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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 10 3271-3277
Copyright © 1997 by The Endocrine Society

Original Studies

Liposomal Thyroxine: A Noninvasive Model for Transplacental Fetal Therapy1

Rekha Bajoria, Nicholas M. Fisk and Soli F. Contractor

Academic Department of Obstetrics and Gynecology, Charring Cross and Westminster Medical School (R.B., S.F.C.); and Institute of Obstetrics and Gynecology, Royal Postgraduate Medical School, Queen Charlotte’s and Chelsea Hospital (R.B., N.M.F.), London, United Kingdom

Address all correspondence and requests for reprints to: Dr. Rekha Bajoria, Ph.D., MRCOG, Academic Department of Obstetrics and Gynecology, St. Mary’s Hospital, Research Floor, The University of Manchester Medical School, Whitworth Park, Manchester, M13 OJH, United Kingdom. E-mail: rbajoria{at}rpms.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Drugs that cross the placenta sparingly are currently given directly to the fetus by invasive procedures. We investigated whether anionic small unilamellar (SUV) liposomes of different lipid compositions enhanced the transfer and uptake of T4 in an in vitro model of perfused human term placenta. T4-encapsulated anionic liposomes were prepared using lecithin (F-SUV) or distearoyl phosphatidylcholine (S-SUV) with cholesterol and dicetylcholine. The size distribution, encapsulation efficiency, and stability were determined in blood-based media. The transfer kinetics of free and liposomally encapsulated T4 were studied in a dually perfused isolated lobule of human term placenta, with creatinine and liposomal carboxyfluorescein as marker substances. Concentrations of T4 and rT3 were measured by RIA. T4 crossed the placenta sparingly (1.9 ± 0.5%) because it was metabolized to rT3 (9.2 ± 1.3%). Transplacental transfer of T4 was significantly increased by F-SUV (15.8 ± 2.1%; P < 0.001) and S-SUV liposomes (7.1 ± 1.2%; P < 0.001), with a concomitant decrease in fetal rT3 levels (P < 0.001). Placental uptake of F-SUV (13.5 ± 2.0%; P < 0.001) was greater than that of S-SUV liposomes (6.7 ± 0.8%; P < 0.001). Our data suggest that anionic liposomes increase transplacental transfer of T4. If confirmed in vivo, liposomes may provide an alternative noninvasive method of drug delivery to the fetus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WITH RECENT advances in diagnostic techniques in fetal medicine, there is a growing need to treat the fetus in utero by administering drugs to the mother (1, 2). Transplacental therapy with corticosteroids is standard clinical practice to prevent respiratory distress syndrome (3) and treat congenital adrenal hyperplasia (4). On the other hand, treatment of fetal arrhythmias by maternal administration of antiarrhythmic agents has had mixed results (5, 6, 7). The main drawback of transplacental fetal therapy is its restriction to drugs that cross the placenta readily. A further limitation is that therapeutic concentrations in the maternal circulation may not necessarily indicate adequate levels in the fetus, because transplacental passage depends upon pharmacological properties of the drug, including protein binding, metabolism by the placenta, and clearance in the fetal circulation. In addition, undesirable maternal exposure to potent therapeutic agents often limits maternal drug administration for fetal therapy (5, 8). Attempts have been made to overcome some of these drawbacks by direct drug administration to fetuses at fetal blood sampling or amniocentesis (6, 7). Direct fetal therapy, however, necessitates invasive procedures and thus the risk of procedure-related morbidity and mortality (9). Its widespread use is further limited by lack of understanding of drug pharmacokinetics in the fetal circulation, including transfer from fetus to mother. There is thus a need for a noninvasive drug delivery system to maintain therapeutic fetal concentrations with minimal maternal effects.

Of the various drug carrier systems available (10), liposomes have been investigated extensively. They can be administered orally or parenterally, and their pharmacokinetics can be modulated by altering their size, surface charge, and lipid composition (11, 12). When administered iv, liposomes are mainly cleared from the circulation by liver, but their tissue distribution can be altered by modifying their physico-chemical characteristics (12). Liposomes have been used clinically for the treatment of infectious diseases (13) and metabolic disorders (14) and to minimize the adverse effects of cytotoxic drugs (15). They are made from naturally occurring phospholipids, and their clinical use is not associated with antigenic or pyrogenic side-effects (16).

Recently, we have shown that anionic small unilamellar (SUV) liposomes made from egg lecithin can enhance uptake in the placenta of inert polar molecules by endocytosis (17, 18, 19). Similar results have been obtained in hepatic and other cell lines (12). Furthermore, for a given size and surface charge, the cellular uptake of liposomes has been shown to depend upon their lipid composition in that fluid liposomes are internalized more readily than solid ones. We hypothesized that SUV liposomes can enhance drug transfer across the placenta and that the rate of transfer depends on lipid composition. We evaluated the transfer and uptake of negatively charged SUV liposomes made from either unsaturated or saturated phospholipids in an in vitro model of dually perfused isolated lobule of human term placenta using T4 as a model drug.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Chromatographically pure egg phosphatidylcholine, distearoyl phosphatidylcholine, and grade 1 dicetyl phosphate in 2:1 chloroform-methanol (2:1) were purchased from Lipid Products (Nutfield, UK). Cholesterol (Chol) was obtained from Sigma Chemical Co. (Pool, UK). Sephadex G-25 was obtained from Pharmacia (Herts, UK). Carboxyfluorescein (CF) was purchased from Eastman Kodak (Hemel Hempstead, UK).

Preparation of liposomes

Anionic SUV liposomes were prepared from unsaturated (F-SUV; phosphatidylcholine-cholesterol-DCP, 7:7:1) and saturated phospholipids (S-SUV; distearoyl phosphatidylcholine-cholesterol-DCP, 7:7:1) by standard techniques of hydration of the dried lipid film with either 250 mmol/L CF or T4 (17, 18, 19). The lipid suspension was sonicated and free T4 or CF were separated from those encapsulated within liposomes by gel filtration on a Sephadex G-25 column (45 x 1 cm) equilibrated with Tris buffer, pH 7.4.

The phospholipid and cholesterol contents of each preparation were determined. The percentage of T4 encapsulated per mol lipid was calculated by determining the latency as described previously (17, 18, 19). Concentration of total T4 or CF was determined by adding 1% Triton X-100 to 10 µL liposomal preparation in buffer. The encapsulation efficiency of T4 or CF was expressed as the percentage of the initial amount added. The specific encapsulation was expressed as the percentage of entrapped solute per nmol liposomal phospholipid. The size and number of lamellae of the liposomes were determined by negative staining with 1% ammonium molybdate under a JEOL 100 CX electron microscope (JEOL, Peabody, MA).

Assessment of permeability of T4-containing liposomes in biological media

The stability of liposomes over 4 h was determined by incubating 1 mL liposomal preparation (0.5 µmol phospholipid) in 5 mL diluted maternal blood or phosphate-buffered saline (PBS) at 37 C (n = 5). Serial 100-µL samples were obtained at 30-min intervals and centrifuged (1500 x g, 10 min) in 4 mL PBS to determine the latency of T4 and CF in the supernatant.

Placental perfusion technique

Placentas were obtained immediately after vaginal or cesarean delivery from uncomplicated pregnancies of more than 37 weeks gestation. Dual closed circuit perfusion of the isolated lobule was commenced within 5–10 min at 37 C under optimal physiological conditions of oxygenation, pressure, flow, osmotic pressure, and acid/base status (20, 21). Closed circuit perfusion of the feto-placental circulation was established with a perfusion pressure of 40–50 mm Hg and a venous outflow of 6–9 mL/min. Fetal perfusates composed of autologous cord blood diluted with modified tissue culture medium 199 with a median hematocrit of 14 (range, 12–18) and a circulating volume of 110–120 mL. Materno-placental circulation was established with an arterial pressure of 15–18 mm Hg and a flow rate of 24–30 mL/min by placing five cannulas in the intervillous space. The maternal perfusates consisted of autologous blood collected from the intervillous space and diluted with modified tissue culture medium 199 (20) with a median hematocrit of 6 (range, 4–9) and a circulating volume of 150–160 mL. Tissue oxygenation was maintained by oxygenating the maternal circulation with 95% oxygen and 5% carbon dioxide. Creatinine was used as a marker to establish juxtaposition of the feto-maternal circulation. CF-encapsulated liposomes were used as liposomal markers.

Experimental protocol

In six sets of control experiments, 100 µg T4 were added to the maternal circulation at the commencement of the experiments. This concentration of T4 (0.1 µg/mL) was selected because it corresponded with the mean concentration of T4 present in liposomal form. In an additional six experiments, either F-SUV liposomally encapsulated T4 or S-SUV containing T4 was added to the maternal circulation. Just before the perfusion experiment, liposomally encapsulated T4 and CF were separated from the free drugs by chromatography on Sephadex G-25.

The free T4 or liposomally encapsulated T4 and CF with 30 mg creatinine was administered to the maternal arterial cannula distribution head over a period of 6 min (the time required for a single maternal circulation). Two-milliliter fetal and maternal samples were taken every 15 min over 2 h. Additional samples (0.5 mL) of maternal and fetal perfusates were taken for determination of acid-base status. All volumes were replaced with fresh perfusate. At the end of the perfusion period, both circuits were drained, and their volumes were measured.

All samples were centrifuged (3000 x g, 15 min), and the supernatants were aliquoted into 0.5- and 1.5-mL volumes. The 0.5-mL aliquot was stored at -20 C for creatinine assay. Liposomal stability was determined at each sample point by measuring CF latency in a 1.5-mL aliquot. Liposomal uptake was determined by homogenizing the perfused placenta as described previously (17, 19). An aliquot of the homogenate was centrifuged (3000 x g, 15 min), and T4 and CF concentrations were measured in the supernatant. The concentrations of T4 in the maternal and fetal circulations and in the placenta were expressed as a percentage of the dose added after correction for background activity, the circuit volume, and the amount removed from the previous sample.

At the end of the experiment, 2 mL maternal and fetal perfusates were applied to a Sephadex G-25 column (45 x 2 cm) to fractionate liposomally encapsulated from free T4/CF at room temperature with Tris-saline buffer. The elution rate was 0.63 mL/min. One-milliliter fractions were collected and assayed for T4, CF, phosphatidylcholine, and cholesterol.

Analytical methods

The concentrations of T4 (Ciba Corning Diagnostic Corp., Medfield, MA) and rT3 (Byk-Sangtec Diagnostics, Germany) were measured by RIA assay kits with coefficients of variation of 8% and 12%, respectively. The minimum amounts of T4 and rT3 detected per mL were 2.5 and 1 pg, respectively. The concentration of CF was measured fluorometrically at excitation and emission wave lengths of 490 and 520 nm, respectively, with a sensitivity of 1 nm/mL and coefficients of variation of 4–7%. The phospholipid and cholesterol contents of the liposomes was assayed by colorimetry with a sensitivity of 5 µg/mL as described previously (17, 18, 19). The creatinine concentration was determined by colorimetric assay (20, 21), with a coefficient of variation of 7–12%.

Data analysis

All values were expressed as the mean ± SEM. Equilibrium between maternal and fetal circuits was determined when fetal/maternal ratios of the drug levels were close to unity. The integrated value of maternal (MAUC) and fetal (FAUC) concentrations of a drug were calculated using the trapezoidal rule (20). The placental transfer rate of T4 was determined by fitting a weighted line for the SD mean fetal concentration between 15–120 min (20). The transfer rate of T4 was calculated from the slope of this line. Two-way ANOVA was used to compare values between groups. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The SUV liposomes of both lipid compositions were unilamellar and had uniform size distribution, with a mean diameter of 73.6 ± 3.8 nm. The percent encapsulation of T4 per mol phospholipid was comparable with that of CF liposomes (Table 1Go). The stability of T4 liposomes in PBS and blood was similar to that of CF liposomes (Table 2Go).


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  		Table 1. Characteristics of carboxylflurosceine- and T4-containing anionic small unilamellar liposomes

 

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  		Table 2. Stability of liposomes in biological medium

 
The transplacental transfer of T4 is shown in Fig. 1aGo and constitutes the control group. The maternal T4 concentration decreased from 100% to 85.2 ± 2.1% at 120 min. The fetal concentration increased linearly (slope = 0.30; r = 0.98) to 1.9 ± 0.5% at 120 min, with a clearance rate of 0.09 ± 0.06 mL/min and a fetal/maternal ratio of 0.02 ± 0.001 (Fig. 2aGo). The MAUC and FAUC values were 9401 ± 173% and 118 ± 28% dose/min, respectively (Fig. 3Go, a and b). The fetal concentration of rT3 increased linearly and was 9.0 ± 1.3% (Fig. 2bGo) with a FAUC of 405 ± 59% dose/min. The placental uptakes of T4 and rT3 were 3.7 ± 0.5% and 9.5 ± 0.9%, respectively (Fig. 3dGo).



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  		Figure 1. Transfer from maternal (•) to fetal ({circ}) circulation of T4 alone (a), F-SUV liposome-encapsulated T4 (b), and S-SUV liposome-encapsulated T4 (c). All values are expressed as a percentage of the initial dose added to the maternal circulation and are the mean of six experiments.

 


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  		Figure 2. The effect of lipid composition of liposomes on the fetal-maternal ratio of T4 (a) and fetal rT3 levels during 2-h perfusion (b). {triangleup}, S-SUV; {circ}, F-SUV; •, free T4.

 


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  		Figure 3. Compares the effects of S-SUV and F-SUV liposomes with free T4 at 120 min on the MAUC (a) and the FAUC (b) of T4 and rT3, the FAUC/MAUC ratio for T4 (c), and the placental concentrations of free T4 and rT3 (d). Solid bar, Free T4 liposomes; hatched bar, S-SUV liposomes; open bar, F-SUV liposomes.

 
The effects of F-SUV and S-SUV liposomes on the placental transfer and uptake of T4 are shown in Fig. 1Go, b and C. The maternal concentration (P < 0.001) and MAUC (P < 0.01) of F-SUV T4 liposomes were lower than those in the control group (Fig. 3aGo). The fetal T4 level increased linearly (slope = 0.21; r = 0.97) and was higher than the control value (15.8 ± 2.1%; P < 0.001). Similarly, the FAUC (P < 0.05), fetal/maternal ratio (P < 0.05), and placental uptake (P < 0.001) of F-SUV were higher than those in the control group (Fig. 3Go). The fetal (2.6 ± 0.5%; P < 0.001) and placental (4.2 ± 0.7%; P < 0.01) concentrations of rT3 were significantly reduced in liposomal group. The latency of CF in the maternal circuit was 98.5 ± 1.1% at 15 min and 93.2 ± 0.8% at 120 min.

The maternal concentration (P < 0.001) of S-SUV T4 liposomes was less than the control value (Fig. 1CGo). Although the MAUC level in the S-SUV group was higher than that in the control group (9086 ± 170% dose/min), the difference was not significant. The fetal concentration (7.1 ± 1.2%; P < 0.001; slope = 0.43; r = 0.98), FAUC (P < 0.001), and fetal/maternal ratio (P < 0.001) were higher than those in the control group. The placental uptake of S-SUV liposome was higher than the control value (P < 0.001). The fetal (2 ± 0.2%; P < 0.001), FAUC (P < 0.01), and placental (P < 0.01) concentrations of rT3 were less than the control values. The stability of liposomes in the maternal circulation was 98.7 ± 1.0% at 15 min and 96.0 ± 1.1% at 120 min.

The maternal concentration (P < 0.01) and MAUC levels (P < 0.01) were higher with S-SUV liposomal T4 than with F-SUV liposomal T4, whereas the fetal concentration (P < 0.001), FAUC (P < 0.01), and fetal/maternal ratio (P < 0.01) were significantly lower. Placental uptake of F-SUV was markedly higher than that of S-SUV (P < 0.001). The fetal concentrations of rT3 in both the groups were comparable (Fig. 3Go).

Chromatography

The chromatogram of the maternal perfusates containing F-SUV liposomes showed two distinct peaks of T4. The peak immediately after the void volume was due to T4-entrapped liposomes, as confirmed by latency, liposomal phospholipid, and cholesterol content. The second peak was due to free T4 (Fig. 4aGo). The liposomal and free T4 concentrations were 68.6 ± 2.9 and 6.1 ± 3.2 µg/dL, respectively. In the fetal perfusate (Fig. 4bGo) no intact liposomes were found, and there was a single peak that corresponded to free T4 (13.0 ± 1.8 µg/dL). The chromatogram of maternal, fetal, and placental homogenate with S-SUV was essentially similar to that with F-SUV.



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  		Figure 4. Chromatograms of maternal (a) and fetal (b) perfusates on Sephadex G-25. The concentration of liposomal lipids is shown as a dotted line. c, Chromatogram of T4 shown for reference.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that T4 does not cross the human placenta in significant quantities, but its transfer from the maternal to the fetal circulation can be enchanced 8-fold by anionic small unilamellar liposomes. We used T4 as a model drug to test our hypothesis because 1) human placenta forms an enzymatic barrier to free passage of T4 (22, 23); and 2) there is a clinical need for intrauterine fetal T4 replacement therapy. This is especially applicable after recent developments made the diagnosis of fetal thyroid dysfunction possible in utero (24). Although T4 therapy in utero has only been shown to be effective in correcting fetal hypothyroidism secondary to propylthiouracil-treated maternal Graves’ disease (25), it also has the potential to be useful in certain fetal conditions, i.e. prevention of respiratory distress syndrome in those at risk of preterm delivery (26). Although in vitro studies indicate that T4 can enhance surfactant production by the human fetal lungs, its use in preventing respiratory distress syndrome in the clinical setting has not been evaluated. This is largely because transplacental fetal therapy with T4 has not been possible. Accordingly, the current approach to fetal thyroid hormone replacement is by intraamniotic administration of T4 or T3 (27). The major drawback of this route is its invasiveness, with multiple administrations cumulatively increasing procedure-related risks. Alternatively, attempts have been made to stimulate the fetal pituitary-thyroid axis by maternal administration of TRH; however, it has only been used acutely. Chronic therapy may be of limited value because TRH is unlikely to produce a sustained fetal thyrotropic response due to negative feedback mechanisms by target hormones (T4 and T3). Notwithstanding this, fetal therapy with TRH can only be effective in the presence of functional thyroid tissue, thereby restricting its use in the treatment of fetal thyroid disorders. Our study suggests a potential role for liposomal encapsulation as a noninvasive approach to correct fetal thyroid deficiency in utero. Although liposomes are currently used clinically to treat systemic fungal disease (13), their safety has not been evaluated during pregnancy. Furthermore, only limited data are available on the transfer kinetics of liposomes in the maternal-placental-fetal unit in vitro.

We studied T4 transport in an in vitro model of an isolated lobule of dually perfused human term placenta. Our finding that T4 crosses the human term placenta sparingly is consistent with previous clinical reports (22) and a recent in vitro study (23). The relatively large molecular size of T4 (890 dalton), extensive protein binding (0.3% present in free form), and minimal lipid solubility may all restrict transplacental transfer. However, it is unlikely that these are the only factors responsible for restricted transfer, because under similar experimental conditions molecules such as inulin (mol wt, 6000) and digoxin (mol wt, 600) (28) cross the placenta at a higher rate than T4. Furthermore, highly protein-bound compounds such as free fatty acid and some drugs are also known to readily cross the placenta (29). Studies using placental slices and homogenates have previously shown that human placenta metabolizes T4 to rT3 by inner ring deiodination (30, 31). Our study confirms that the human placenta converts T4 to rT3, suggesting that enzymatic degradation of T4 by the placenta also contributes to its minimal transfer. However, our data contradict those from the in vivo study, which indicate substantial amounts of transplacental passage of T4 into the fetal circulation of infants with thyroid agenesis (32). Although the reason for the discrepant observations remains unclear, one possibility may be that the use of dilute maternal and fetal blood-based perfusate in our experiments has impaired protein binding of T4. Given the evidence that transfer of a drug across the placenta is inversely related to its protein-binding function (29), and that T4 binds extensively to T4-binding globulin (>99%), the net transfer of T4 in our system is expected to be higher than that observed in vivo. Similarly, using a nonblood-based perfusate, Mortimer et al. (23) also found that T4 crosses the human placenta sparingly. Hence, the more logical explanation for the passage of T4 observed in clinical studies may be that fetal thyroid status probably regulates transfer of T4 by modulating placental enzymatic activity. An alternative explanation can be that transfer of T4 occurs via an extraplacental chorio-amniotic route.

We next studied the effect of anionic liposomes on transfer and used CF as a liposomal marker. This approach enabled us to monitor the stability of liposomal T4 indirectly by simply measuring the latency of CF liposomes, particularly when the stabilities of T4- and CF-containing liposomes were comparable in blood-based medium. This study demonstrates that liposomes can significantly increase the transfer of T4 from the maternal to the fetal circulation. Enchanced transfer of T4 could not be attributed to direct placental transfer of intact liposomes, because chromatography of the fetal perfusate showed no liposomes or liposomal lipids. Another possibility is that encapsulation of T4 within the liposomes increases placental uptake and transfer by preventing enzymatic conversion of T4 to rT3. Low fetal rT3 levels in the liposomal group substantiate this proposition. We speculate that after placental uptake, liposomes are directed to lysosomes, where they are degraded with intracellular release of encapsulated T4. Instead of being inactivated to rT3 by microsomal monodeiodinase enzyme, T4 diffuses down the concentration gradient into the fetal circulation. However, we made no attempt to dissect intracellular pathways of liposomal uptake by syncytiotrophoblast. Recent studies using CV1 monkey kidney cells suggested that the anionic liposomes are internalized by coated pits, with migration to lysosomes and subsequent release of the encapsulated material into the cytoplasm (33).

Our data also suggest that transplacental uptake and transfer of anionic fluid liposomes were significantly higher than the solid liposomes. Although this study fails to elucidate the mechanism by which lipid composition can influence the kinetics of liposomal T4, increased leakiness of fluid liposomes in blood-based medium may be one explanation (34). The latency of fluid liposomes of more than 95% make it unlikely that differential transfer was due to relatively higher maternal levels of free T4. The possibility remains that the difference in the transfer rate of T4 by liposomal lipid composition could be due to the difference in placental uptake. We are unaware of any work evaluating the mechanism by which the lipid composition of liposomes can influence placental uptake. In vitro and in vivo studies indicate that fluid liposomes are internalized by hepatocytes and macrophages more avidly than solid liposomes (35) because of higher binding affinity to factors such as {alpha}2-macroglobulin, IgG, and fibronectin (12, 36, 37).

We appreciate that as we used an in vitro model of perfused human term placenta, it is difficult to extrapolate this finding in terms of clinical usage, where T4 is likely to be given in the midtrimester either to treat fetal goiter (25) or enhance fetal lung maturity (26). Although no information is available on the effect of gestational age on transplacental transfer of T4, observation of transplacental transfer of T4 in the first trimester (38) raises the possibility that deiodination of T4 to rT3 may be gestational age dependent. Similarly, it is plausible that uptake of liposomes by the placenta increases with gestation. As this study did not address the fetal metabolism of T4, it is difficult to extrapolate any therapeutic relevance of our findings. It is possible that high levels of T4 attained in the fetal circulation by using liposomes may not be effective to treat the fetus in utero because of the rapid metabolism of T4 to rT3. Although compartmental modelling of our experimental data could have provided some information on the effect of increased levels of T4 on fetal clearance, in this paper we choose to use the simple trapezoidal method to determine whether transplacental transfer of T4 can be facilitated by SUV liposomes. However, based on the clinical evidence that intraamniotic administration can increase circulating fetal levels of T4, we envisage that increased transplacental transfer of T4 by liposomes is likely to achieve therapeutic levels in clinical settings. Further studies are necessary to evaluate the effects of gestational age and variable maternal dose on transplacental transfer of liposomal T4. Perhaps it would be then more appropriate to analyze the experimental data using the complex compartmental model to determine the ideal/optimal therapeutic maternal dose necessary to achieve desirable therapeutic levels in the fetus. This knowledge is crucial before undertaking clinical studies to determine the materno-fetal pharmacokinetics of liposomally encapsulated T4 and its therapeutic efficacy. As the human fetal circulation is now accessible through invasive procedures, this has become a realistic possibility.

In conclusion, this study shows that anionic liposomes significantly increase the transfer of T4 across the human term placenta and may provide a novel noninvasive method by which the fetus can be treated in utero by pharmacological agents with minimal maternal exposure.


   Acknowledgments
 
We thank Prof. G. Gregoriadis, School of Pharmacy (London, United Kingdom), for his assistance in developing the liposomal methodology in our Laboratory.


   Footnotes
 
1 This work was supported in part by a project grant from Action Research. Back

Received January 16, 1997.

Revised April 1, 1997.

Revised June 6, 1997.

Accepted June 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Batshaw M, Brusilo S, Waber L. 1982 Treatment of inborn errors of urea synthesis: activation of alternative pathways of waste nitrogen synthesis and excretion. N Engl J Med. 306:1387–1392.[Abstract]
  2. Packman S, Cowan MJ, Golbus MS, et al. 1982 Prenatal treatment of biotin responsive multiple carboxylase deficiency. Lancet. 1:1435–1438.[CrossRef][Medline]
  3. Crowley P, Chalmers I, Keirse MJ. 1990 The effects of corticosteroid administration before preterm delivery: an overview of the evidence from controlled trials. Br J Obstet Gynaecol. 97:11–25.[Medline]
  4. Levine LS, Pang S. 1994 Prenatal diagnosis and treatment of congenital adrenal hyperplasia. J Pediatr Endocrinol. 7:193–200.[Medline]
  5. van-Engelen AD, Weijtens O, Brenner JI, et al. 1994 Management outcome and follow-up of fetal tachycardia. J Am Coll Cardiol. 24:1371–1375.[Abstract]
  6. Hallak M, Neerhof MG, Perry R, Nazir M, Huhta JC. 1991 Fetal supraventricular tachycardia and hydrops fetalis: combined intensive, direct, and transplacental therapy. Obstet Gynecol. 78:523–525.[Medline]
  7. Jouppila P, Makarinen L, Rasanen J, Valkaman M, Paavilainen T. 1993 Aggressive direct treatment of a fetus with supraventicular tachycardia and hydrops fetalis. Ultrasound Obstet Gynecol. 3:279–283.[CrossRef][Medline]
  8. Pang S, Clark AT, Freeman LC, et al. 1992 Maternal side effects of prenatal dexamethasone therapy for fetal congenital adrenal hyperplasia. J Clin Endocrinol Metab. 75:249–253.[Abstract]
  9. Bernaschhek G, Yildiz A, Kolankaya A, Stuempflen I, Deutinger J. 1995 Complications of cordocentesis in high risk prenancies: effects on fetal loss or preterm delivery. Prenat Diagn. 15:995–1000.[Medline]
  10. Gregoriadis G, Florence AT. 1993 Liposomes in drug delivery. Clinical, diagnostic and ophthalmic potential. Drugs. 45:15–28.
  11. Gregoriadis G, Florence AT. 1993 Recent advances in drug targeting. Trends Biotechnol. 11:440–442.[CrossRef][Medline]
  12. Senior J. 1987 Fate and behaviour of liposomes in vivo: a review of controlling factors. CRC Crit Rev Ther Drug Carrier Systems. 3:123–193.
  13. Ralph ED, Barber KR, Grant CW. 1993 Clinical experience with multilamellar liposomal amphotericin B in patients with proven and suspected fungal infections. Scand J Infect Dis. 25:487–496.[Medline]
  14. Gregoriadis G, Weereratne H, Blair H, Bull GM. 1982 Liposomes in Gaucher type I disease: use in enzyme therapy and the creation of an animal model. Prog Clin Biol Res. 95:681–701.[Medline]
  15. Gabizon A, Isacson R, Libson E, et al. 1994 Clinical studies of liposome-encapsulated doxorubicin. Acta Oncol. 33:779–786.[Medline]
  16. Eichler HG, Senior J, Stadler A, Gasic S, Pfundner P, Gregoriadis G. 1988 Kinetics and disposition of fluorescein-labelled liposomes in healthy human subjects. Eur J Clin Pharmacol. 34:475–479.[CrossRef][Medline]
  17. Bajoria R, Contractor-SF. Effect of surface charge of small unilamellar liposomes on uptake and placental transfer of carboxyfluoreseine. Pediatr Res. In press.
  18. Bajoria R, Sooranna-SR, Contractor-SF. 1997 Endocytotic uptake of small unilamellar liposomes by trophoblast cells in culture. Hum Reprod. 12:1343–1348.
  19. Bajoria R, Contractor SF. 1997 Effect of size of liposomes on transplacental transfer of carboxyfluorescein across the perfused human term placenta. J Pharm Pharmacol. 49:675–681.[Medline]
  20. Bajoria R, Contractor SF. 1992 Transfer of heparin across the human perfused placental lobule. J Pharm Pharmacol. 44:952–959.[Medline]
  21. Bajoria R, Oteng-Ntim E, Fisk NM. 1996 Transfer and metabolism of thyrotropin releasing hormone across the perfused human term placenta. J Clin Endocrinol Metab. 81:3476–3482.[Abstract]
  22. Myant NB. 1959 Passage of thyroxine and tri-iodothyronine from mother to fetus in pregnant women. Clin Sci. 17:75–79.
  23. Mortimer RH, Galligan JP, Cannell GR, Addison RS, Roberts MS. 1996 Maternal to fetal thyroxine transmission in the human term placenta is limited by inner ring deiodination. J Clin Endocrinol Metab. 81:2247–2249.[Abstract]
  24. Avni EF, Rodesch F, Vandemerckt C, Vermeylen D. 1992 Detection and evaluation of fetal goitre by ultrasound. Br J Radiol. 65:302–305.[Abstract]
  25. Noia G, De Santis-M, Tocci A, et al. 1992 Early prenatal diagnosis and therapy of fetal hypothyroid goiter. Fetal Diagn Ther. 7:138–143.[Medline]
  26. Ballard PL. 1989 Hormonal regulation of pulmonary surfactant. Endocr Rev. 10:165–181.[CrossRef][Medline]
  27. Mashiach S, Barkai G, Sack J, et al. 1978 Enhancement of fetal lung maturity by intra-amniotic administration of thyroid hormone. Am J Obstet Gynecol. 130:289–293.[Medline]
  28. Reynolds F, Knott C. 1989 Pharmacokinetics in pregnancy and placental drug transfer. Oxf Rev Reprod Biol. 11:389–449.[Medline]
  29. Bajoria R, Contractor SF. 1994 Maternal-fetal transfer of warfarin across an in vitro model of perfused placenta; effect of albumin binding. Br J Haematol. 84:59.
  30. Roti E, Fang SL, Green K, Emerson CH, Braverment LE. 1981 Human placenta is an active site of thyroxine and 3,3–5 tri-iodothyronine tyrosyl ring de-iodination. J Clin Endocrinol Metab. 53:498–501.[Abstract]
  31. Yoshida K, Suzuki M, Sakurada T, et al. 1985 Human placental thyroxine inner ring monodeiodinase in complicated pregnancy. Metabolism. 34:535–538.[CrossRef][Medline]
  32. Vulsma T, Gons MH, de-Vijlder JJ. 1989 Maternal-fetal transfer of thyroxine in congenital hypothyroidism due to a total organification defect or thyroid agenesis. N Engl J Med. 321:13–16.[Abstract]
  33. Straubinger RM, Papahadjopoulos D, Hong KL. 1990 Endocytosis and intracellular fate of liposomes using pyranine as a probe. Biochemistry. 29:4929–4939.[CrossRef][Medline]
  34. Senior J, Gregoriadis G. 1982 Stability of small unilamellar liposomes in serum and clearance from the circulation: the effect of the phospholipid and cholesterol components. Life Sci. 30:2123–2136.[CrossRef][Medline]
  35. Spanjer HH, van-Galen M, Roerdink FH, Regts J, Scherphof GL. 1986 Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition. Biochim Biophys Acta. 863:224–230.[Medline]
  36. Moghimi SM, Patel HM. 1989 Serum opsonins and phagocytosis of saturated and unsaturated phospholipid liposomes. Biochim Biophys Acta. 984:384–387.[Medline]
  37. Moghimi SM, Patel HM. 1988 Tissue specific opsonins for phagocytic cells and their different affinity for cholesterol-rich liposomes. FEBS Lett. 233:143–147.[CrossRef][Medline]
  38. Contempre B, Jauniaux E, Calvo R, Jurkovic D, Campbell S, Morreale de Escobar G. 1993 Detection of thyroid hormones in human embryonic cavities during the first trimester of pregnancy. J Clin Endocrinol Metab. 77:1719–1722.[Abstract]




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