Teratogenicities I. Antiviral
of
Ophthalmic Drugs
Ophthalmic Drugs
Motokazu Itoi, MD; Jeffrey W. Gefter; Noboru Kaneko, MD; Yasuo Ishii; Richard M. Ramer, PhD; Antonio R. Gasset, MD
Nonradioactiveidoxuridine(IDU,5-iodo2\m='\-deoxyuridine), while not teratogenic to rats, does produce fetal malformations in
rabbits when administered topically to the eye in doses similar to those used clinically, 0.1% four times a day for 12 days. These malformations include exophthalmos and clubbing of the forelegs. By
contrast, trifluorothymidine (F3 TdR), another highly effective antiherpetic agent
currently under investigation but not avail able for general use, was found not to be teratogenic to rabbits, even when given in concentrations tenfold greater than the doses used to produce idoxuridine teratogenicity.
studies on mammalian tera¬ in the 1930s, the science of teratology practiced in lower classes of animals was at least 100 years old. But it was doubted by many investigators of hu¬ man congenital malformations that the results obtained on eggs of lower classes could be applied to mamma¬ lian or human situations. It was thought that mammalian embryos
Whentologyexperimental began
Submitted for publication May 25, 1973. From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville (Drs. Ramer and Gasset and Mr. Gefter); and Juntendo University, Tokyo (Drs. Itoi and
Kaneko). Reprint requests
to Department of Ophof Medicine, University of Florida, Gainesville, FL 32601 (Dr. Gasset).
thalmology, College
and fetuses were so well protected by the maternal organism that they could not be modified by methods teratogenic to embryos of lower classes. In 1948, two definite teratogens, the nitrogen mustards and trypan blue, were found. Haskin1 described severe malformations in the off¬ spring of rats treated with a radiomimetic and cytotoxic nitrogen mus¬ tard, and similar results were later reported in mice.- Gillman et al,1 using trypan blue as a teratogen, found hydrocephalus, spina bifida, and other congenital anomalies in the young of treated rats. Many hormones have been used as teratogens.1 Notable among those drugs is cortisone which was first found to be teratogenic to mice by Baxter and Fraser in 1950. Of even greater importance is the work by Fraser and Faintstat and Kalter,68 who demonstrated the inheritance of susceptibility to the teratogenic ac¬ tion of cortisone. A review of the field of experimen¬ tal mammalian teratology disclosed that a huge mass of facts has been
gathered; however, some entire branches such as teratogenicities of ophthalmic drugs have failed to de¬ velop. Teratogenicities of ophthalmic medications, eg, corticosteroids, antimetabolites, edetic acid (EDTA),
Downloaded From: http://archopht.jamanetwork.com/ by a New York University User on 05/27/2015
heavy metals and others, which are clearly implicated in the etiology of congenital malformations when ad¬ ministered systemically, have not yet been evaluated when applied topically to the eye. Such studies are of the ut¬ most importance in view of the wide
and
frequent use of some of these preparations as ophthalmic drugs or preservatives. The purpose of this study is to eval¬ uate the teratogenic effect of a vari¬ ety of ophthalmic drugs when given topically to the eye in doses equiva¬ lent to those used clinically. In this report, the teratogenic effect of idoxuridine (IDU, 5-iodo-2'-deoxyuridine) and trifluorothymidine (F,TdR), when given topically to the eye in doses equivalent to those used clini¬ cally against herpetic keratitis, was
studied. In addition to the evaluation of external malformations, dissection of the abnormal specimens, serial histological sectioning, drug accumula¬ tion and thin-layer chromatography studies were done. Materials and Methods
Idoxuridine-FjTdR Teratogenesis in Rab¬ bits.—A total of 31 Japanese albino rabbits were used in these studies. Fifteen rabbits received 1 drop of 0.1% idoxuridine in each eye six times on the sixth day of gestation and thereafter four times a day from the seventh to the 18th day of gestation. Eight
20-
Subcutaneous
njection Topical Eye Drops
Fig 1.—Exophthalmus in rabbit fetus, in 28th day of development, malformed by Idoxuridine administration to mother.
4 5 6 Hours After Application
Fig 4.—Idoxuridine ,25l accumulation in blood of adult rabbits receiving subcutaneous injection vs topical administration of drug, (cpm indicates counts per minute.)
Fig 2.—Fetus "on 28th day of gestation showing clubbing of forelegs.
•-·
(3 Rabbits)
1/2 1
Fig 3.—Composite photograph of nor¬ mal and malformed rabbit fetus after idox¬ uridine administration (IDU) stained with Alizarin red S, showing that clubbing (ar¬ row) is due to soft-tissue deformities rather than defective bone development. treated with 1.0% F.TdR and rabbits with 0.9% physiologic saline following exactly the same schedule as in the group treated with 0.1% idoxurirabbits
eight
were
more
Fig
Ito 2
Fetal Blood Mother's Blood
(3)
21/2 3
31/2 4 41/2 5 51/2 6 61/2 Hours After Injection
7
7V2 8
81/2 9
5.—Idoxuridine l«*l accumulation in maternal and fetal blood after maternal subcu¬ injection of drug on 28th day of gestation.
taneous
dine. All rabbits were killed on the 28th day of gestation, and the fetuses were re¬ moved by cesarean section and carefully examined for external malformations. In addition, dissection of abnormal specimens for further examination of skeletal malfor¬ mation and serial histological sectioning were done.
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Accumulation Studies of Idoxuridine La¬ beled With Iodine 125.—Route of Adminis¬
tration.—A total of six healthy adult New Zealand albino rabbits weighing 4.0 to 4.4 kg were used. Each rabbit received 150 mi¬ crocuries of idoxuridine 125I in a 0.1% cold idoxuridine solution either topically as eye drops or subcutaneously. Three (two males
103 cpm/gm
103 cpm/gm I
—X
Idoxuridine
·—·
lodouracil
> — Idoxuridine •—·
lodouracil Iodide
Iodide
-14
-r
2
Hours
Hours
Fig 6.—Thin-layer separation of idoxuridine 121 from its metabo¬ lites in maternal blood (left) and in fetal blood (right) after materand one nonpregnant female) received idoxuridine ''"I topically, and three (two males and one nonpregnant female) re¬ ceived the same amount of idoxuridine 12T subcutaneously. Blood samples were drawn from each rabbit of both groups at succes¬ sive time intervals after administration. The samples were then counted for radio¬ activity in a gamma well-type scintillation counter calibrated for '-T emissions. Maternal vs Fetal Accumulation.—In the first of these studies, 11 healthy 6month-old New Zealand albino rabbits were each mated twice with the same male, and the day of conception was deter¬ mined by the presence of sperm on vaginal smear. Twenty-eight days after mating, each of the 11 females was given 30 microcurie of idoxuridine 12T in a 0.1% cold idox¬ uridine solution subcutaneously, and they were then killed at different time intervals from 45 minutes to seven hours after ad¬ ministration. After blood samples were collected, brain, kidney, and liver tissues of both mothers and fetuses were excised, weighed, and counted. In a second study, to determine the rela¬ tive metabolic breakdown of the idoxuri¬ dine ,2T into iodouracil 125I and iodide ,25I, four healthy 6-month-old Japanese albino rabbits in their 28th day of gestation, were each given a subcutaneous injection of 150 microcuries of idoxuridine 125I in a 0.1% cold idoxuridine solution. The rabbits were killed at intervals of 1, 3, 5V2, and 8 hours after administration, respectively. Blood samples from each time period were pre¬ pared by removing the cells and deproteinizing the serum. Thin-layer Chromato¬ graphie separation of idoxuridine 12T from its metabolic breakdown products as de¬ scribed by Clarkson et al" was performed on the fetal and maternal blood samples prepared as above. After extraction of idox-
nal subcutaneous injection of drug indicates counts per minute.)
uridine, iodouracil and iodide, the radio¬ activity of each fraction was counted and
compared. In addition,
two healthy 6-month-old New Zealand albino rabbits in their 12th day of gestation, as determined by vaginal smear, were each subcutaneously injected with 300 microcuries of idoxuridine 12T in a 0.1% cold idoxuridine solution. Both rabbits were killed 4Vè hours after administration, and their fetuses were removed by cesarean section. Determinations of radio¬ active accumulation in maternal blood, pla¬ centa, and whole fetus were made by com¬ parison of specific activity in tissues. Autorudiography.—A healthy 6-monthold New Zealand albino rabbit in the 28th day of gestation was subcutaneously in¬ jected with 300 microcuries of idoxuridine 12T in a 0.1% cold idoxuridine solution, and killed one hour after administration. A fetus from this rabbit was removed, sec¬ tioned, and the sections mounted on glass slides. Several mounted sections were directly applied to the sensitive surface of recording autoradiographic film, fixed to the film unmoved, and kept in darkness for three weeks. The mounts were then re¬ moved, and the sensitized film developed.
on
28th
day of gestation, (cpm
Results
Teratogenicity Studies.—The re¬ sults on 272 fetuses obtained by cesarean section from 31 pregnant rabbits treated with idoxuridine, F,TdR, or saline are summarized in the Table. The idoxuridine-treated group had a fivefold greater incidence of dead fetuses than did the control or the F:,TdR-treated group. In addition, 23% of the live fetuses from the idoxuridine group demonstrated con¬ genital anomalies such as the one shown in Fig 1 to 3. Figure 1 shows an exophthalmus-like malformation which was found in approximately 16% of the fetuses from idoxuridinetreated mothers. Figure 2 shows club¬ bing of the forelimbs, another con¬ genital abnormality, found in about 9% of the fetuses. Further examina¬ tion of the clubbed forelegs by skele¬ tal staining with Alizarin red S dis¬ closed no defective bone development. Accumulation Studies of Idoxuridine
12SL—A comparison of subcutaneous
Teratogenicities of Antiherpetic Drugs: Rabbit Studies No. of
No. of
Topical Agent Physiologic saline 0.1% IDU
Pregnant Rabbits
No. of Fetuses
No. of Dead Fetuses
8 15
121
3(3.5%) 23(19%)
Living
Anomalous Fetuses
28(23%)
Description of Anomaly
0
Exophthalmus 20
Clubbed
foreleg 13
1.0%
F, TdR
65
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1(1.5%)
higher in fetal tissues than in mater¬ nal tissues, as reflected in blood con¬
centrations. It was noted that the idox¬ uridine 125I concentration peaked in fetal blood at approximately 2V2 hours after maternal administration. These findings were consistent in all fetal tissues studied. The results obtained in thin-layer Chromatographie separation studies are shown schematically in Fig 6, left and right. A significant breakdown of idoxuridine can be seen in both mother and fetus; however, a signifi¬ cantly large amount of idoxuridine was still present at each time interval in both maternal and fetal blood. The results obtained after the in¬ jection of idoxuridine 12T into preg¬ nant rabbits on the 12th day of gesta¬ tion, and killed 4% hours after administration are summarized in Fig 7. A significant accumulation of idoxuridine 125I in all fetal tissues is evident in comparison with maternal blood concentration. A representative sample of an autoradiograph of a 28-day-old fetus, obtained one hour after maternal ad¬ ministration of idoxuridine ,2T, is shown in Fig 8. A significant fetaltissue uptake of radioactive material can be seen. Of some importance is the especially high accumulation in tissues such as eye (E), brain (BR), liver (L), kidney (K), intestines (I), and bones (B). Comment
Antimetabolites have long been
suspect teratogens. During the past
ten years, the
long list of teratogenic compounds'" that produce congenital malformations and mon¬ strosities in fetuses after a single chemical
concentration of idoxuridine 2 in maternal blood placenta and whole fetus 414 hours after maternal subcutaneous injection of drug on 12th day of gestation (height of teratogenic period), (cpm Indicates counts per minute.)
Fig 7.—Relative
topical administration of idoxuri¬ dine 12T is schematically illustrated in Fig 4. As can be seen, topically applied idoxuridine was readily ab¬ sorbed in the blood stream; as ex¬ pected, however, subcutaneously ad¬ ministered idoxuridine 12T reached a slightly higher level of concentration more rapidly than did the topically vs
applied drug.
The results obtained from the 11 pregnant rabbits that were given 30 microcuries of idoxuridine ,2T via subcutaneous injection on the 28th day of gestation, are shown in Fig 5. A definite pattern can be seen, in that the concentration of idoxuridine 12! at each time interval is consistently
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dose administered to a pregnant ro¬ dent were found to be alkylating agents, antimetabolites, and triacene. In addition, antimetabolites are known to be powerful teratogenic tools which make possible conversion of long-term nutritional experiments into short-term chemical experi¬ ments. It is understandable that the new methods shorten teratogenic ex¬ periments greatly; but the new sharp tools also permit the possible produc¬ tion of a large number of certain congenital malformations which pre-
viously had seldom been obtained. example, in contrast to cleft pal¬ ate, cleft lips are only rarely produced in rat fetuses by teratogenic proce¬ For
dures. However, Nelson et al" ob¬ tained cleft lips in more than 90% of the young of rats subjected, with the help of an anti metabolite, to a transi¬ tory folie acid deficiency during the ninth to 11th day of gestation. On the basis of this study, it can be concluded that idoxuridine is tera¬ togenic to rabbits in doses similar to those used clinically, causing exophthalmus-like deformities and clubbed forelegs. In addition, it was shown that idoxuridine is absorbed systemically after topical application, crosses the blood-brain barrier and placenta, and is accumulated in important amounts in fetal tissue. Although the mechanism of this malformation is still unexplored, the teratogenicity of idoxuridine is most probably related to the fact that the drug is incorpo¬ rated into DNA during DNA syn¬ thesis in mammalian cells. Moreover, the thin-layer chromatography stud¬ ies determine only the concentration of free non-protein-bound idoxuridine in fetal blood after maternal adminis¬ tration, allowing for the possibility of a much greater total accumulation of idoxuridine in the fetal tissue. Thus the presence of these antimetabolites in the fetus seems to correlate with its teratogenic effects. In 1965, Mur¬ phy12 found idoxuridine not to be teratogenic when given systemically to Wistar rats. However, this is not surprising since some of the features of thalidomide" were that this drug, which appeared completely harmless when given for a limited time in post¬ natal life, proved to be very dele¬ terious in minimal doses in prenatal life. Moreover, it was also unusual that the rat embryo, used so exten¬ sively in previous teratogenic experi¬ ments, proved very resistant to thal¬ idomide. The systemic absorption of the topically administered idoxuri¬ dine could, however, be high. One drop of 0.1% idoxuridine four times a day, if "fully absorbed" by the eye, compounds to about 0.2 mg/day. In a 4.0-kg rabbit it constitutes about 0.05 mg/kg of body weight, which is com¬ parable to an adult dose of 0.9 mg/kg
Fig 8.—Autoradiograph of 28-day-old fetus obtained one hour after maternal adminis¬ tration of idoxuridine '- . Note high accumulation in eye (E), brain (BR), liver (L), kidney (K), intestine (I), and bones (B). of idoxuridine or about three drops every hour around the clock. Although such amounts are rarely given to hu¬ mans, still they represent a low embryopathic dose if one considers that, for example, the 50% teratogenic dose (TD:>„) of orally administered aspirin is 650 mg/kg, at which level it almost approaches its 50% lethal dose of 1,528 mg/kg in adult rats. Therefore, idox¬ uridine appears capable of inducing teratogenic effects at levels of about 0.05 mg/kg in rabbits when applied topically to the eye. On the basis of this study, 0.05 mg/kg appears to be the minimum dose required. How¬ ever, because of the fact that not all of the topically applied idoxuridine
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be assumed to be absorbed, it is difficult to completely rule out the possibility that this drug as well as thalidomide has no minimum embryopathic dose. In contrast to idoxuridine, F,,TdR was found not to be teratogenic to rabbits, at least under the conditions of these studies, when given in con¬ centrations tenfold greater than doses used to verify idoxuridine teratogenicity. This is consistent with re¬ cent findings that F,TdR shows a lack of mutagenicity to mammalian cells in in vitro studies.13 The practical importance for man of the teratogenic effect of idoxuri¬ dine in rabbit is unclear. In addition, can
definite hazard from idoxuridine in has been established. However, the lack of teratogenicity of F:1TdR, another antiherpetic agent currently under investigation but not available for general use, must be considered in selecting the ideal antiherpetic agent for the treatment of herpetic keratitis in man. no
man
This investigation was supported by Public Health Service grant EY-1098-01 from the Na¬ tional Eye Institute, and in part by Public Health Service grants EY-52868 (Dr. Gasset) and EY-00446, and by National Institutes of Health medical student research fellowship grant RR05362-10 (Mr. Gefter). Herbert E. Kaufman, MD, made helpful con¬ tributions to this study through discussions.
Keywords.—Teratology; idoxuridine (IDU, 5-iodo-2'-deoxyuridine);trifluorothymidine;
herpetic keratitis; blood-brain barrier; idox¬ uridine 125I.
Nonproprietary Names and Trademarks of Drugs Idoxuridine—Dendrid, Herplex, Stoxil. Thalidomide-ÄeOTdora.
References 1. Haskin D: Some effects of nitrogen mustard the development of external body form in the foetal rat. Anat Rec 102:493-511, 1948. 2. Danforth CH, Center E: Nitrogen mustard as a teratogenic agent in the mouse. Proc Soc Exp Biol Med 86:705-707, 1954. 3. Gillman J, et al: A preliminary report on hydrocephalus, spina bifida and other congenital anomalies in the rat produced by trypan blue. S Afr J Med Sci 13:47-90, 1948. 4. Katter H, Warkany J: Experimental production of congenital malformations in mammals by metabolic procedures. Physiol Rev 39:69\x=req-\ on
115, 1959.
5. Baxter H, Fraser FC: Production of condefects in offspring of female mice treated with cortisone. McGill Med J 19:245-251,
genital
1950. 6. Fraser
FC, Faintstat TD: Production of congenital defects in offspring of pregnant mice treated with cortisone. Pediatrics 8:527-533,
1951. 7. Kalter H: The Genetics and Physiology of Susceptibility to the Teratogenic Effects of Cortisone in Mice, thesis. McGill University, Montreal, 1953. 8. Fraser FC, Faintstat TD, Kalter H: Experimental production of congenital defects with particular reference to cleft palate. Neo-Natal Studies 2:43-49, 1953. 9. Clarkson DR, Oppelt WW, Byvoet P: The fate of 5-iodo-2\m='\-deoxyuridine (IUDR) in plasma and cerebrospinal fluid of dogs. J Pharmacol Exp Ther 157:581-588, 1967.
Downloaded From: http://archopht.jamanetwork.com/ by a New York University User on 05/27/2015
10. Murphy ML: Teratogenic effects of tumor inhibiting chemicals in the foetal rat, in Ciba Foundation Symposium on Congenital Malformations. 1960, pp 78-107. 11. Nelson MM, et al: Multiple congenital abnormalities resulting from transitory deficiency of pteroylglutamic acid during gestation in the
rat. J Nutr 56:349-363, 1955. 12. Murphy ML: Factors influencing teratogenic response to drugs in teratology, in Wilson JG, Warkany J (eds): Teratology: Principles and Techniques. Chicago, University of Chicago Press, 1965, chap 7, p 196. 13. Lenz W, et al: Thalidomide and congenital abnormalities. Lancet 1:45-46, 1962.
of
Ophthalmic Drugs
Ophthalmic Drugs
Motokazu Itoi, MD; Jeffrey W. Gefter; Noboru Kaneko, MD; Yasuo Ishii; Richard M. Ramer, PhD; Antonio R. Gasset, MD
Nonradioactiveidoxuridine(IDU,5-iodo2\m='\-deoxyuridine), while not teratogenic to rats, does produce fetal malformations in
rabbits when administered topically to the eye in doses similar to those used clinically, 0.1% four times a day for 12 days. These malformations include exophthalmos and clubbing of the forelegs. By
contrast, trifluorothymidine (F3 TdR), another highly effective antiherpetic agent
currently under investigation but not avail able for general use, was found not to be teratogenic to rabbits, even when given in concentrations tenfold greater than the doses used to produce idoxuridine teratogenicity.
studies on mammalian tera¬ in the 1930s, the science of teratology practiced in lower classes of animals was at least 100 years old. But it was doubted by many investigators of hu¬ man congenital malformations that the results obtained on eggs of lower classes could be applied to mamma¬ lian or human situations. It was thought that mammalian embryos
Whentologyexperimental began
Submitted for publication May 25, 1973. From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville (Drs. Ramer and Gasset and Mr. Gefter); and Juntendo University, Tokyo (Drs. Itoi and
Kaneko). Reprint requests
to Department of Ophof Medicine, University of Florida, Gainesville, FL 32601 (Dr. Gasset).
thalmology, College
and fetuses were so well protected by the maternal organism that they could not be modified by methods teratogenic to embryos of lower classes. In 1948, two definite teratogens, the nitrogen mustards and trypan blue, were found. Haskin1 described severe malformations in the off¬ spring of rats treated with a radiomimetic and cytotoxic nitrogen mus¬ tard, and similar results were later reported in mice.- Gillman et al,1 using trypan blue as a teratogen, found hydrocephalus, spina bifida, and other congenital anomalies in the young of treated rats. Many hormones have been used as teratogens.1 Notable among those drugs is cortisone which was first found to be teratogenic to mice by Baxter and Fraser in 1950. Of even greater importance is the work by Fraser and Faintstat and Kalter,68 who demonstrated the inheritance of susceptibility to the teratogenic ac¬ tion of cortisone. A review of the field of experimen¬ tal mammalian teratology disclosed that a huge mass of facts has been
gathered; however, some entire branches such as teratogenicities of ophthalmic drugs have failed to de¬ velop. Teratogenicities of ophthalmic medications, eg, corticosteroids, antimetabolites, edetic acid (EDTA),
Downloaded From: http://archopht.jamanetwork.com/ by a New York University User on 05/27/2015
heavy metals and others, which are clearly implicated in the etiology of congenital malformations when ad¬ ministered systemically, have not yet been evaluated when applied topically to the eye. Such studies are of the ut¬ most importance in view of the wide
and
frequent use of some of these preparations as ophthalmic drugs or preservatives. The purpose of this study is to eval¬ uate the teratogenic effect of a vari¬ ety of ophthalmic drugs when given topically to the eye in doses equiva¬ lent to those used clinically. In this report, the teratogenic effect of idoxuridine (IDU, 5-iodo-2'-deoxyuridine) and trifluorothymidine (F,TdR), when given topically to the eye in doses equivalent to those used clini¬ cally against herpetic keratitis, was
studied. In addition to the evaluation of external malformations, dissection of the abnormal specimens, serial histological sectioning, drug accumula¬ tion and thin-layer chromatography studies were done. Materials and Methods
Idoxuridine-FjTdR Teratogenesis in Rab¬ bits.—A total of 31 Japanese albino rabbits were used in these studies. Fifteen rabbits received 1 drop of 0.1% idoxuridine in each eye six times on the sixth day of gestation and thereafter four times a day from the seventh to the 18th day of gestation. Eight
20-
Subcutaneous
njection Topical Eye Drops
Fig 1.—Exophthalmus in rabbit fetus, in 28th day of development, malformed by Idoxuridine administration to mother.
4 5 6 Hours After Application
Fig 4.—Idoxuridine ,25l accumulation in blood of adult rabbits receiving subcutaneous injection vs topical administration of drug, (cpm indicates counts per minute.)
Fig 2.—Fetus "on 28th day of gestation showing clubbing of forelegs.
•-·
(3 Rabbits)
1/2 1
Fig 3.—Composite photograph of nor¬ mal and malformed rabbit fetus after idox¬ uridine administration (IDU) stained with Alizarin red S, showing that clubbing (ar¬ row) is due to soft-tissue deformities rather than defective bone development. treated with 1.0% F.TdR and rabbits with 0.9% physiologic saline following exactly the same schedule as in the group treated with 0.1% idoxurirabbits
eight
were
more
Fig
Ito 2
Fetal Blood Mother's Blood
(3)
21/2 3
31/2 4 41/2 5 51/2 6 61/2 Hours After Injection
7
7V2 8
81/2 9
5.—Idoxuridine l«*l accumulation in maternal and fetal blood after maternal subcu¬ injection of drug on 28th day of gestation.
taneous
dine. All rabbits were killed on the 28th day of gestation, and the fetuses were re¬ moved by cesarean section and carefully examined for external malformations. In addition, dissection of abnormal specimens for further examination of skeletal malfor¬ mation and serial histological sectioning were done.
Downloaded From: http://archopht.jamanetwork.com/ by a New York University User on 05/27/2015
Accumulation Studies of Idoxuridine La¬ beled With Iodine 125.—Route of Adminis¬
tration.—A total of six healthy adult New Zealand albino rabbits weighing 4.0 to 4.4 kg were used. Each rabbit received 150 mi¬ crocuries of idoxuridine 125I in a 0.1% cold idoxuridine solution either topically as eye drops or subcutaneously. Three (two males
103 cpm/gm
103 cpm/gm I
—X
Idoxuridine
·—·
lodouracil
> — Idoxuridine •—·
lodouracil Iodide
Iodide
-14
-r
2
Hours
Hours
Fig 6.—Thin-layer separation of idoxuridine 121 from its metabo¬ lites in maternal blood (left) and in fetal blood (right) after materand one nonpregnant female) received idoxuridine ''"I topically, and three (two males and one nonpregnant female) re¬ ceived the same amount of idoxuridine 12T subcutaneously. Blood samples were drawn from each rabbit of both groups at succes¬ sive time intervals after administration. The samples were then counted for radio¬ activity in a gamma well-type scintillation counter calibrated for '-T emissions. Maternal vs Fetal Accumulation.—In the first of these studies, 11 healthy 6month-old New Zealand albino rabbits were each mated twice with the same male, and the day of conception was deter¬ mined by the presence of sperm on vaginal smear. Twenty-eight days after mating, each of the 11 females was given 30 microcurie of idoxuridine 12T in a 0.1% cold idox¬ uridine solution subcutaneously, and they were then killed at different time intervals from 45 minutes to seven hours after ad¬ ministration. After blood samples were collected, brain, kidney, and liver tissues of both mothers and fetuses were excised, weighed, and counted. In a second study, to determine the rela¬ tive metabolic breakdown of the idoxuri¬ dine ,2T into iodouracil 125I and iodide ,25I, four healthy 6-month-old Japanese albino rabbits in their 28th day of gestation, were each given a subcutaneous injection of 150 microcuries of idoxuridine 125I in a 0.1% cold idoxuridine solution. The rabbits were killed at intervals of 1, 3, 5V2, and 8 hours after administration, respectively. Blood samples from each time period were pre¬ pared by removing the cells and deproteinizing the serum. Thin-layer Chromato¬ graphie separation of idoxuridine 12T from its metabolic breakdown products as de¬ scribed by Clarkson et al" was performed on the fetal and maternal blood samples prepared as above. After extraction of idox-
nal subcutaneous injection of drug indicates counts per minute.)
uridine, iodouracil and iodide, the radio¬ activity of each fraction was counted and
compared. In addition,
two healthy 6-month-old New Zealand albino rabbits in their 12th day of gestation, as determined by vaginal smear, were each subcutaneously injected with 300 microcuries of idoxuridine 12T in a 0.1% cold idoxuridine solution. Both rabbits were killed 4Vè hours after administration, and their fetuses were removed by cesarean section. Determinations of radio¬ active accumulation in maternal blood, pla¬ centa, and whole fetus were made by com¬ parison of specific activity in tissues. Autorudiography.—A healthy 6-monthold New Zealand albino rabbit in the 28th day of gestation was subcutaneously in¬ jected with 300 microcuries of idoxuridine 12T in a 0.1% cold idoxuridine solution, and killed one hour after administration. A fetus from this rabbit was removed, sec¬ tioned, and the sections mounted on glass slides. Several mounted sections were directly applied to the sensitive surface of recording autoradiographic film, fixed to the film unmoved, and kept in darkness for three weeks. The mounts were then re¬ moved, and the sensitized film developed.
on
28th
day of gestation, (cpm
Results
Teratogenicity Studies.—The re¬ sults on 272 fetuses obtained by cesarean section from 31 pregnant rabbits treated with idoxuridine, F,TdR, or saline are summarized in the Table. The idoxuridine-treated group had a fivefold greater incidence of dead fetuses than did the control or the F:,TdR-treated group. In addition, 23% of the live fetuses from the idoxuridine group demonstrated con¬ genital anomalies such as the one shown in Fig 1 to 3. Figure 1 shows an exophthalmus-like malformation which was found in approximately 16% of the fetuses from idoxuridinetreated mothers. Figure 2 shows club¬ bing of the forelimbs, another con¬ genital abnormality, found in about 9% of the fetuses. Further examina¬ tion of the clubbed forelegs by skele¬ tal staining with Alizarin red S dis¬ closed no defective bone development. Accumulation Studies of Idoxuridine
12SL—A comparison of subcutaneous
Teratogenicities of Antiherpetic Drugs: Rabbit Studies No. of
No. of
Topical Agent Physiologic saline 0.1% IDU
Pregnant Rabbits
No. of Fetuses
No. of Dead Fetuses
8 15
121
3(3.5%) 23(19%)
Living
Anomalous Fetuses
28(23%)
Description of Anomaly
0
Exophthalmus 20
Clubbed
foreleg 13
1.0%
F, TdR
65
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1(1.5%)
higher in fetal tissues than in mater¬ nal tissues, as reflected in blood con¬
centrations. It was noted that the idox¬ uridine 125I concentration peaked in fetal blood at approximately 2V2 hours after maternal administration. These findings were consistent in all fetal tissues studied. The results obtained in thin-layer Chromatographie separation studies are shown schematically in Fig 6, left and right. A significant breakdown of idoxuridine can be seen in both mother and fetus; however, a signifi¬ cantly large amount of idoxuridine was still present at each time interval in both maternal and fetal blood. The results obtained after the in¬ jection of idoxuridine 12T into preg¬ nant rabbits on the 12th day of gesta¬ tion, and killed 4% hours after administration are summarized in Fig 7. A significant accumulation of idoxuridine 125I in all fetal tissues is evident in comparison with maternal blood concentration. A representative sample of an autoradiograph of a 28-day-old fetus, obtained one hour after maternal ad¬ ministration of idoxuridine ,2T, is shown in Fig 8. A significant fetaltissue uptake of radioactive material can be seen. Of some importance is the especially high accumulation in tissues such as eye (E), brain (BR), liver (L), kidney (K), intestines (I), and bones (B). Comment
Antimetabolites have long been
suspect teratogens. During the past
ten years, the
long list of teratogenic compounds'" that produce congenital malformations and mon¬ strosities in fetuses after a single chemical
concentration of idoxuridine 2 in maternal blood placenta and whole fetus 414 hours after maternal subcutaneous injection of drug on 12th day of gestation (height of teratogenic period), (cpm Indicates counts per minute.)
Fig 7.—Relative
topical administration of idoxuri¬ dine 12T is schematically illustrated in Fig 4. As can be seen, topically applied idoxuridine was readily ab¬ sorbed in the blood stream; as ex¬ pected, however, subcutaneously ad¬ ministered idoxuridine 12T reached a slightly higher level of concentration more rapidly than did the topically vs
applied drug.
The results obtained from the 11 pregnant rabbits that were given 30 microcuries of idoxuridine ,2T via subcutaneous injection on the 28th day of gestation, are shown in Fig 5. A definite pattern can be seen, in that the concentration of idoxuridine 12! at each time interval is consistently
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dose administered to a pregnant ro¬ dent were found to be alkylating agents, antimetabolites, and triacene. In addition, antimetabolites are known to be powerful teratogenic tools which make possible conversion of long-term nutritional experiments into short-term chemical experi¬ ments. It is understandable that the new methods shorten teratogenic ex¬ periments greatly; but the new sharp tools also permit the possible produc¬ tion of a large number of certain congenital malformations which pre-
viously had seldom been obtained. example, in contrast to cleft pal¬ ate, cleft lips are only rarely produced in rat fetuses by teratogenic proce¬ For
dures. However, Nelson et al" ob¬ tained cleft lips in more than 90% of the young of rats subjected, with the help of an anti metabolite, to a transi¬ tory folie acid deficiency during the ninth to 11th day of gestation. On the basis of this study, it can be concluded that idoxuridine is tera¬ togenic to rabbits in doses similar to those used clinically, causing exophthalmus-like deformities and clubbed forelegs. In addition, it was shown that idoxuridine is absorbed systemically after topical application, crosses the blood-brain barrier and placenta, and is accumulated in important amounts in fetal tissue. Although the mechanism of this malformation is still unexplored, the teratogenicity of idoxuridine is most probably related to the fact that the drug is incorpo¬ rated into DNA during DNA syn¬ thesis in mammalian cells. Moreover, the thin-layer chromatography stud¬ ies determine only the concentration of free non-protein-bound idoxuridine in fetal blood after maternal adminis¬ tration, allowing for the possibility of a much greater total accumulation of idoxuridine in the fetal tissue. Thus the presence of these antimetabolites in the fetus seems to correlate with its teratogenic effects. In 1965, Mur¬ phy12 found idoxuridine not to be teratogenic when given systemically to Wistar rats. However, this is not surprising since some of the features of thalidomide" were that this drug, which appeared completely harmless when given for a limited time in post¬ natal life, proved to be very dele¬ terious in minimal doses in prenatal life. Moreover, it was also unusual that the rat embryo, used so exten¬ sively in previous teratogenic experi¬ ments, proved very resistant to thal¬ idomide. The systemic absorption of the topically administered idoxuri¬ dine could, however, be high. One drop of 0.1% idoxuridine four times a day, if "fully absorbed" by the eye, compounds to about 0.2 mg/day. In a 4.0-kg rabbit it constitutes about 0.05 mg/kg of body weight, which is com¬ parable to an adult dose of 0.9 mg/kg
Fig 8.—Autoradiograph of 28-day-old fetus obtained one hour after maternal adminis¬ tration of idoxuridine '- . Note high accumulation in eye (E), brain (BR), liver (L), kidney (K), intestine (I), and bones (B). of idoxuridine or about three drops every hour around the clock. Although such amounts are rarely given to hu¬ mans, still they represent a low embryopathic dose if one considers that, for example, the 50% teratogenic dose (TD:>„) of orally administered aspirin is 650 mg/kg, at which level it almost approaches its 50% lethal dose of 1,528 mg/kg in adult rats. Therefore, idox¬ uridine appears capable of inducing teratogenic effects at levels of about 0.05 mg/kg in rabbits when applied topically to the eye. On the basis of this study, 0.05 mg/kg appears to be the minimum dose required. How¬ ever, because of the fact that not all of the topically applied idoxuridine
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be assumed to be absorbed, it is difficult to completely rule out the possibility that this drug as well as thalidomide has no minimum embryopathic dose. In contrast to idoxuridine, F,,TdR was found not to be teratogenic to rabbits, at least under the conditions of these studies, when given in con¬ centrations tenfold greater than doses used to verify idoxuridine teratogenicity. This is consistent with re¬ cent findings that F,TdR shows a lack of mutagenicity to mammalian cells in in vitro studies.13 The practical importance for man of the teratogenic effect of idoxuri¬ dine in rabbit is unclear. In addition, can
definite hazard from idoxuridine in has been established. However, the lack of teratogenicity of F:1TdR, another antiherpetic agent currently under investigation but not available for general use, must be considered in selecting the ideal antiherpetic agent for the treatment of herpetic keratitis in man. no
man
This investigation was supported by Public Health Service grant EY-1098-01 from the Na¬ tional Eye Institute, and in part by Public Health Service grants EY-52868 (Dr. Gasset) and EY-00446, and by National Institutes of Health medical student research fellowship grant RR05362-10 (Mr. Gefter). Herbert E. Kaufman, MD, made helpful con¬ tributions to this study through discussions.
Keywords.—Teratology; idoxuridine (IDU, 5-iodo-2'-deoxyuridine);trifluorothymidine;
herpetic keratitis; blood-brain barrier; idox¬ uridine 125I.
Nonproprietary Names and Trademarks of Drugs Idoxuridine—Dendrid, Herplex, Stoxil. Thalidomide-ÄeOTdora.
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