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Objective To assess the pharmacokinetics of anti-D IgG in pregnant Rhesus D-negative women after intramuscular and intravenous administration of 300 μg of Rhophylac.
Design An open, randomised, multicentre study.
Setting Seven gynaecological practices in Germany.
Sample Fourteen RhD-negative pregnant women at risk of becoming Rhesus D immunised received study drug at 28th week of pregnancy either by intramuscular or intravenous route.
Main outcome measures Anti-D IgG concentrations of serum samples obtained up to 11 weeks following antenatal Rhesus D prophylaxis were quantified by flow cytometry.
Results Mean anti-D IgG concentrations after intravenous and intramuscular administration differed up to seven days post-injection, from two weeks onwards they were comparable to each other. Irrespective of the administration route, anti-D IgG in serum was detectable in all women up to at least nine weeks post-administration.
Conclusions The serum concentrations of anti-D IgG measured after administration of Rhophylac were very similar to those obtained with 300 μg of a different anti-D immunoglobulin product.
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Routine postnatal administration of anti-D immunoglobulin to RhD-negative mothers who have given birth to an RhD-positive infant has been shown to be effective in preventing RhD alloimmunisation and has significantly reduced the incidence of haemolytic disease of the fetus and newborn1. However, without prophylaxis, anti-D antibodies may develop in a small proportion of women during pregnancy owing to occult fetomaternal haemorrhage, which is known to occur in about 45% of women during the last trimester2. Results of clinical studies have suggested that routine antenatal prophylaxis, in addition to postnatal prophylaxis, further decreases the risk of RhD alloimmunisation3–5. In North America and several European countries, guidelines are in place to provide antenatal Rhesus prophylaxis with anti-D immunoglobulin for all non-sensitised RhD-negative pregnant women unless the father of the baby is known to be RhD-negative. Dosage of anti-D, dosing schedule and methods of administration differ as a result of divergent clinical practises in individual countries. For instance, the United States and Germany favour a single antenatal injection of 300 μg anti-D at pregnancy week 286,7, whereas in the United Kingdom, two injections of at least 100 μg anti-D one at week 28 and one at week 34 are recommended8.
Previous studies have addressed the post-injection kinetics of anti-D immunoglobulin administered at gestation week 28. Anti-D serum concentrations were measured serologically9,10 or quantitatively by the autoanalyser method11,12. The half-life of anti-D was determined only in one study11, but other pharmacokinetic characteristics of anti-D immunoglobulin were not assessed. In all those studies, anti-D was given by intramuscular administration, and in one study12, it was also given by intravenous injection.
Recently, an anti-D immunoglobulin product (Rhophylac) has been developed for intramuscular and intravenous use. It is produced by an ion exchange chromatography fractionation procedure to enrich anti-D from hyperimmune plasma13. In the present study, we quantified the anti-D IgG serum concentrations by flow cytometric analysis (FACS) after intravenous and intramuscular administration of 300 μg of anti-D immunoglobulin at the 28th week of pregnancy and calculated the pharmacokinetic parameters of anti-D IgG.
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The study was designed as an open-label, multicentre, randomised trial to be conducted under outpatient conditions in seven gynaecological practices in Bavaria, Germany. RhD-negative women at 28 weeks of gestation were randomised to receive a single antenatal injection of 300 μg of Rhophylac by intravenous or intramuscular route. The goal was to obtain pharmacokinetic data from six pregnant women per administration route. To account for potential dropouts, it was planned to enroll 16 women, whereby eight women each were to receive anti-D either by the intramuscular or by the intravenous route, respectively. The administration route for the postnatal prophylaxis with Rhophylac, if required, was not randomised but was up to the discretion of the investigator.
The study protocol was approved by the Ethics Committee of the Bavarian Physicians' Chamber (‘Bayerische Ärztekammer’). The study was conducted in compliance with the Declaration of Helsinki and the ICH Guideline of Good Clinical Practice (CPMP/ICH/135/95).
Anti-D immunoglobulin [Rhophylac lot no. 01902-00002, ZLB Central Laboratory (now ZLB Bioplasma), Berne, Switzerland] was supplied in a ready-for-use syringe containing 1500 iu (equivalent to 300 μg) of anti-D in 2 mL solution.
RhD-negative pregnant women were enrolled in the study after giving voluntary written informed consent if they were at less than 28 weeks of gestation, at least 18 years old and if the biological father of the child was RhD-positive. Women were excluded from study participation if they met one of the following exclusion criteria: RhD-positive (including RhDweak-positive), RhD-negative but already immunised against the RhD antigen, history of anaphylactic or other severe systemic reaction to immunoglobulins, administration of anti-D immunoglobulin before week 28 of current pregnancy, IgA deficiency, transfusion of RhD-positive blood or administration of any other blood-borne products six months prior to enrollment, clinically relevant abnormal laboratory parameters (haematology, biochemistry, coagulation, urinalysis), which in the opinion of the investigator were not acceptable.
Prior to antenatal injection of anti-D immunoglobulin, blood samples were taken for measurement of irregular red blood cell antibodies (for details, see below). Then anti-D immunoglobulin was administered as a single bolus intramuscularly or intravenously according to a randomisation scheme. There was no attempt to conceal treatment allocation from women and clinicians. The allocation of the women to the treatment groups was determined by block randomisation with a block size of two. The investigators had to contact the project manager at AAI (Contract Research Organisation) each time when randomisation of a woman was upcoming, and the next available random number was assigned to the respective woman. The protocol did not stipulate the site of the intramuscular injection.
Blood samples (2 mL) for measurement of serum concentrations of anti-D IgG were taken immediately prior to antenatal administration of anti-D immunoglobulin and at defined intervals up to 11 weeks thereafter. After delivery, information on the newborn (date and time of birth, AB0 blood group, Rh-factor, gender, birthweight, height) was obtained. The blood group and RhD status of the newborn was determined according to the routine techniques of each participating centre. If the child was RhD-positive, a dose of 300 μg Rhophylac was administered to the mother within 72 hours after delivery.
Blood samples (2 mL) for measurement of serum concentrations of anti-D IgG were taken shortly prior to antenatal administration of anti-D immunoglobulin and 1, 2, 3, 4, 7, 14, 21, 35, 49, 63 and 77 days afterwards. Serum samples were frozen at −20°C until they were shipped to ZLB for analysis.
Serum concentrations of anti-D IgG were analysed by flow cytometry. Briefly, R2R2 (cDE/cDE) red blood cells were incubated with low ionic strength solution and patient's serum sample or a serial dilution of standard samples. Standard samples were prepared by spiking normal human AB serum pool with defined concentrations of Rhophylac lot 01902-00002. The anti-D concentration of Rhophylac lot 01902-00002 had been previously determined by an autoanalyser against the WHO reference anti-D standard 68/419. To detect cell-bound antibody, cells were washed with phosphate buffered saline, and then incubated with phycoerythrin-conjugated F(ab′)2-anti-human IgG (Jackson Immuno Research, West Grove, Pennsylvania, USA). Then, cells were washed and fluorescence intensity was measured by flow cytometry. The median fluorescence intensity of the stained red blood cells is dependent on the concentration of anti-D IgG in the sample/standard.
Pharmacokinetic parameters were determined from measured data without assuming any specific pharmacokinetic model. The calculation of the pharmacokinetic parameters was programmed in SAS. The following pharmacokinetic parameters were determined for each woman in both treatment groups: the maximum anti-D IgG serum concentration (Cmax) and the sampling time of the sample with maximum concentration (tmax) were directly obtained from measured data by observation. The area under the serum concentration–time curve (AUC(0–tz)) from time zero to the last sampling time point tz with a quantifiable anti-D IgG concentration Cz was calculated by the linear trapezoidal rule. The area under the serum concentration–time curve from time zero to infinity (AUC(0–∞)) was obtained by extrapolating the course of measured data to infinity according to the equation AUC(0–∞) = AUC(0 –tz) +Cz/λz. The terminal disposition rate constant (λz) was calculated by log-linear regression of the last portion of the serum concentration–time curve by fitting the function ln (C(t)) = A −λzt to the data points. The terminal half-life (t1/2) of anti-D IgG was calculated as t1/2= ln 2/λz.
After intravenous administration, in addition, the following parameters were determined: The half-life of the distribution phase (t½,α= ln 2/α), where α was obtained by log-linear regression of concentration data in the initial distribution phase after stripping for the elimination component, the total clearance of the drug from the body (CLtot= dose/AUC), and the apparent volume of distribution calculated as Vz= CLtot/λz. After intramuscular administration, the absorption rate constant (ka) was determined by log-linear regression of concentration data in the initial absorption phase after stripping for the elimination component. In addition, the half-life of the absorption phase (t½,a= ln(2)/ka), the total apparent clearance of the drug from the body (CLtot/f= dose/AUC) and the total apparent volume of distribution (Vz/f= (CLtot/f)/λz) were calculated.
The study was analysed in an exploratory manner (i.e. no statistical hypothesis was tested and a formal sample size calculation was not done). Six women per administration route seemed to be a reasonable sample to gather information about the pharmacokinetics of anti-D immunoglobulin. The trial was evaluated using descriptive statistics only. For continuously distributed parameters, the number of observations, mean, standard deviation, minimum, maximum and coefficient of variation were determined. The bioavailability after intramuscular administration and the corresponding 95% confidence intervals were estimated based on non-parametric U-statistics comparing AUC(0–∞) obtained after intramuscular administration to AUC(0–∞) obtained after intravenous administration.
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Eighteen women were screened at seven study centres. Two women had already received anti-D immunoglobulin during the present pregnancy prior to gestation week 28 and one woman tested positive for anti-D antibody. Fifteen women were randomised for antenatal administration of anti-D immunoglobulin at pregnancy week 28 (Table 1). One woman (no. 11) was excluded. In view of her strikingly low serum concentrations of anti-D IgG, her Rhesus factor was re-analysed at the ZLB laboratory and was found to be RhDweak-positive (phenotype CcDuee). Six women received the antenatal Rhesus prophylaxis by intravenous administration, and eight by intramuscular administration.
Table 1. Women randomised for antenatal administration of anti-D immunoglobulin.
|Woman no.||Age||Previous full term births and prematures||Weight (kg)||Height (cm)||Administration route||Site of intramuscular administration|
The 14 pregnant RhD-negative women were 21 through 37 years old (Table 1). All women were Caucasians. Seven out of 14 women were multiparae, with six women already being treated with anti-D during previous pregnancies. None of the women required additional anti-D immunoglobulin administration due to antenatal bleed or massive postnatal fetomaternal haemorrhage.
From each woman, a predose and 9 to 11 postdose samples were obtained up to 9 or 11 weeks post-administration. Four women gave birth prior to the last scheduled sampling time. All scheduled blood samples except one (week two sample of woman no. 9) could be obtained. One sample (week five sample of woman no. 9) was taken one week later than planned. Altogether, 176 serum samples were analysed for their anti-D IgG concentration by flow cytometry.
Mean anti-D IgG concentrations after intravenous and intramuscular administration differed up to seven days; however, from two to three weeks post-administration onwards, they were comparable to each other (Fig. 1). Woman no. 11, who was later found to be RhDweak-positive, received anti-D immunoglobulin by intravenous administration. The course of her anti-D IgG serum concentration–time curve was similar to those of the RhD-negative women who received the study drug intravenously, however, her serum anti-D IgG concentrations consistently were approximately 90% lower.
Figure 1. Mean (SD) anti-D IgG serum concentrations after intravenous and intramuscular administration of 300 μg anti-D immunoglobulin at 28th week of pregnancy.
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Irrespective of the administration route, quantifiable anti-D IgG serum concentrations were observed in all women up to at least nine weeks after administration. Quantifiable anti-D IgG serum concentrations were also found at week 11 post-administration in six (two intravenous, four intramuscular) of the 10 women who gave birth after week 11 post-administration. Trace concentrations (<0.4 ng/mL) of anti-D IgG were found in another three women (one intramuscular, two intravenous) at week 11 post-administration, whereas one woman of the intramuscular group had no detectable anti-D IgG at week 11 post-administration. Anti-D IgG serum concentrations above 1 ng/mL were observed for at least 63 days in seven out of the eight patients receiving anti-D immunoglobulin intramuscularly and in five out of the six patients receiving anti-D immunogloblin intravenously. The period [mean (SD)] with anti-D IgG concentrations above 1 ng/mL was 66.5 (9.9) days for the intramuscular treatment and 65.3 (10.5) days for the intravenous treatment.
The pharmacokinetic parameters of anti-D IgG are listed in Table 2. In both treatment groups, serum concentrations of anti-D IgG decreased with a half-life of about 17 days. The half-life of anti-D IgG was independent of the Rhesus D status of the newborn, which was 16.9 (5.2) days (10.5–26.6 days) for mothers with RD-positive babies (n= 8), and 17.4 (3.7) days (11.0–22.4 days) for mothers with RD-negative babies (n= 6). The relative bioavailability after intramuscular administration was estimated at 77.8%. The distribution half-life after intravenous administration could be estimated in four women only. In the other cases, the initial phase could not sufficiently be characterised.
Table 2. Pharmacokinetic parameters of anti-D immunoglobulin after intravenous (n= 6) and intramuscular administration (n= 8) in RhD-negative pregnant women.
|Mean (SD)||Range||CV (%)||Mean (SD)*||Range||CV (%)|
|AUC(0–tz)||day ng/mL||1015 (146)||870–1290||14.4||689 (251)||292–930||36.5|
|AUC(0–∞)||day ng/mL||1074 (195)||886–1447||18.1||738 (253)||369–893||34.3|
|Cmax||ng/mL||70.9 (8.2)||62.0–84.4||11.5||22.1 (12.0)||6.9–46.1||54.1|
|CL or CL/f||mL/hour||11.9 (1.8)||8.64–14.1||15.3||17.2 (7.5)||11.3–30.0||43.3|
|Vz or Vz/f||L||6.6 (0.8)||5.4–7.4||12.4||11.0 (7.6)||4.8–27.7||68.9|
|t1/2||day||16.4 (4.0)||11.0–22.4||24.3||17.6 (5.0)||10.5–26.6||28.5|
|t1/2 (absorption)||hours||–||–||–||2.5 (0.7)||1.6–3.5||28.1|
|t1/2 (distribution)+||hours||2.3 (0.7)||1.4–3.2||30.9||–||–||–|
Eight RhD-negative women gave birth to a RhD-positive child and received another 300 μg dose of Rhophylac within 72 hours of delivery. All six months follow up serum samples tested negative when the indirect antiglobulin tests were performed with native red blood cells. The serum sample of one woman (no. 10) gave a weakly positive result in the enzyme test. Another serum sample from this woman taken two months later tested negative in both tests.
A total of seven (three in the intravenous group, four in the intramuscular group) adverse events occurred in five women (two in the intravenous group, three in the intramuscular group). All adverse events were considered by the investigator to be not related to study drug. One woman complained about oesophagitis. Influenza-like symptoms were reported for three women. One of them also suffered from neuritis. This was the only adverse event that was reported following postpartal administration of study drug. All these adverse events were of mild to moderate severity and resolved completely within a maximum of 13 days.
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The main purpose of this study was to determine the pharmacokinetic parameters of anti-D IgG in pregnant women and thus to provide a rationale for the dose of anti-D immunoglobulin for routine Rhesus prophylaxis at the 28th week of gestation.
In the present study, we measured the anti-D IgG serum concentrations after intramuscular (eight women) and intravenous (six women) antenatal administration of anti-D immunoglobulin. The intravenous administrations resulted in mean peak serum anti-D IgG concentrations, which were 3.2 times higher than those after intramuscular administrations. Our results are not far off from calculations by Smith et al.14, who claimed the theoretically expected peak serum levels of anti-D IgG to be 2.5 times as high after intravenous as after intramuscular administration. The peak concentrations after intramuscular administration showed pronounced variations and ranged between subjects from 6.9 to 46.1 ng/mL. In contrast, peak concentrations after intravenous administration varied only from 62 to 84 ng/mL. Following intramuscular administration of anti-D immunoglobulin, peak serum concentrations of anti-D IgG were achieved after seven days (four women), four days (two women), three days (one woman) and two days (one woman). The variation in the time to reach peak levels of anti-D IgG probably reflects differences in the speed of uptake of antibody from muscular deposits. Interestingly, the two women (no. 9 and no. 12) of the intramuscular treatment group, who weighed more than 80 kg, achieved peak concentrations rather late (on day seven). Also, their maximum serum anti-D IgG concentrations were noticeably lower than those of the other women of the intramuscular group (6.9 and 10.0 ng/mL, respectively, versus a mean of 26.6 ng/mL for the six other women of the intramuscular group). In spite of the low peak anti-D IgG serum concentrations, these women had quantifiable anti-D IgG levels up to the last scheduled blood sample (weeks 9 and 11, respectively). The earliest tmax was observed in the woman who had the highest maximum concentration of anti-D IgG in the intramuscular group.
Mean serum anti-D IgG concentrations following intravenous and intramuscular administration were grossly different up to seven days after administration [36.1 (2.6) ng/mL iv; 19.8 (8.7) ng/mL im on day seven]. From two weeks post-administration onwards, however, there was no relevant difference in the mean serum levels after intramuscular and intravenous administration and mean curves were virtually superimposable from day 21 onwards. These data are in agreement with data from Bowman and Pollock12, who found that by 10 to 14 days after injection and thereafter, there was no difference in the levels in women given anti-D IgG intravenously versus given the same preparation intramuscularly. The bioavailability of anti-D IgG after intramuscular administration was estimated at 77.8% with a 95% confidence interval reaching from 41% to 96%. The large size of the confidence interval is partially due to the low sample size but also is influenced by low results observed for patients 9 and 12 who weighted more than 80 kg. Thus, for overweight women, the intravenous administration of Rhophylac may be more advantageous. Local loss of intramuscularly injected gammaglobulin has been recorded in earlier studies15,16.
The mean terminal elimination half-lives of anti-D after intramuscular and after intravenous administration of Rhophylac were almost equal in both treatment groups. The overall mean elimination half-life of ≈17 days determined in this study is shorter than the mean half-life of anti-D IgG found by Eklund et al.11 (≈21 days), however, in both studies, the half-lives varied considerably between subjects. The half-live ranged from 16.6 to 29.6 days in Eklund's study and from 10.5 to 26.6 days in our study. One reason for the shorter mean elimination half-life in our study might be that we used a flow cytometric assay rather than an autoanalyser. We chose flow cytometry as this method is more convenient, is found to be reliable and is sufficiently sensitive17–19. In our hands, the autoanalyser measures higher anti-D IgG levels than the flow cytometric method in the low concentration range. This is relevant for the samples taken late in pregnancy and may attribute to the apparently longer half-life when autoanalyser data are used. Furthermore, differences in half-life may be also due to the way it is determined as it is somewhat subjective which time points are selected for calculating the half-life. For our calculations, we consistently chose data points from week three onwards.
The low serum concentrations after intravenous administration of Rhophylac in woman no. 11 (maximum concentration 5.9 ng/mL) may be explained by absorption of anti-D IgG by the RhDweak-positive blood cells (CcDuee) in vivo. The anti-D serum concentration profile found in woman no. 11 with a half-life of 21.9 days is characteristic for an IgG molecule. It is astonishing that not all of the administered anti-D IgG is absorbed in vivo. Obviously, the flow cytometric assay detected an IgG subpopulation in Rhophylac that binds to the test red blood cells (R2R2) in vitro but not to the woman's red blood cells in vivo and circulates free in serum. Perhaps, the presence of low ionic strength solution in the incubation mixture facilitates binding of weakly reacting anti-D IgG in the test tube. The underlying reason for our findings was not investigated further.
The levels of serum anti-D IgG concentrations found towards the end of pregnancy in our study are very similar to those in Bowman and Pollock's study12, who found that as late as 9–10 weeks after injection, all maternal samples tested at that time had anti-D IgG serum levels ranging between 0.7 and 2.6 ng/mL. In our study, the anti-D IgG concentrations in the week nine samples ranged from 0.58 to 4.92 ng/mL. In Bowman's study, thereafter, although some women in both groups had detectable anti-D IgG as late as 88 to 98 days and as high as 1.9 ng/mL, others had no detectable anti-D IgG (<0.5 ng/mL). In our study, the week 11 samples had anti-D concentrations as high as 2.22 ng/mL. In one woman, there was no anti-D detectable any more. Both in Bowman's study and in our study, the anti-D IgG concentrations at the end of pregnancy were considerably lower than those in Eklund's study even though Eklund's patients received 50 μg less anti-D. In Eklund's study, after intramuscular injection of 250 μg, the mean (SD) value for the time of the lowest detectable concentration of anti-D IgG (1 ng/mL) was 119.7 (14.8) days in women with RhD-positive babies. Possibly, this discrepancy can be ascribed to differences and/or variations of the analytical methods used.
By multiplying the observed serum concentrations by 8400, Bowman calculated the mean residual anti-D IgG at 12 weeks after antenatal administration to be 6.7 μg (range <4 to 16.8 μg). In our study, taking into account the elimination half-life of anti-D IgG and using the same assumptions for calculating residual total anti-D IgG as Bowman did, the mean residual anti-D IgG at 12 weeks post-administration would be 10.1 μg (range: 1.7–24.3 μg). In theory, 10 μg of anti-D IgG would be sufficient to neutralise up to 0.5 mL fetal red blood cells, but the small proportion (≈2%)12,20 of women with fetomaternal haemorrhage of more than 0.5 mL at 30–39 weeks would not be protected. However, in Bowman's study, even women with residual anti-D below 4 μg did not get immunised. The compelling clinical success of the widely used antenatal Rhesus prophylaxis with a single 300 μg dose of anti-D immunoglobulin at week 28 underlines that this dosing schedule is sufficient to prevent Rhesus immunisation during the last trimester of pregnancy21. Alternatively, a dose of 100 μg given at week 28 and at week 34 would theoretically result in slightly more residual anti-D IgG at the end of pregnancy, however, for logistical reasons, it is not assured that all eligible women would get both administrations at the correct time22.