Developmental toxicity of ibutilide fumarate in rats after oral administration

TERATOLOGY M157-164 (1996)

Developmental Toxicity of Ibutilide Fumarate in Rats After Oral Administration THOMAS A. MARKS AND ROGER D. TERRY Inuestigatiue Toxicology (TAM) and Drug Development Toxicology II (RDT), Pharmucia & Upjohn, Inc., Kalamazoo, Michigan 49001

ABSTRACT In two Segment II Teratology studies, timed-pregnant Crl:CD[BR] (Sprague- Dawley) rats were treated orally (gastric intuba- tion) on days 6-15 of gestation with ibutilide fumarate (ibutilide), a class Ill antiarrhythmic that has been shown to increase the refractory period and action potential duration of myocardial cells. In the first study, ibutilide does of 20, 40, and 80 mg/kg/ day were tested. Although maternal toxicity was equivocal in the 80 mg/kg/day group, all 23 rats that conceived had entirely resorbed liters when the animals were killed on day 20 of gestation. Similarly, 12 of 24 litters were completely resorbed in the 40 mg/kg/day group, with an 87.7% postimplantational loss. Of the surviving fetuses in this group, 48.6% had at least one malformation. The incidences of malformed pharynx and malformed palate, along with adactyly, were statistically significantly higher in this group than in the control group. In addition, a significant (P< 0.05) increase in total malformations (5.7% of the fetuses), relative to the controls (0.8%), was found for the 20 mg/kg/day group. Since a no observed adverse effect level (NOAEL) was not found, a second teratology study was performed. In this study, the ibutilide doses were 5, 10, and 20 mg/kg/day. The 20 mg/kg/day dose was again teratogenic with 9.2% of the fetuses malformed, as compared to a control value of 1 .O%. Also, the incidences of scoliosis and interventricular septa1 defect were statistically significantly higher in this group. Although statistically significant differences were not detected, scoliosis was also found in the 10 mg/kg/day group (3 fetuses in 2 litters), along with a significant dose-response trend for this malformation. As the result, the NOAEL for ibutilide teratogenicity in rats was set at 5 mg/kg/day. This dose is 4 times the proposed maximum clinical dose (two 1 mg doses, each infused over 10 minutes, or 0.033 mg/kg for a 60 kg person), when corrected for 2.6% oral bioavailability in the rat at a dose of 10 mg/kg, as determined in separate studies. o 1996 WiIey-tiss, Inc.

Ibutilide fumarate (ibutilide), N-[4-[4-(ethylheptyl- amino) - 1 - hydroxybutyll phenyll methanesulfonamide-

(E)-2-butenedioate, is a class 111 antiarrhythmic agent (Buchanan et al., '92; Cimini et al., '92; Hester et al., '91). However, ibutilide has a unique mechanism of action in that a t therapeutic levels to lop6 M) it has been shown to increase an inward, depolarizing sodium current, prolonging the duration of the action potential of atrial and ventricular myocytes isolated from guinea pigs (Lee, '92). At such concentrations, ibutilide could further prolong the action potential duration that already had been prolonged by a potassium channel blocker, whereas a higher concentration ( M) reversed such a prolongation (Lee et al., '93). An- tiarrhythmic effects were demonstrated after acute, intravenous administration of ibutilide, in canine models of atrial flutter, atrial fibrillation, ventricular tachy- cardia, and ventricular fibrillation that were dependent upon reentrant cardiac conduction (Buchanan et al., '92; Nabih et al., '93). Animal studies have shown that this drug produced significant class I11 antiarrthy- mic actions including increased refractory periods, increased QT intervals, and/or increased duration of the monophasic action potential. Ibutilide also significantly reduced the energy necessary for defibrillation in canine models (Wesley et al., '93). In addition, this agent produced substantially less of the proarrhythmic phenomenon torsade de pointes than other class I11 agents when tested in a rabbit model of proarrhythmia dependent upon early afterpolarizations (Buchanan et al., '93).

Clinical trials have demonstrated that intravenous administration of ibutilide, in a monitored setting, rap- idly and effectively terminated sustained atrial flutter in a high proportion of studies (DiMarco, '91). As a result, the United States Food and Drug Administra- tion has recently approved the use of this drug for rapid conversion of atrial fibrillation and atrial flutter of re- cent onset to normal sinus rhythm. However, before approval was granted, it was necessary to carry out extensive preclinical drug safety studies, including developmental and reproductive toxicology experiments.

May 23, 1996; accepted August 9, 1996. Address reprint requests to Thomas A. Marks, Pharmacia & Upjohn, Inc., 7228-300-224, Kalamazoo, MI 49001.

0 1996 WILEY-LISS, INC.

158 T.A. MARKS AND R.D. TERRY

The results of the rat segment I1 teratology studies are reported here. Since a no observed adverse effect level (NOAEL) for developmental toxicity could not be established in the first study, a second experiment was carried out. Some of the data from these studies were presented in a preliminary communication (Marks and Terry, '94).

MATERIALS AND METHODS Animal and animal husbandry

All rats were housed in individual stainless steel wire-bottom cages in a room with a 12-hour light cycle; the temperature was maintained at approximately 74°F with a relative humidity of approximately 50%. Purina Certified Rodent Chow@ 5002 was fed, and fresh tap water was supplied in water bottles attached to the cages (both ad libitum). Inseminated 8-10-week-old Crl:CD(SD)BR rats arrived on day 0 of gestation from Charles River Laboratories (Portage, MI). The dams were randomly assigned to four groups of 24.

Drug formulation and administration Ibutilide fumarate was synthesized at The Upjohn

Company and dissolved in Purified Water USP containing 0.1% sorbic acid NF (preservative), at concentrations of 2.0,4.0, and 8.0 mg/ml (first experiment) or 0.5, 1.0, and 2.0 mglml (second experiment). The for- mulations were administered orally (gastric intuba- tion) on days 6-15 of gestation, in a volume of 10 mlkg (adjusted daily according to the individual body weight of each dam), giving doses of 20,40, and 80 mg/kg/day (first experiment) or 5, 10, and 20 mg/kg/day (second experiment).

Observations Body weights were recorded on days 0, 6, 10, 12, 15,

and 20 of gestation. The dams were observed daily during and after treatment for unusual effects. Im- mediately after death (COz inhalation on day 20 of gestation), a laparotomy was performed and the uterus was removed, weighed, and incised; the number and location of live, dead, and resorbed con- cepti were recorded. All fetuses were examined grossly. Live fetuses from each litter were randomly divided into two approximately equal groups, one of which was examined for visceral anomalies (Staples, '74) while the fetuses in the other group were fixed, eviscerated, stained, and subjected to skeletal examinations (Staples and Schnell, '64). Anomalies were classified as malformations or variations (Black and Marks, '92). Uteri that showed no signs of conception were examined for the presence of implantation sites as previously described (Marks and Poppe, '88).

Statistical methods Maternal body weights and the numbers of corpora

lutea and implantation sites, as well as the numbers of

live, dead, and resorbed fetuses, were subjected to analysis of variance on raw and ranked data (Conover and Iman, '81). When significant differences between dose groups were found, comparisons among the group means were made using the Least Significant Differ- ence Method. Fetal body weights were subjected to a weighted analysis of variance employing the litter as the experimental unit for sexes separately and sexes combined (Healy, '72). Group proportions for malformations, preimplantatiodpostimplantation losses, and livetresorbed conceptuses were analyzed using the Modified Jonckheere Ordered Alternatives Test for significant dose response (Lin and Haseman, '76). When significant effects were found, jackknife methods were employed to estimate group proportions and make pair- wise comparisons (Gladen, '79).

RESULTS First experiment

Oral administration of ibutilide resulted in postdosing salivation in 8, 20, and 24 dams in the 20,40, and 80 mg/kg/day groups, respectively. Red vaginal dis- charges also were observed in the 40 (10 dams) and 80 (6 dams) mg/kg/day groups; all of these rats were found to have resorptions only, when sacrificed on day 20 of gestation. Incidental findings included soft stools, observed in one dam each in the mid- and high-dose groups, and alopecia of the ventral abdomen, found in one dam in the high-dose group. Although statistically significant (P < 0.01) reductions in body weight gain were found during and aRer treatment in the 40 and 80 mg/kg/day groups, as compared to the vehicle control group, when the body weight means during gestation (Day 20 dam body weight minus gravid uterine weight minus Day 0 weight) were compared, significant differences were not evident (Table 1).

On day 20 of gestation, 91.7, 91.7, 100, and 95.7% of the dams (24/group) in the control, 20, 40, and 80 mg/ kglday groups, respectively, were identified as pregnant or having been pregnant (resorptions). The means obtained for the number of implantations, as well as the number of corpora lutea, were comparable in all groups. However, only regressing corpora lutea were found in the uteri of the dams in the high-dose group, as all 23 of the dams that conceived had resorption sites only. Although 12 viable litters were found in the 40 mg/kg/day group, the litters of 12 other dams in this group were completely resorbed by day 20. Thus, as compared to the controls, statistically significant (P < 0.01) decreases in mean number of live fetuses were found in the mid- and high-dose groups, with corre- sponding significant increases in the means for resorptions and postimplantational losses. In addition, mean fetal body weight in the 40 mg/kg/day group was statistically significantly less than the control mean.

Statistically significant increases, as compared to the vehicle control group, in the total number of fetuses

DEVELOPMENTAL TOXICITY OF IBUTILIDE FUMARATE 159

TABLE 1. Experiment 1: summary of reproductive data'

Observations Ibutilide (mg/kg/day)

0.0 20 40 80 Number of pregnant dams Mean weight gain during treatment (days 6-15) Mean weight gain during gestation (days 0-20)' Number of litters Number of dams with resorptions only Mean number of corpora lutea/dam3 Mean number of implantationddam Mean number of live fetuseddam Number of dams with resorptions Mean number of resorptionsldam Mean body weight of live fetuses (g) G ~ U D mean Dostimdantational loss (%)'

22 22 44.0 42.3 84.6 78.7 22 22 0 0 14.0 13.5 11.5 12.1 11.1 11.2 6 10 0.3 0.9 3.3 3.2 2.8 7.5

24 36.5* 89.4 12 12 13.4 12.5 1.5* 24 11.0* 2.3* 87.7*

23 37.1* 83.5 0 23

11.1 o.o* 23 11.1*

-

- 100.0

'Nonpregnant dams were excluded from all calculations. 'Minus gravid uterine weight (where live fetuses were present). 31ncludes only dams with live fetuses; dams with resorptions only had regressing corpora lutea. '(Total No. of Implantations - Total No. of Live FetusedTotal No. of Implantations) x 100. *Statistically significantly different from the control group, P < 0.01.

with one or more malformations were observed in the 20 (P < 0.05) and 40 (P < 0.01) mgkglday groups (Ta- ble 2). Significant (P < 0.01) increases also were observed, in the 40 mglkglday group, in the mean number of fetuses with either gross, visceral, or skeletal malformations. The incidences of adactyly , malformed palate, and malformed pharynx in this group were also significantly higher than the incidences found in the control group (Table 3). In addition to the statistically significant increasing dose-response trends found for the malformations listed in this paragraph, a significant (P < 0.01) dose-related trend was detected for scoliosis.

A statistically significant (P < 0.01) increase in the proportion of fetuses with a skeletal variation was found on comparison of the 40 mgkglday group with the vehicle control group (Table 2). Similar comparisons resulted in significant (P < 0.05) increases in the following skeletal variations: bipartite sternebrae, incomplete ossification of centra, incomplete ossification of sternebrae other than no. 5,27 presacral vertebrae, sternebrae no. 5 unossified, and sternebrae other than no. 5 unossified (Table 4). The incidence of incomplete ossification of sternebrae no. 5 also was statistically significantly increased in the low-dose group.

Second experiment Since significant increases in the incidences of mal-

formations, along with other indications of developmental toxicity, were observed in the first experiment, in both of the groups that had live fetuses, a second experiment was performed. In this experiment, ibutilide was again administered orally to pregnant rats on days 6-15 of gestation; the doses were 0,5, 10, and 20 mgkg/day. Such treatment did not result in clear evidence of maternal toxicity, although incidental findings, including porphyrin staining, soft stools, and

scabbing, occurred as single incidences in the groups receiving ibutilide. All dams appeared to be normal at necropsy, and maternal body weight means were comparable in all groups during the dosing period, as well as over the entire gestational period (Table 5). How- ever, there was a slight, but statistically significant (P < 0.01), decrease in weight gain, as compared to the controls, in the high-dose group between days 15 and 20 of gestation. Also, mean fetal body weight in this group was statistically significantly less than that of the control group.

The incidences of gross or visceral, as well as total, malformations were significantly (P < 0.05) increased in the high-dose group, as compared to the controls (Table 6). Individual malformations that were statistically significantly higher in the high-dose group, as compared to the control group, were interventricular septa1 defect and scoliosis (Table 7). The latter anomaly also was found in three fetuses (two litters) in the mid- dose group, as compared to only one fetus in the controls; however, the difference was not statistically significant, although a significant positive dose-response trend was found for this anomaly. In addition, the incidences of the skeletal variations accessory ribs and unossified pubes were statistically significantly higher in the high-dose group than in the controls (Table 8).

DISCUSSION At the time developmental and reproductive toxicol-

ogy (DART) studies on ibutilide were initiated, it was hoped that this antiarrhythmic agent could be administered orally in the clinics, as well as intravenously. However, oral single-dose animal studies indicated that bioavailability was low, although plasma levels increased with dose (Hsu and Walters, manuscript in preparation). Toxicology studies estimated that the intravenous single-dose LD, for ibutilide was 85.1 mg/


TABLE 2. Experiment 1: summary of malformations and variations after oral treatment with ibutilide'

Malformations' Variations2 Observations 0.0 20.0 40.0 0.0 20.0 40.0 In fetuses

Gross* 1/245 (0.4) 3/247 (1.2) 10/37 (27.0** 0/245 (0.0) 0/247 (0.0) 0/37 (0.0) 1/118 (0.8) 3/122 (2.5) 5/14 (35.7)** 7/118 (5.9) 9/122 (7.4) 1/14 (7.1) Visceral*

Skeletal*** 11127 (0.8) 81125 (6.4) 6/23 (26.1)** 71/127 (55.9) 69/125 (55.2) 23/23 (loo)** Total 37*** 2/245 (0.8) 14/247 (5.7)**** 18/37 (48.6)** 78/245 (31.8) 781247 (31.6) 24/37 (64.9)**

Gross 1/22 (4.5) 3/22 (13.6) 6/12 (50.0) 0/22 (0.0) 0/22 (0.0) 0/12 (0.0) Visceral 1/22 (4.5) 1/22 (4.5) 316 (50.0) 7/22 (31.8) 7/22 (31.8) 1/6 (16.7) Skeletal 1/22 (4.5) 5/22 (22.7) 4/12 (33.3) 21/22 (95.5) 20/22 (90.9) 12/12 (100) Total 2/22 (9.1) 8/22 (36.4) 7/12 (58.3) 21/22 (95.5) 21/22 (95.5) 12/12 (100)

In litters

'There were no fetuses in the 80.0 mg/kg/day group as all dams that conceived had resorption sites only. 2Number foundhumber examined (%). 3Fetuses with multiple malformations/variations were counted only once in each category. *The Modified Jonckheere Ordered Alternatives Test for dose response was statistically significant (P < 0.05) for malformations. **Statistically significantly higher than the control group, P < 0.01. ***The Modified Jonckheere Ordered Alternatives Test for dose response was statistically significant (P < 0.05) for variations, as well as for malformations. ****Statistically significantly higher than the control group, P < 0.05.

TABLE 3. Experiment 1: incidence of individual malformations'

Ibutilide (mg/kg/day)' 0.0 20 40

Gross Ablepharia 0 Adactyly* 0 Agnathia 1 Cleft palate 0 Edema 0

Interventricular septa1 defect 1 Malformed pharynx* 0 Malformed palate* 0 Retroesophageal right subclavian 0 Portion of lung absent 0

Visceral

0 2/2 0 1 0

0 0 0

3/1 0

1 6/5**

0 3/2

1

0 4/3** 4/3**

0 1 -

Skeletal Metacarpal($ absent or fused 0 2/1 1 Rib(s) bent 0 1 0 Rib($ bifurcated 0 1 1 Rib(s) absent 0 1 0 Scoliosis* 1 5/5 413

'Number of fetuses malformedhumber of litters with this malformation. 'All pregnant dams in the 80 mg/kg/day group had resorptions only; thus, there were no fetuses to examine. *Statistically significant dose-response effect, P < 0.05. **Statistically significantly greater than the control group, P < 0.01.

kglday in rats, while a lethal oral single dose in rats was not obtained at doses up to 500 mglkg. First-pass metabolism most likely was responsible for the low oral bioavailability (2.6-12.8% in rats) as studies with ra- dio-labelled ibutilide showed that intestinal absorption was reasonable. As the result of these findings, the decision was made to discontinue clinical trials with the oral formulation, concentrating on acute intrave-

nous trials in hospitals. Since toxicological effects were clearly evident in preclinical animal studies employing the oral route, it was concluded that additional DART studies employing the i.v. route were unnecessary.

Minimal maternal toxicity was evident in the two experiments summarized in this manuscript. The dose- related salivation, observed in the first experiment after oral administration, most likely was due to the ir- ritability of the drug. Other indications of maternal toxicity appeared to be the result of the embryolethal effects of ibutilide at 40 and 80 mglkglday. Thus, the decreases during the fetal period in mean weight gain, in these groups as compared with the controls, were not evident when the final weight of each dam was determined by weighing her remains, after removing the uterus and its contents, before calculating weight gain during gestation. This conclusion is also supported by the fact that differences in weight gain were not found during early treatment (days 6-12 of gestation), when the body weights of the embryos would be expected to be negligible.

All 23 dams in the 80 mgkglday dose group that conceived suffered complete losses of their embryos. Al- though 12 of 24 dams in the 40 mgkglday dose group did not experience 100% loss of their litters, in utero, a total of only 37 live fetuses were found at sacrifice on day 20 of gestation; this resulted in an 87.7% postimplantational loss for this group. The fact that 18 of the surviving fetuses in this group (48.6%) had malformations suggests that more severe anomalies contributed to the in utero losses. Jbutilide also was teratogenic at 20 mglkglday, as evidenced by the statistically significant increase in the incidence of malformations in this group, on comparison with the controls. In addition, the statistically significant decrease in fetal body weight


TABLE 4. Experiment 1: incidences of individual variations'72

Ibutilide (mgkglday) Observations 0.0 20 40 Visceral

Grade 0 kidney Hydroureter Left umbilical artery No innominate

Centra: incomplete ossification* Hyoid: body unossified* Frontal(s): incomplete ossification InterparietaKs): incomplete ossification Ilia: incomplete ossification Ischia: incomplete ossification Ischia: unossified Nasal(& incomplete ossification Parietab): incomplete ossification Pubes: unossified Rib(s): accessory Rib(+ cervical no. 7 Squamosal(s): incomplete ossification Sternebrae: bipartite* Sternebrae: lack of apposition Sternebrae: no. 5 unossified* Sternebrae other than no. 5: incomplete

Sternebrae other than no. 5: unossified Vertebrae: 25 presacral Vertebrae: 27 presacral* Supraoccipital(s): incomplete ossification

Skeletal

ossification*

0 0 414 414

0 1117

1 25/13 1 212 1 1 515 1 1 0 512 0 0

48/16 414

715 0 0

20110

312 312

1 616

0 714 0

24/13 0 0 0 0

412 0 1

212 412

1 1

55/18 19/10***

1518 1 0

1017

0 0 0 1

7/7** 0** 0 615 0 212 0 0 1 212 311 0 0

8/85 2/22 22/11** 14/9**

2011 1 0 6/5** 414

Zygoma(sj: incomplete issification 211 1 0 'There were no fetuses in the 80.0 mgkglday group as all dams that conceived had resorption sites only. 'Number of fetuses malformdnumber of litters with this malformation; no gross variations were detected. *The Modified Jonckheere Ordered Alternative Test for dose response was statistically significant, P < 0.05. **Statistically significantly different than the control group, P < 0.01. ***Statistically significantly higher than the control group, P < 0.05.

TABLE 5. Experiment 2 s u m m a r v of reproductive data' ~ ~~

Ibutilide (mgkglday) Observations 0.0 5 10 20 Number of pregnant dams (litters) 23 23 24 22 Mean weight gain during treatment (days 6-15) 46.6 45.5 50.4 45.9 Mean weight gain posttreatment (days 15-20) 65.7 65.7 65.2 54.2* Mean weight gain during gestation2 82.3 80.3 84.0 81.0 Mean number of corpora lutealdam 14.1 14.4 14.5 13.0 Mean number of implantationsldam 13.0 13.2 13.3 11.7 Mean number of live fetusesldam 12.4 12.4 12.7 10.9 Number of dams with resorptions 9 12 12 9 Mean number of resorptionsldam 0.6 0.8 0.6 0.8 Mean body weight of live fetuses (g) 3.5 3.4 3.4 3.2* Group mean postimplantational loss (%I3 4.5 5.7 4.6 7.2 'Nonpregnant dams were excluded from all calculations. 2Minus gravid uterine weight (where live fetuses were present). 3(Total No. of Implantations - Total No. of Live FetusesITotal No. of Implantations) x 100. *Statistically significantly less than the control group, P < 0.01.

and the significant increases in the incidences of skel- eta1 variations, in the 40 mg/kg/day dose group, indicated that this drug was fetotoxic, as defined by Black and Marks ('86).

Since a NOAEL could not be established in the ini- tial developmental toxicology study, a second experiment was performed. In this study, the 20 mg/kg/day dose was repeated, along with groups dosed at 5 or 10


TABLE 6. Experiment 2 summary of malformations'

Ibutilide (mdkeldav) Observations In fetuses

Gross* Visceral* Skeletal* Total2.*

In litters Gross Visceral Skeletal Total2

0.0

01286 (0.0) 1/142 (0.7) 21144 (1.4) 31286 (1.0)

0123 (0.0) 1/23 (4.3) 2/23 (8.7) 3/23 (13.0)

5.0

11286 (0.3) 11141 (0.7) 01145 (0.0) 21286 (0.7)

1/23 (4.3) 1/23 (4.3) 0123 (0.0) 2/23 (8.7)

10.0 20.0

01304 (0.0) 61239 (2.5)** 0/152 (0.0) 6/116 (5.2)** 41152 (2.6) 131123 (10.6)** 41304 (1.3) 221239 (9.2)**

0124 (0.0) 5/22 (22.7) 0124 (0.0) 5/21 (23.813 3/24 (12.5) 7/22 (31.8) 3/24 (12.5) 9/22 (40.9)

'Number foundhumber examined (%I. 2Fetuses with multiple malformations were counted only once. 30ne litter containing one fetus was not examined viscerally. *The Modified Jonckheere Ordered Alternatives Test for Dose Response was statistically significant, P < 0.05. **Statistically significantly higher than the control group, P < 0.05.

TABLE 7. Experiment 2 incidences of individual malformations'

Ibutilide (mg/kg/day) 0.0 5 10 20

Gross Adactylyl Agnathia Anal atresia Anasarca Syndactyly Threadlike tail

Interventricular septal defect* Microphthalmia Retroesophageal subclavian Right-sided aortic arch Situs inversus Vestigial pulmonary truncus

Metacamal(s) absent or fused

Visceral

Skeletal

0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0

0 0 0

212 0 1

212 1 1

4/4** 0

2l2 1 0 1

212 Rib(s) bent 1 0 1 1 Scoliosis* 1 0 312 10/5**

'Number of fetuses malformdnumber of litters with this malformation. *Statistically significant dose-response effect, P < 0.05. **Statistically significantly greater than the control group, P < 0.05.

mglkglday. Otherwise the protocol was the same as the one employed in the first study. No biologically mean- ingful adverse effects were observed in ibutilide- treated dams, and they all appeared to be normal on necropsy. The slight, but statistically significant, decrease in maternal weight gain during the postdosing period, in the high-dose group, most likely was a con- sequence of the decreases in the means for the number of implantations, the number of live fetuses per pregnant dam, and fetal body weight, also found in this group. It was unlikely that ibutilide affected litter size as the reduction in mean number of corpora lutea, relative to the other groups, found in the 20 mglkglday

group appeared to be responsible for this finding. Also, the relatively higher mean fetal weight for the controls most likely accounted for the statistically significant difference found, since the means obtained in the 20 mgkglday groups for this parameter were the same in both experiments.

Ibutilide was again teratogenic at 20 mglkglday, as evidenced by the statistically significant increases in the incidences of gross, skeletal, and total malformations. The fact that the malformations interventricular septal defect and scoliosis were each significantly higher in this group than in the controls further supported this conclusion. Ibutilide also appeared to be fetotoxic a t 20 mgkglday in the second experiment, as was the case in the first study, since the incidences of the skeletal variations accessory ribs and unossified pubes were each significantly increased in this group. Although no malformation or variation in the 5 and 10 mg/kg/day groups had a statistically significantly higher incidence than that found in the control group, the incidence of scoliosis was higher in the mid-dose group (3 fetuses in 2 litters), than in the controls (1 fetus). Since a statistically significant dose-response trend was found for this malformation in both experiments, the NOAEL for ibutilide's teratogenicity and developmental toxicity was set at 5 mglkglday.

Lacking evidence that effects on maternal homeosta- sis were indirectly responsible for the results obtained in the studies reported here, it is assumed that ibutilide was embryolethal, fetotoxic, and teratogenic as the result of effects on the conceptuses. Such effects may be consistent with the pharmacological actions of this drug as another class I11 antiarrhythmic agent L-691,121 also showed evidence of developmental toxicity in rats (Ban et al., '92). It was concluded that L-691,121 administration resulted in decreased embry- onic heart rates, which were associated with the mor- phological abnormalities and the subsequent embryo- lethality.


TABLE 8. Experiment 2 incidences of individual variations

Ibutilide (mgkglday) Observations' 0.0 5 10 202 Visceral

Grade 0 kidney Hydroureter Left umbilical artery No innominate

Femurb): incomplete ossification Fibula(s): incomplete ossification Frontal(s): incomplete ossification Hyoid: unossified Interparietal: incomplete ossification Interparietal: unossified Ischia: incomplete ossification Metacarpal(s): incomplete ossification Metatarsalb): incomplete ossification Parietalb): incomplete ossification Pubeb): unossified* Rib(s): accessory* Rib(s): 7th cervical Rib($: incomplete ossification Rib(s): rudimentary Squamosal(s): incomplete ossification Sternebrae: bipartite* Sternebrae: lack of apposition Sternebrae: no. 516 unossified Sternebrae other than 516: incomplete ossification Sternebrae other than 516: unossified Supraoccipital: incomplete ossification Tibia(s): incomplete ossification Vertebrae: incomplete ossification Vertebrae: 25 presacral Zvgoma(s): incomdete ossification

Skeletal

1 0 1

212

0 0

412 1418 412 0 0 0

412 1115 0 1

414 0

413 716 0 0

56/21 312 613

1118 0 0 1

914

2/2 212 212 212

0 0 0

2211 1 312 0 0 0 0

712 0 0 0

211 1

1014 0 0

53/17 514 413

1218 0 1 0

714

313 313 0 0

0 0 0

18112 513 0

211 0 0

212 212 0 1 1

513 1 0 1

6612 1 414 1

1017 0 0 0

313

1 1 0

414

1 1 0

1418 716

1 1 1 0

413 4/4** 8/6**

0 0 1

212 4 3

1 60121 1117 413

12/10 1

313 1

212

'Number of fetuses with variationslnumber of litters with this variation; no gross variations were detected. 'Fetuses from only 21 of the 22 litters in the group were subjected to visceral examinations. *Statistically significant dose-response effect, P < 0.05. **Statistically significantly higher than the control group, P < 0.05.

Since the NOAEL for developmental toxicity in the study reported here was set a t 5 mg/kg/day, and since the highest dose tested (80 mg/kg/day) did not show clear indications of maternal toxicity, the ratio (A/D) of toxicity in the adult (A) to the lowest dose that was toxic to the developing rat (D) was at least 80/10 or 8. Although an A/D of 8 is clearly reason for concern (Johnson, '87), the administration of ibutilide gener- ally will be short term (1-2 days), in a hospital setting, in older patients. In addition, the NOAEL (5 mg/kg/ day) is 4 times the proposed maximum clinical dose (two 1 mg doses, each infused over 10 minutes, or 0.033 mgkg for a 60 kg person), when corrected for 2.6% bioavailability in the rat a t a dose of 10 mg/kg (Hsu and Walters, Jungbluth et al., manuscripts in preparation). Thus, it is unlikely that clinical use of ibutilide will be a threat to the human conceptus.

ACKNOWLEDGMENTS The technical assistance of Daniel Black, Diann Mor-

ris, and Charles Shaw is greatly appreciated. Further thanks go to Joseph Haas and John Weaver for per-

forming the statistical analyses, and to Rodney Wal- ters for providing input in the toxicokinetics area.

LITERATURE CITED Ban, Y., R. Konishi, K. Kawana, T. Nakatsuka, and T. Fqjii (1992)

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Developmental toxicity of ibutilide fumarate in rats after oral administration