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Department of Non-Clinical Development (T.M.R.), Ferring Pharmaceuticals A/S, 2300 Copenhagen S, Denmark; Sierra Division (W.H.B., J.C.R., J.K.M., G.J.C.), Charles River Discovery and Development Services, Sparks, Nevada 89431; and Oregon National Primate Research Center (G.J.H.), Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Dr. Gary J. Chellman, Department of Toxicology, Charles River Laboratories, Preclinical Services, 587 Dunn Circle, Sparks, Nevada 89431. E-mail: gchellman{at}sbi.criver.com.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OT receptor antagonists comprise a comparatively new class of tocolytics under investigation. Endogenously generated OT is thought to play an important role in the initiation and maintenance of preterm and term labor (4, 5, 6). Both endocrine and paracrine sources of OT are involved, emanating from the hypothalamic-pituitary axis and uterus, respectively (7). Levels of OT receptor mRNA in the uterine myometrium increase substantially around the onset of labor (8). Also, it is well established in nonhuman primates and pregnant women that a circadian rhythm exists in myometrial contractility during late gestation, with peak activity occurring during periods of darkness between approximately 1800 and 0200 h. This rhythm of uterine activity appears to be driven by maternal OT (9, 10, 11). A crescendo in the intensity of this uterine activity rhythm occurs in the last 3–5 d before parturition and heralds the onset of labor. Collectively these data support a role for OT and its receptor in PTL and term labor (12). Thus, OT antagonists are potentially useful therapeutic agents to delay or prevent premature labor or delivery.
Atosiban (TRACTOCILE) is a combined vasopressin (V1a) and OT receptor antagonist and the first tocolytic developed and approved (in Europe) specifically for management of PTL (13). In clinical trials, atosiban has been shown to delay delivery in PTL (14). Atosiban can be used to delay imminent preterm birth between 24 and 33 wk of gestational age, but the delay is typically of modest duration (
48 h).
Barusiban (FE 200440) is a new OT receptor antagonist under development for PTL. Barusiban has high affinity for the human cloned OT receptor, approximately 300-fold that for the vasopressin V1a receptor, whereas atosiban binds well to both receptors (15). Barusiban also demonstrates more selective inhibitory effects than atosiban in studies of contractile patterns in isolated human myometrial tissue (15, 16). Furthermore, pharmacokinetics in animal models indicate a longer half-life for barusiban, compared with atosiban, suggesting that the duration of action may be prolonged. This could provide the convenience of less frequent administration in the clinic.
A previous study (17) reported a radiotelemetric model in conscious, preterm cynomolgus monkeys for evaluating the efficacy and pharmacokinetic/pharmacodynamic (PK/PD) relationships of new tocolytics. As a prelude to clinical trials for efficacy and potency in PTL, this model has been used in the present study to compare the PK/PD of barusiban and atosiban, including efficacy, potency, onset of action, duration of action, and reversibility.
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Materials and Methods |
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Eight pregnant female cynomolgus monkeys were assigned to the study, experimentally nonnaive, 3.9–7.3 yr of age, and weighing 3.4–5.1 kg at study start (within 3 d of surgery). Surgical procedures for implantation of telemetry units [to measure intrauterine pressure (IUP) and electromyograms (EMGs)] and infusion catheters (for dosing and blood sampling) were as described previously (17).
Briefly, during the third trimester of each animal’s pregnancy (on gestation d 120 ± 3), two venous catheters and an arterial catheter were implanted for dosing and blood sample collection. The catheters were routed through a jacket and tether to the outside of the cage, through the adjacent wall, and into the next room. This allowed remote techniques for dosing and blood sampling to be used, thereby reducing iatrogenic influence on uterine contractions. Heparinized saline was used to help maintain bidirectional catheter patency. A telemetry transmitter (TL11M2-D70-PCT, Data Sciences International, St. Paul, MN), IUP catheter, and biopotential electrodes were also surgically implanted into each animal for continuous recording of IUP and EMG. All surgical procedures were conducted by a veterinarian and conducted under isofluorane gas (approximately 0.5–1.0%) anesthesia. Analgesia was provided before and after surgery as appropriate for control of postsurgical discomfort. Cefazolin (systemic antibiotic; 50 mg/kg im) was administered intraoperatively and postoperatively for 4 d. Other antibiotics and antiinflammatory and pain medications including oxymorphone hydrochloride (Numorphan, 0.15 mg/kg), indomethacin (50 mg/dose), and buprenorphine (Buprenex, 0.01 mg/kg) were administered as needed postoperatively. Terbutaline sulfate (Brethine; 0.091–0.75 mg/h) was administered iv as needed during the first 5–6 d postoperatively to control postsurgical uterine activity; doses were based on the level of uterine activity monitored via the telemetric intraamnionic pressure catheter. A topical antibiotic ointment (bacitracin/neomycin/polymyxin or equivalent) was applied to the dorsal catheter exit site and the femoral surgical incision sites as deemed appropriate by the veterinarian. The animals were allowed to recover for at least 1 wk before conducting the evaluations of barusiban and atosiban. The study plan was reviewed and approved by the laboratory’s Animal Care and Use Committee, and the study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (18).
Test article administration
Each treatment group consisted of two to three pregnant females (total of eight females in study) and participated in up to four phases of treatment. The treatment phases included: OT control treatment; OT + barusiban; OT + atosiban; and barusiban + OT rescue therapy. The phases were separated by a 24- to 48-h washout period. The first treatment phase generally started on gestation d 128 ± 4. Dose levels of barusiban and atosiban were 10–50 and 100–500 µg·kg–1 (bolus) and 2.5–150 and 50–250 µg·kg–1·h–1 (infusion), respectively. These dose levels were selected to be in the range of maximal to half-maximal efficacy, based on preexisting PK and/or PD data in animals, including monkeys. Intravenous infusion dosing (barusiban, atosiban, and OT) was accomplished using clinical-grade Baxter Auto Syringe AS50* infusion pumps.
OT control infusions were given to determine the lowest dose required to give stable and submaximal contractions in each individual female. OT was initially given at 20 ± 10 mU·kg–1·h–1 and gradually adjusted (generally increased) approximately every 30 min by approximately 5–10 mU·kg–1·h–1 to find the lowest dose required to give contractions of approximately 30 ± 10 mm Hg approximately every 2–4 min. Once the maintenance dose was identified, it was continuously dosed for up to 15 h to see whether subsequent changes in IUP occurred and ensure there was no desensitization to OT. Maintenance doses of OT for the eight pregnant females ranged from 5 to 90 mU·kg–1·h–1.
Effects of the two OT receptor antagonists (barusiban and atosiban) were compared by first giving OT at the individual infusion rate required to elicit submaximal uterine contractions. One or the other of the antagonists was then administered to achieve maximal inhibition (high-dose bolus and infusion doses) or approximately 25–30% inhibition (low-dose bolus and infusion doses) of uterine contractions.
A rescue therapy phase evaluated the ability of high-dose OT to reverse the inhibitory effect of barusiban. After obtaining maximal inhibition of uterine contractions with a high-dose bolus (50 µg·kg–1) of barusiban, OT was given at escalating dose levels (133–2000 mU·kg–1·h–1) until predose contraction levels were restored.
Overall health of the animals and any adverse effects from the implanted devices or test articles were monitored by recording clinical signs and food consumption (daily) and body weight (periodically). Fetal health was monitored by ultrasound. IUP and EMGs were monitored continuously by telemetry. The telemetry data output was monitored remotely from a room adjacent to the animal room. The data generated during the recording periods were computed by data acquisition software (Data Sciences International), recorded, and plotted graphically. Telemetric recordings of the IUP data were further evaluated by integrating the data as area under the curve for half-hour intervals, using ChromPerfect Spirit software (Justice Laboratory Software, Denville, NJ). EMG data are not tabulated in the present report.
Blood samples for analysis of barusiban and atosiban pharmacokinetics were collected from all animals. The samples were collected predosing and at 15 min, 45 min, 1.25, 1.75, 2.25, 2.75, 3.25, 3.75, 4.75, and 5.75 h after the start of dosing with each OT receptor antagonist. Samples were analyzed by liquid chromatography/tandem mass spectrometry methods, and noncompartmental PK analysis of the resulting data was performed using WinNonlin software (Pharsight Corp., Mountain View, CA). The PK data were also used in conjunction with the PD data for effects on IUP to develop a comparative PK/PD simulation model for barusiban and atosiban (19). This model was used to calculate antagonist potency, i.e. the estimated plasma concentration of each of the OT receptor antagonists that resulted in a 50% reduction of OT-induced IUP levels.
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Results |
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As described previously (17), constant iv infusion of OT at 5–90 mU·kg–1·h–1 to pregnant cynomolgus monkeys mimicked PTL by creating a stable pattern of contractions (20–40 mm Hg IUP) occurring approximately every 2–4 min. Against this controlled background of OT-induced contractions, iv administration of either barusiban or atosiban was effective in reducing uterine contractions to baseline levels (Fig. 1
). As shown by the IUP tracings, barusiban had a longer-lasting effect than atosiban, even when given at a lower dose by bolus injection instead of infusion.
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. Barusiban (150 µg·kg–1·h–1, n = 3) immediately (within 30 min) reduced IUP from 6–7.5 kmm Hg · h to approximately 1 kmm Hg · h. The duration of maximum action (Tmax period) occurred from 0.5 to 9 h after barusiban administration, during which time IUP ranged primarily from approximately 0.3 to 1.2 kmm Hg · h (79–98% inhibition, compared with OT control levels). [For all experiments, the OT control level was defined as the average IUP determined during OT infusion for 1 h before the start of dosing with OT antagonist.] During the subsequent period of inhibition (9–14 h after the start of barusiban infusion), IUP was reduced to approximately 1–2 kmm Hg · h (55–86% inhibition). High-dose infusion of atosiban (250 µg·kg–1·h–1, n = 3) caused immediate (within 30 min) reductions in IUP levels. During the 2-h period of maximum inhibition (0.5–2.5 h post treatment initiation, Tmax period), IUP levels were approximately 0.3 kmm Hg · h, corresponding to 94–96% inhibition, compared with OT control. The total duration of action lasted for 3 h from the start of atosiban infusion. Pharmacokinetics associated with these effects (and others as discussed below) are presented below the PD (IUP) results in Fig. 2 and summarized at the end of this Results section.
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. Barusiban (50 µg·kg–1, n = 3) immediately (within 30 min) reduced IUP from 4–5 kmm Hg · h to approximately 1 kmm Hg · h. The Tmax period for inhibition of IUP was up to 8 h duration post treatment. During this time, IUP was inhibited 71–92%, compared with OT control. During the subsequent response period (8–15 h post treatment), IUP ranged primarily from approximately 1 to 2 kmm Hg · h (53–94% inhibition). Pharmacokinetics associated with these effects are also shown in Fig. 3.
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). However, the duration of action of atosiban was only 1.5 h (compared with > 15 h for high-dose bolus barusiban), after which time IUP returned to OT control levels.
Efficacy of the OT receptor antagonists given by low-dose infusion is shown in Fig. 4. Barusiban was given in a dose-escalating manner to determine the lowest dose needed to cause a 25–30% decrease in OT-induced contractions within 1 h, starting with 2.5 µg·kg–1·h–1 in the first hour and 7.5 µg·kg–1·h–1 in the second hour. In the third hour, one animal received 22.5 µg·kg–1·h–1, and the other two animals continued at 7.5 µg·kg–1·h–1. The onset of action in all three animals occurred within 1.5 h after the start of the low-dose infusion. Maximal reduction in IUP (values primarily < 1 kmm Hg · h, 88–96% inhibition, compared with OT control) was observed 2.5–6.5 h after initiation of treatment (Tmax period). IUP levels during the period of subsequent inhibition (6.5–13 h after treatment initiation) remained low (primarily < 3 kmm Hg · h and 71–100% inhibition). Atosiban low-dose infusion (50 µg·kg–1·h–1, n = 3) reduced IUP within 1 h of the start of infusion. During the 2-h Tmax period (1.0–2.5 h post treatment initiation), IUP was inhibited 40–65%, compared with OT control levels. After this 2-h duration of action, IUP returned to OT control values.
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). Both antagonists inhibited OT-induced contractions within 0.5–1 h of dosing. Maximum inhibition of IUP was 43–76% for barusiban (10 µg·kg–1, n = 1–2) and 72–90% for atosiban (100 µg·kg–1, n = 3). The duration of action was approximately 8 h for barusiban, compared with 1 h for atosiban.
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). The results demonstrated that barusiban-induced inhibition of uterine contractions could be reversed by administration of high-dose OT. In the two primary animals evaluated, IUP was reduced to 16% of control values (OT controls) within 0.5 h of administration of barusiban. Administration of OT to these two animals in a dose-escalating fashion resulted in mean recovery to 51% of OT control within 1.5 h. One of these animals reached maximally 74% of OT control within 1.5 h; the other reached maximally 88% of OT control within 2.5 h. A third animal recovered to 100% of OT control within 1.5 h of starting dose-escalating OT but was excluded from group mean calculation because of delivery before the end of the study.
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. The onset of action and the efficacy (percentage decrease in IUP) were comparable between the two antagonists, but barusiban had a much longer duration of action and greater potency.
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. Consistent with the more prolonged PD effects of barusiban, compared with atosiban, for both iv bolus and iv infusion dosing, barusiban had a longer elimination half-life and lower clearance than atosiban. Overall for both routes combined, the half-life was increased approximately 2- to 5-fold for barusiban, compared with atosiban, and clearance was decreased approximately 8- to 37-fold.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary response to barusiban and atosiban treatment in this study was similar, i.e. there was an immediate and marked reduction in uterine contractions, compared with OT-control levels. At maximum effect, both compounds showed approximately 90–98% inhibition of OT-induced contractions. However, barusiban was more potent than atosiban, producing the same level of IUP inhibition at approximately 4-fold lower blood levels. Also, the duration of action of barusiban (>13–15 h) markedly exceeded that of atosiban (1–3 h). Overall, because barusiban is more potent and longer acting than atosiban, it could provide important therapeutic advantages, e.g. ability to dose less frequently and eventually use of nonparenteral administration.
Inhibition of uterine contractions by both atosiban and barusiban was dose dependent. This was true for both induction and maintenance of reduced IUP levels and for both routes of administration (iv bolus and infusion). For example, for barusiban given by bolus injection, there were differences between the high dose (50 µg·kg–1) and low dose (10 µg·kg–1) in onset of action (
0.5 h vs. 1 h) and range of maximum inhibition (71–92 vs. 43–76%). Similarly, when atosiban was administered by infusion, the high dose (250 µg·kg–1·h–1) elicited a shorter onset of action (0.5 h vs. 1 h), greater maximum IUP inhibition (94–96 vs. 40–65%), and longer duration of action (3 vs. 2 h), compared with the low dose (50 µg·kg–1·h–1).
Barusiban achieved the same level of inhibition of OT-induced contractions at up to 10-fold lower doses than atosiban. This is consistent with its preferential affinity for the OT receptor, in contrast to atosiban. Unlike atosiban, which binds well to both vasopressin (V1a) and OT receptors, barusiban has approximately 300-fold higher affinity for the OT receptor than for the V1a receptor (15). Evaluation of the PD (IUP effects) and PK data from this study showed that the potency of barusiban was almost 4-fold greater than that of atosiban. The IC50 for inhibition of OT-induced contractions was 12.7 ng·ml–1± 14% (mean ± coefficient of variation) for barusiban, compared with 47.4 ng·ml–1± 20% for atosiban (19).
There is cross-reactivity between OT and vasopressin and their respective receptors (20, 21, 22). The animal model in this study used OT to induce uterine contractions and therefore predominantly evaluated efficacy associated with OT-induced PTL and the OT-antagonistic properties of barusiban and atosiban. However, during pregnancy there may be a complex interplay between OT and vasopressin and their interactions with each other’s receptor. For example, even if the OT receptor is blocked, OT may be able to influence uterine activity by its cointeraction with vasopressin receptors. If this is the case, a less selective drug like atosiban that interacts with vasopressin (V1a) receptors in addition to OT receptors may afford some advantage in maintaining uterine quiescence. This remains to be determined through clinical experience. Vasopressin is even more promiscuous than OT in terms of cross-receptor activation, and its role in PTL is less well understood.
Although PK data in the present study showed the half-life of iv administered barusiban to be approximately 2 h, the PD effects of barusiban persisted much longer than expected (>13–15 h; Figs. 2 and 3). This was in contrast to atosiban, in which PK/PD paralleled each other much more closely in time (Fig. 2). The reason(s) for the prolonged activity of barusiban may include such factors as kinetics of receptor binding, intracellular signaling, and/or kinetics of receptor recycling. Simulation modeling for barusiban has shown that there is a delay from the time plasma concentrations are achieved until onset of action occurs. This has been accounted for by a PK/PD model that includes a separate compartment for the PD effect (19). Further cellular research is needed to determine the mechanisms underlying barusiban’s prolonged duration of action.
Attempts were made in this study to quantitate plasma levels of OT. However, neither the available antibody-based nor liquid chromatography/tandem mass spectrometry methods were sensitive enough to measure these levels in the small plasma samples that were available. Although these data could be useful, it is known that endogenous OT comes from both pulsatile endocrine (hypothalamic-pituitary axis) and paracrine (uterine) sources (23, 24, 25). Ultimately the local concentrations of OT in the receptor compartment may be more important to biological activity than the levels circulating systemically in plasma that would be measured by bioanalysis.
The reversibility of barusiban’s OT antagonism was verified by rescue treatment consisting of administration of OT in a dose-escalating fashion (133–2000 mU·kg–1·h–1). The three animals evaluated in this phase of the study showed recovery of IUP levels to 74–100% of control levels between 1.5 and 2.5 h after beginning the high-dose OT rescue. This is important because in the event that emergency procedures (cesarean section) need to be used clinically to deliver a baby, effective control of postpartum uterine bleeding requires the ability to alleviate any existing OT antagonism so that the uterus can be constricted through administration of OT, vasopressin, or other vasoconstrictors. The ability to rapidly reverse barusiban’s pharmacological activity is an important aspect of ensuring the ability of this drug to be used safely and effectively in the clinic. Although not investigated in this study, higher doses of OT from the beginning of rescue therapy would be expected to result in even faster recovery, compared with the dose-escalating OT regimen used in this study. Also related to safety, several preclinical distribution and autoradiography studies with barusiban demonstrated a low placental transfer of about 10% (comparable with atosiban) and therefore only a minor exposure of the fetus (data not published).
In the cynomolgus monkey model used for this study there does not appear to be any desensitization or sensitization to OT-induced contractions after multiple hours, several days, or even several weeks of repeated daily dosing with OT (17). Thus, the infusions of OT used in the present study (up to 15-h duration) were known to elicit a relatively stable elevation in IUP levels, compared with baseline levels, and it was against these induced contractions simulating PTL that the effects of the OT receptor antagonists were evaluated.
In conclusion, the OT system is known to play a key role in the initiation and maintenance of labor, including PTL. In consequence, OT antagonists are logical candidates for pharmacological management of PTL. The results of the present study show that two different OT receptor antagonists, barusiban and atosiban, both have high efficacy and rapid onset of action when evaluated for inhibition of OT-induced uterine contractions in a cynomolgus monkey model of PTL. Furthermore, the inhibitory effects of barusiban were reversible by administration of high-dose OT, which confers control over the duration of the inhibition and thereby facilitates safe use in the clinic, especially in the case of emergency delivery by cesarean section. Barusiban had higher potency and longer duration of action than atosiban, consistent with its greater affinity for the OT receptor and improved PK profile, respectively.
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Acknowledgments |
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Footnotes |
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Results from this work were presented in part previously at the 45th Spring Meeting of the German Society for Experimental and Clinical Pharmacology and Toxicology, Mainz, Germany, 2004, and the 51st Annual Meeting of the Society for Gynaecologic Investigation, Houston, Texas, 2004.
First Published Online January 25, 2005
Abbreviations: EMG, electromyogram; IUP, intrauterine pressure; OT, oxytocin; PD, pharmacodynamic; PK, pharmacokinetic; PTL, preterm labor; Tmax period, duration of maximum action.
Received October 28, 2004.
Accepted January 19, 2005.
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