
2 INTRODUCTION O n e o f t h e first p r o b l e m s
facing the experimentally
working pharmacologist
when
screening water-insoluble drugs is to transfer them into a hqmd water-mixable phase Therefore some common drug solvents were tested for their applicability m the pharmacological laboratory The solvents were screened for acute Lp. toxicity, influence on behavior as well as for their sedative and muscle relaxant properties m mice 3
MATERIALS
AND
METHODS
3.1 ANIMALS All tests except the hexobarbRone sleeping tune test were performed m male NMRI m l c e ( 1 8 - 2 2 g) F o r d e t e r m i n a t i o n o f h e x o b a r b l t o n e s l e e p i n g t i m e f e m a l e N M R I m l c e ( 1 8 - 2 2 g ) w e r e u s e d All a n i m a l s w e r e b r e d at t h e Z e n t r a l i n s t i t u t f u r V e r s u c h s -
tterzucht, Hannover. They were kept m air=conditioned rooms at 20-30°C tern= perature and 50-60% relative humidity in groups of 20 anunals per cage (Macrolon Type III). The animals were fed with a commercial diet ("altrominmR 1324", Altrogge) and tap water ad libitum. In case of oral drug admmtstratlon fasted animals were used.
3.2
DRUG SOLVENTS
The chemical and physical data and the origin of the solvents tested are listed m Table 1 467
78 13
92 10
133 15
DImelhylsulfoxlde
Glycerine
N-(O-hydroxyethyl)-Iactamlde
190 23
Tetrahydrofurfurylalcohol dlethyleneglycolether (Tetraglycoi®)
Polyoxyethylene sorbltanemonooleat (Tween® 80)
76 i0
i ,2-Propanedlol
400
115 18
N,N-dlethylacetamlde
Polyethylene glycol 400 /(Lutrol9®)
134 18
Dmthyleneglycoimouoethylether (Transcutol®)
Solvent
Molecular wetght
CsHjsO4
C~HsOz
HO(C2H,O).H
CsH.NO3
C3HsO3
C2H6OS
C6Ht3NO
C6HI,Oj
Molecular formula
OH
H
O
I
OH
I
OH
I
H2C C H--C H2--O--C Hz--CHz--O--CH2-- CH2 \/ I O OH
I
OH OH
H2C'-CH--CH~
HO--(C H2--C H 2---O--),- - H
N H--'CH2---CH2--'OH
I
OH
H2C--CH--CH2
H~C~ S--CH3
f/° CH3-- C H - - - C \
Hz(~-~Hz
/CH~--CH3
H3C--C--N \CH2---CH~
O II
HO--"C H z--C H 2"-'-O-'-CH~----C H 2--O--C H z---C H ~-
Structural formula
TABLE I Chemical, Physical and Ongm Data of the Tested Drug Solvents
I 079
1 086
I 036
I 126
I 144
! 221
1099
0 907
0990
(d 20°C)
Specdic gravtty Ongm
Pfannenschmldt
Merck
BASF
C H Boehrmger & Sohn, lng
Merck
Fluka
Merck
Gattefoss~, Lyon
DAB7 Atlas-Chemle /USP XVIi
chem pure
USP XVII
DAB 7
pharm qual
DAB 7
purlss
pro synth
pharm qual
Purity
o m z
Toxic. sedative and muscle relaxant potency of vartous drug solvents m m,ce
469
3.3 METHODS 3.3 1. Acute mtrapentoneal toxiczty T h e solvents w e r e diluted with w a t e r and administered In geometrically increasing d o s e s (dose f a c t o r 2) at a v o l u m e of 10ml/kg, exceptionally at 2 0 m l / k g in high d o s e s Six animals w e r e used per dose. The LDs0 values were d e t e r m i n e d by problt analysis.
3 3 2 CNS-profile test according to Campbell and Richter, reclined screen and balance rod tests The solvents were administered as described in Section 3.3.1 T h r e e animals were used per dose T h e p e r f o r m a n c e of the CNS-profile test and the classtficatlon of the solvents were m a d e according to the criteria of Campbell and Richter (1967) S u b s e q u e n t to the CNS-profile test the inchned screen test w a s p e r f o r m e d In the s a m e animals T h e animals w e r e set d o w n at the b o t t o m end of an inclined screen (a = 30 °) consisting of a wtre grating ( a p p r o x 40 × 30 c m side length, square m e s h e s of 2 m m side length) Animals not c h m b m g up the screen within 5 m m were regarded as reactors. T h e EDs0 value was d e t e r m i n e d by the m e t h o d of Behrens and K h r b e r (1935) S u b s e q u e n t to the inchned screen test the balance rod test was p e r f o r m e d In the s a m e a m m a l s (30-35 rain p.l.) T h e mice had to run on a horizontal metal bar 40 cm long and d i a m e t e r 8 ram. Animals that d r o p p e d off or did not run at all w e r e designated as reactors. The ED~0 value was calculated b y the Behrens and K a r b e r (1935) m e t h o d 3.3.3 Potentiation of hexobarbitone sleeping time T h e solvents were administered to fasted mice as a 6.25-100 per cent a q u e o u s solution at a v o l u m e of 10 ml/kg p.o Sixty*minutes later the animals w e r e given hexobarb~tone at a dose of 64 mglkg, intravenously. The sleeping antrnals were put on a w a r m plate (37°C) The time untd r e a p p e a r a n c e of the righting reflex was measured. By m e a n s of probit analysis the d o s e of the drug solvent w a s calculated wh,ch caused a 1 2 ( E D ~ ) or 1 "4 (EDs0b) potenUatlon of sleeping Ume in half of the a m m a l s as c o m p a r e d with the control group (aqua bidest. 10 mllkg) Six animals w e r e used per dose 3.3 4 Rota-rod test Mice w e r e t r a m e d to run on a rotating rod (dla 4 cm; 12 rev/mln) without d r o p p m g off for a 5 min period T h e drug solvents were administered orally at a v o l u m e of 10 ml/kg T h e time until dropping off (max 60 sec) w a s d e t e r m i n e d 40, 60, 90 and 135 m m p a. Five d o s e s (four a m m a l s per dose) w e r e tested
4 RESULTS 4.1
ACUTE INTRAPERITONEAL TOXICITY
T h e L D values, with hterature data and r e f e r e n c e s , are listed In Table 2 As part of the literature data were given in ml/kg they w e r e c o n v e n e d mto g/kg by m e a n s of their density 4.2
CNS-PROFILE TEST ACCORDING TO CAMPBELL AND RICHTER, INCLINED SCREEN AND BALANCE ROD TESTS
T h e r e s u l t s are s u m m a r i z e d in T a b l e 3 I n the C N S - p r o f i l e t e s t the f o l l o w m g p h e n o m e n a w e r e o b s e r v e d w i t h i n 30 m l n p l at JPT
~ i ~
~F
LDse (gJkg) I p
65 43
78 (7 54-8 06) 76 (7 22-7 98)
Tetrahydrofurfurylalcohol dlethyleneglycolether (Tetraglycol ®) Polyoxyethylene sorbltanemonooleat (Tween ® 80)
Polyethylene 81ycol 400 (Lutroi 9 ° ) 1,2-Propanedlol 83
10 I
13 8
16 3 (157-170) 13 2 (12 7-13 5) II 2 (11 0-11 5)
N -(/3-hydroxyethyl)-Iactamlde
73
98
14
I7
LDos (8]kg) t p
88 (8 49-9 04)
(12 6-13 3)
130
(1 58-1 67)
ib
(2 25-2 34)
23
(confidence hmtts)
Glycenne
Dtmethylsuifoxtde
I~ethyleneglycol monoethylether (Transcutol ®) N,N-dlethylacetamtde
Solvent
II 4 17 2 3 8(t v )
42 14.5 97
13 8 14 7 18.0 6.9 8.7 12 2 (s c ) 15 8 (s c )
--
--
LDso(gJklg), p (hteraturc)
Davis and Jenner (1959) Zaroshnskl el a/ (19"/I) Sptegelberg el al (1956)
Spector (1956) Beurtsch el ai (19/6) i_~mpc and Easlerday (1953)
Bartsch el a/ (1976) Farrant (1964) Caujolle et al (1967) Farrant (1964) Bartsch et a/ (1976) Latven and Mohtor (1939) Neumann and Vlehmann (1959)
--
Reference
TABLE 2 Acute lntrapentoneal Toxlc,ty o[ the Tested Drug Solvents m Mice (LDso, 95% Conj~dence Limits, LDos, and Literature Data) Number ol Animals per Dose n = 6
4~
u~
O
3 3 3 3
N -(0-hydroxyethyl)-Iactamtde
Polyethylene glycol 400 (Lutrol 9~)
Tetrahydrofurfurylalcohol dtethyleneglycolether (Tetraglycol ®) Polyoxyethylenesorbltanemonooleat (Tween®80) No classification, abduced hind legs ante mortem
3
Glycerine
3
SympatholyUc AntJconvulsant, weak analgeslc No classtflcatton, pdoerectmn and convulsions ante mortem No classlficatmn, ataxla, hypothermia, convulsions ante re®nero Antidepressant AntidepresSant, skeletal muscle relaxant NeurolepUc
3 3
1,2-Propanedaol
Neuroleptic
3
CNS-profile classdicatlon, symptoms
Dtethyleneglycol monoethylether (Transcutol ®) N,N -dlethylacetamlde DImethylsulfoxide
Solvent
No of animals per dose (n)
10 8
07
28 50
II 4
60
06 28
50
Mm sympt dose (g/kg) I p
• > 10 0
I4
99 77
> 50
> 50
> I ! 11 0
50
Inclined screen ED~o(g/kg) i p
> 10 0
I4
II 3 45
> 50
- 50
08 36
3I
Balance rod EDse(g/kg) I p
TAnLE 3 Results of CNSoprolile Test (Campbell and Richter. 1967), Inclined Screen Test and Balance Rod Test (Classl~cation, Symptoms, Mm Sympt Dose. ED~o(g/kg) for Inclined Screen and Balance Rod Tests Determined by Method of Behrens and K~rber (1935)) All Tests were Performed with Mtce
4~
.s
,if ft
e~
O
¢f
g~ f~
O
g_
e~
go
e~
R BUDDEN et al
472
the mmzmum symptomatic dose Dlethyleneglycol ethylether
mollo-
N , N -dtethylacetamtde
Dlmethylsulfoxlde Glycerine N -(~ -hydroxyethyl)-Iactamtde 1,2-Propanedml Tetraglycol® Tween ® 80
ptos~s, plloerectlon, hypothermta, mus-
cle relaxation, mhlbmon of Hatfner reflex, cyanosls increased paw temperature, mmsts, mhtbttmn of Hafiner reflex muscle relaxation pdoerect~on hypothermm, ataxm Straub tad phenomenon muscle relaxation exRus letahs
E x c e p t for polyethylene glycol 400, all preparatmns caused the ammals to react more sensitively m the balance rod test than m the lnchned screen test Yet the mimmum symptomatic doses of N,N-dtethylacetamtde, dimethylsulfoxtde, polyethylene glycol 400 and Tetraglycol ® m the CNS-profile test are even below the EDs0 values m the balance rod test 4 3 POTENTIATION OF HEXOBARBITONE SLEEPING TIME
The results are presented in Table 4 Except for N-(//-hydroxyethyl)-lactamlde and polyethylene glycol 400 all drug solvents caused at least a 1:2 potentiatmn of sleeping t~me in the dose range tested. It is noteworthy that m all solvents except polyethylene glycol 400 the minimum dose leading to a potentmtion of sleeping time is even below the mtmmum symptomatic dose In the CNS-profile test 4 4. ROTA-ROD TF.ST
The results are presented in Table 5. Dlethyleneglycol monoethylether, dtmethylsulfoxlde, and glycenne possessed the highest, N-(//-hydroxyethyl)-lactamtde the lowest potency of the tested solvents m producing premature drop off. It is remarkable that the lowest dose of diethyleneglycol jnonoethylether (0.3 g/kg) produced premature drop off by excRatmn, not by muscle relaxant effects. TAaLE 4 Potentmtion o f Hexobarbltone Sleeping Time in Mice by Drug Solvents (EDos..*EDso~,*EDosb,* ED~ob,* 95% Confidence Lzm~ts) Number of Ammals per Dose n = 6 All Numbers m g/kg p o
Solvent
ED05.
ED~ (confidence hmRs)
EDosb
ED~, (confidence hmRs)
l~ethyleneglycol monoethylether (Transcutol ®) N,N-chethylacetamtde
0 07
07 (0 43- ! 2 I)
0 42
24 (I 37-4 25)
0 03
0 12
DImethylsulfoxlde
009
Glycenne
078
06 (0 45--0 77) 25 (1 38-4 51) >50
N *(~-hydroxyethyl)-Iactamtde Polyethylene glycol 400 (Lutroi 9° )
62t 56t
02 (0 11-0 22) 08 (0 52-1 3) 36 (2 45-5 18) >114 >113
1,2-Propanedtol
2 03
Tetrahydrofurfurylalcohol dlethyleneglycolether (Tetraglycoi ®) Polyoxyethylene sorbltanemonooleat (Tween ® 80)
067 0 68
52 (4 26-6 42) 14 (1 11-1 71)
043
~i14 ~.113 > 104 15
22 (I 71-2 79)
*Index "'a" means i 2 and mdex "b" means 1 4 potenttatlon of sleepmg ttme tApprox values, no 1 2 potentlatton w a s seen m the htghest dose
29 (247-3 34) > 50
Toxic, sedative and muscle relaxant potency of various drug solvents m mice
473
T^aLe 5 Muscle Relaxant EMects o f Drug Solvents, Results From Rota-Rod Experiments m Mice Number o f Ammals per Dose n = 4
Mm dose producingdrop off (g/kg p o )
Solvent Dlethyleneglycolmonoethylether(Transcutol®) N , N -dIeth ylacetamIde
Uqmethyisulfoxlde Glycerine N-(fl-hydroxyethyl)-iactamlde Polyethyleneglycol400 (Lutrol 9®) 1.2-Propanediol Tetrahydrofurfurylalcohold~ethyleneglycolether (Tetraglycol®) Polyoxyethylenesorbltanemonooleat(Tween® 80)
03* 13"t
03 03 50 06 25 25 13
*Strong stimulation of ammals at lowest dose tLDtee at 50 g/kg p o
5 DISCUSSION Except for dlmethylsulfox,de and 1,2-propanedtol, knowledge about drug solvent effects in screening tests ~s rare Most reference data are on toxic effects The reference values of Lp. LD~0 are in agreement with our results. Only the LDs0 value of 4.2 g/kg for polyethylene glycol 400 reported by Spector (1956) and the value of 17.2 g/kg found by Zaroslmski e t a l (1971) for 1,2-propanediol differ notably from our results The latter LD~0 value also differs from the other reference values for 1,2-propanediol (Lampe and Easterday, 1953; Davis and Jenner, 1959) The LDs0 value for polyethylene glycol 400 reported by Spector (1956) is not congruent w,th the value found by Bartsch e t a l (1976) and our own results. No reference data were found concerning the effects of drug solvents m psycopharmacological screemng tests (e.g CNS-profile tests according to Campbell and P,achter (1967) or to Irvm (1962), mchned screen test, and balance rod test) As the effects on behavior predominantly occurred m subtoxic doses, we assume that they are toxic effects rather than specific actions Zaroslinski e t a l (1971) mvestigated the action of 1,2-propanediol on hexobarbttone sleeping tune. The authors found a significant potentiation of sleeping time by more than 4 0 ~ at 4.16g/kg 1,2-propaned,ol p.o Our E D ~ value for this compound (5.2 g/kg) is consistent with the above result. Potenuatton of hexobarbitone sleepmg tune m all solvents except one ,s effectuated m doses below the mmunum symptomatic dose m the CNS-profile test The relative potency of the drug solvents in the hexobarbltone sleepmg tune as compared with the other tests is underhned by the fact that the solvents were administered orally Except for glycenne, N-(fl-hydroxyethyl)-lactamlde and polyethylene glycol 400, thts test seems to be even more sensmve than the rota-rod test, as most ED05, values were below the mmtmum dose producing drop off. In contrast to our results with dunethylsulfoxtde, Gergely (1970) found a shortening of hexobarbltone sleepmg time m female and a potenUaUon only in male m~ce We have no apparent explanation for this dlscrepancy The conclusions from our results are summartzed m Table 6, where recommendations for the h,ghest concentrations of the tested drug solvents are g~ven Except for glycerine, N-(13-hydroxyethyl)-lactamide and polyethylene glycol 400, these data correspond to the ED05a values m the hexobarbitone sleeping time test For the above compounds the recommended value corresponds to the mmunum effecUve dose m the rota-rod test These data were calculated assuming a volume of 10 ml/kg ~s not exceeded m drug admm~stration m m~ce The results presented emphaslze that the drug solvents tested cannot be regarded as "inert" cosolvents in pharmacologic studies, whether the oral or lntrapentoneal route Is employed Possible rarefactions w~th the dissolved drugs must be kept m m i d .
R BUDDEN et al
474 TAet~
6 Recommendation for Tolerable Concentrations of Psychopharmacologtcal Screemng Tests
Drug
Solvents
m
Tolerable concentration m % (w/v)
Solvent Dtethyleneglycol monoethylether (Transcutol ®) N,N-dlethylacetamlde l~methylsulfoxlde Glycerme N -(~ -hydroxyethyl)-lactam,de Polyethylene glycol 400 (Lutroi 9®) 1,2-Propanedlol Tetrahydrofurfurylalcoholdaethyleneglycolether (Tetraglycoi©) Polyoxyethylene sorbRanemonooleat (Tween ® 80)
07 03 09 30 50 0 60 200 67 68
W h e n e v e r a d r u g s o l v e n t is u s e d , Rs c o n c e n t r a t i o n m u s t be s p e c i f i e d a n d l a c k o f a c t w l t y m t h e r e s p e c U v e t e s t s y s t e m h a s to be d e m o n s t r a t e d
REFERENCES BARTSCH, W , SPONER, G , DIEYMANN, K and FUCHS. G (1976) Acute toxicity of vartous solvents in the mouse and rat Arzneun-Forsch. (Drug Res ) 26, 1581-1583. BEUI~NS, B and K ~ I g R , G (1935) Wte stud Rmhenversuche for btolo~sche Auswertungen am zweckmaBtgsten anzuordnen "t Naunyn-$chmledeberg's Arch Pharmac 177, 379-388 CAMPaSLL, D E. S and 17dct-rr~ W (1967) An observattonai method esttmatmg toxlc:ty and drug actions m mice apphed to 68 reference drugs Acta Pharmac Toxicoi 25, 345-363 CAUJOL[.~ F, M. E , CAUJOLLI~, D H , CROS, S B and CALvL~r M-M J (1967) LimRs of toxic and teratogemc tolerance of dimethyl sulfoxtde Ann. N Y Acad Scz 141, 110-125 DAVIS. K J and JENNER,P M (1959) Toxtclty of three drug solvents Toxicol appl Pharmac 1,576-578 FARRAIcr, J (1964) Pharmacolotccal acUons and toxicity of dlmethyl sulphoxlde and other compounds which protect smooth muscle during freezmg and thawing J Pharm Pharmac 16, 472-483 GERGELY, J (1970) Injekc16k old6szeremek hatisa a hexobarbR~ti haUtstartamira Acta pharmaceut huns 40, 97-101 IRVlN, S (1962) Drug screemng and evaluaUve procedures Science, N Y 136, 123-128 LAMPE, K. F and EASTERDAY,O D (1953) A note on a contramdzcatlon to propylene glycol, as a solvent m toxicity stud,es J Pharmaceut S o 42, 445 L^TVeN, A R and MoLrroa, H (1939) Comparison of the toxic, hypnoUc and trntatmg propemes of eight orgamc solvents J Pharmac exp Ther 6S, 89-95 NEUMANN,H and Vlth~tlANN, P (1959) Stabtle wissrtige l..6sungen yon Antiblottka der Tetracychn-Gruppe mlt verhesserter Gewebsvertraghchkett A~nelm -Forsch 9. 711-713 SPECTOR, W S (1956) Quoted m SPIIgGt~Land NOSEWORTHY(1963). q v SPXEGEL, A J and NOSEWOttTHY, M M (1963) Use of nonaqueous solvents m parenteral products 2" Pharm Scl !2, 917-927 SPlELGELBERG, H , SCHL,gPFER, R , ZaiNDEN, G and STUD~a, A (1956) Em neues mjtzterbares LosungsmRtel (Glycofurol) Arznelm -Forsch 6, 75-77 ZAROSLINSKI, J F , BROWNIL R K and POSSLEY, L H (1971) Propylene glycol as a drug solvent m pharmacologic stu&es Toxwol appl Pharmac 19, 573--578
NOTE
ADDED
IN PROOF
W h i l e th,s r e p o r t w a s in p r o d u c t i o n , f u r t h e r i n v e s t i g a t i o n s w , t h the a b o v e d r u g s o l v e n t s w h e r e p e r f o r m e d ( e s p e c i a l l y c a r d i o v a s c u l a r ) , the r e s u l t s o f whzch w e r e p u b l i s h e d ,n A r z n e ~ m . - F o r s c h ( D r u g Res.) 28. 1571-1593 (1978)