Small-Bowel Length and the Dose of Cyclosporine in Children after Liver Transplantation

Patients

The population consisted of 56 patients, 3 months to 20 years of age, who received liver transplants at the University of Chicago between January 1985 and January 1989. A precise description by the surgeons of the amount of intestine resected and excluded before and during liver transplantation was available for 53 patients. Pharmacokinetic studies were performed in 17, for 14 of whom there was also information concerning intestinal length. Children receiving liver transplants were excluded from the study if neither accurate information about bowel length nor results of pharmacokinetic studies were available (n = 6) or if there was marked cholestasis that could impair the absorption of cyclosporine (n = 4).

The amount of drug required was recorded as the total daily intake of cyclosporine that was necessary to achieve whole-blood levels of 200 ng per milliliter (therapeutic range, 150 to 250), as determined by high-performance liquid chromatography 12 hours after the dose was given. This method of measuring cyclosporine was chosen for clinical monitoring as well as pharmacokinetic studies because radioimmunoassay with polyclonal antibody does not distinguish native cyclosporine from metabolites, which can accumulate in the serum of patients with liver disease. Its only major disadvantage is low sensitivity; the lower limit of detection is 62 ng per milliliter. The intravenous doses were recorded on the day before the route of administration was changed, usually one to three weeks after transplantation. The oral doses (expressed as milligrams per kilogram per day) were recorded 10 to 16 weeks after transplantation, after recovery from surgery and discharge from the hospital. The results of liver-function tests (levels of total bilirubin, conjugated bilirubin, alkaline phosphatase, and alanine aminotransferase) and precise height and weight measurements were recorded at the same time as the cyclosporine dose. Body-surface area was computed with the equation of Haycock et al. for children who weighed less than 15 kg¹⁵ and with that of Dubois and Dubois for children who weighed 15 kg or more.¹⁶ The total daily dose was recomputed after correction for body size. Although the dose is most commonly expressed per kilogram of body weight, its expression per square meter of body-surface area is considered to reflect drug distribution more accurately.

Estimation of Bowel Length

Functional residual bowel length — that is, the length of small intestine available for the absorption of cyclosporine — could not be measured directly and was therefore estimated. Seibert's data on intestinal length obtained from 183 autopsies in children between 30 weeks of gestation and 15 years of age¹⁴ were used to estimate expected total bowel length at the time of study by interpolation from the height. From this value we subtracted the length of any intestine excised before or during liver transplantation plus that made unavailable for cyclosporine absorption during liver transplantation. The length of the intestine to be excised was measured in situ. Patients who had had intestine removed in previous operations were not included unless the resected bowel had been accurately measured in a similar manner. Bowel excluded from cyclosporine absorption was considered to include both limbs of the Roux-en-Y anastomosis because our previous work in rats indicated that cyclosporine absorption by intestine proximal to the site of entry of bile is negligible.^{4 , 5} The length of excluded intestine was measured along the antimesenteric border in situ. In patients with primary anastomosis of the bile ducts, the value for excluded bowel was zero.

Cyclosporine Pharmacokinetics

Studies of cyclosporine pharmacokinetics were performed three months after liver transplantation in 17 children and in 6 adults between 18 and 50 years of age. They were repeated six to nine months after liver transplantation in seven of the children. These studies were performed in the clinical research center with use of a protocol approved by the University of Chicago Clinical Investigation Committee. Full informed consent was obtained from the parents of the children studied and from adult patients.

The patients were hospitalized for two days to complete the pharmacokinetic studies. A Teflon catheter was placed in a vein in an arm or leg for repeated blood sampling. On the first day, cyclosporine was administered intravenously over a two-hour period. The intravenous dose used was one third of the patient's regular oral dose because we expected the fractional availability to be approximately 33 percent.^{6 7 8} On the second day, the patient received the regular oral dose. Blood cyclosporine levels were determined just before the oral dose was given or the intravenous infusion was begun and 0.5, 1, 1.5, 2, 4, 6, 8, and 12 hours afterward.

Whole-blood samples were placed in tubes treated with heparin and analyzed while fresh or stored at −80°C until assayed. High-performance liquid chromatography of cyclosporine was performed according to a modification of the method of Lensmeyer and Fields.¹⁷ One-milliliter aliquots of whole blood were diluted with 2 ml of water:acetonitrile (70:30 by vol) and centrifuged (2500 RCF for 20 minutes at 4°C). The supernatants were applied to Bond Elut cyanopropyl columns (Analytic-Chem International, Harbor City, Calif.) and extracted in a vacuum. Columns were washed with 2 ml of 0.5 M acetic acid:acetonitrile (80:20) and then with 0.1 ml of 0.5 M acetic acid:acetonitrile (60:40). Cyclosporine was eluted in 0.6 ml of acetonitrile, which was evaporated under a stream of air at room temperature. The samples were reconstituted with 200 μ1 of the mobile phase, of which 60 μ1 was injected.

A modular chromatography system (Waters Division, Millipore, Milford, Mass.) was used with a guard column (5 cm by 4.6 mm) packed with Permaphase ETH (Dupont, Wilmington, Del.) and a Zorbax cyanopropyl analytical column (25 cm by 4.6 mm, Dupont) maintained at 58°C. The mobile phase was acetonitrile:water (50:50) at a flow rate of 1 ml per minute, with detection at 214 nm. The peak height and area were computed and compared with an internal reference to cyclosporin D, and the concentration was computed from a standard curve of authentic cyclosporine (cyclosporin A) provided by Sandoz (East Hanover, N.J.).

The fractional availability of orally administered cyclosporine was computed as follows: fractional availability = (AUC_oral/(3 × AUC_intravenous), where AUC_oral is the area under the concentration curve after oral administration, and AUC_intravenous is the area under the concentration curve after intravenous administration, multiplied by 3 to compensate for the difference in the doses.

The areas under concentration curves were computed by the trapezoidal method from the whole-blood concentrations.⁸ The zero-order terminal clearance rate was determined as the slope of the line described by the concentrations 6 to 12 hours after the intravenous dose, as estimated by least-squares regression. The values for the area under the concentration curve were corrected by subtracting the contribution predicted by the zero-time value and the zero-order terminal clearance rate.

Statistical Analysis

Cyclosporine doses expressed as milligrams per day and as milligrams per square meter per day were plotted against residual bowel length, the terminal clearance rate, age, height, and weight. After correction of the dose for body-surface area, the oral cyclosporine doses were converted to log(dose) before regression analyses were performed because they demonstrated marked kurtosis when plotted against age or any variable related to body size. A microcomputer-based program of least-squares regression was used to establish possible linear relations between the dependent and individual independent variables. Then, multiple regression analyses were performed that included each dependent and all independent variables. The significance of regressions was determined by the F test. The significance of the differences between means was determined by Student's t-test for unpaired and paired populations as appropriate. The null hypothesis was rejected at a P value of less than 0.05. Data are presented as means ±SD except where indicated.

The mean (±SD) dose of intravenous cyclosporine required by the 56 children evaluated was 69±10 mg per day, 5.9±3.0 mg per kilogram per day, or 140±67 mg per square meter per day. The required oral dose was 461±206 mg per day, 43.2±35.2 mg per kilogram per day, or 944±672 mg per square meter per day. The ratio of the oral to the intravenous required dose was 9.5±10.1 (range, 1.67 to 50). The mean functional residual bowel length in 53 children was 341±49 cm and ranged from 217 to 450 cm. The results of liver-function tests in 56 children were as follows: total bilirubin, 15.2±7.0 μmol per liter (0.89±0.41 mg per deciliter), with a range of 3.4 to 34.2 μmol per liter (0.2 to 2.0 mg per deciliter); conjugated bilirubin, 2.4±2.9 μmol per liter; alanine aminotransferase, 60±43 IU per liter; and alkaline phosphatase, 323±159 IU per liter.

Table 1. Table 1. Pharmacokinetics of Cyclosporine.*

Pharmacokinetic studies were performed in 17 children and 6 adults. The results are shown in Table 1. The fractional availability of oral cyclosporine in the children, which ranged from 0.3 to 28.2 percent, averaged about one third of that in adults, which ranged from 9.8 to 34.5 percent (P = 0.002). The rate of terminal clearance of cyclosporine was no greater in children than adults. In eight children, the pharmacokinetic studies were repeated after three to six months. The fractional availability of cyclosporine increased in all patients between measurements (from 7.9±8.8 to 14.0±11.7 percent; P = 0.05), but there was no change in the rate of terminal clearance (from 0.18±0.10 to 0.15±0.07 ng per milliliter per minute; P not significant).

Factors Contributing to the Cyclosporine Requirements of Children

Figure 1. Figure 1. Effect of Intestinal Length on the Oral Dose of Cyclosporine Required by 53 Children Who Had Received Liver Transplants.

In Panel A, the total oral daily doses of cyclosporine are plotted against the length of functional residual bowel. The dose declines as bowel length increases (r = −0.36, P<0.01). In Panel B, the oral cyclosporine doses, corrected for body size, are plotted against bowel length. The dose decreases rapidly with increasing bowel length. In Panel C, the data are plotted as log(oral dose) against bowel length. Least-squares linear regression analysis demonstrated an association between dose and bowel length (r = −0.77, P = 0.0001).

The required intravenous dose increased as all variables related to body size increased. The dose was significantly related to age (r = 0.75, P = 0.0001), weight (r = 0.81, P = 0.0001), height (r = 0.79, P = 0.0001), and the length of the bowel (r = 0.71, P = 0.0001). This indicates that bigger children need larger doses of cyclosporine, as expected, and provides a rationale for correcting the dose according to the patient's size. In contrast, only bowel length was found to be significantly related to the oral dose: there was a decline in dose with increasing functional bowel length (r = −0.36, P<0.01; Fig. 1A). There was a slight but not significant decrease in the dose with increasing age (r = −0.20), height (r = −0.24), and weight (r = −0.16) — findings that are all the opposites of the expected relations. These data suggest that although body size is the chief determinant of the required intravenous dose of cyclosporine, the required oral dose is possibly determined by a set of factors, among which bowel length is central.

We further analyzed the drug requirement after correction for changing body size by expressing it as milligrams per square meter of body-surface area per day. The intravenous doses were normally distributed and not significantly related to age (r = 0.13), weight (r = 0.16), height (r = 0.22), or bowel length (r = 0.21). This indicates that the correction for body-surface area adequately compensated for the effect of increasing size on the dose.

Table 2. Table 2. Multiple Regression Analyses of the Relation of Log(Cyclosporine Dose) to Independent Variables.

In sharp contrast, the required oral dose decreased with increasing patient size. The oral cyclosporine dose plotted against functional bowel length is shown in Figure 1B. As shown in Figure 1C, the log(dose) was inversely related to bowel length (r = −0.77, P = 0.0001). It was also inversely related to age (r = −0.68, P = 0.0001), height (r = −0.74, P = 0.0001), and weight (r = −0.65, P = 0.0001). When we corrected the oral dose for the effects of body size on the amount of drug required by expressing it as a ratio of the intravenous dose, the results were similar. The log(ratio) was related to bowel length (r = −0.68, P = 0.0001), age (r = −0.62, P = 0.0001), height (r = −0.71, P = 0.0001), and weight (r = −0.59, P = 0.0001). Multiple regression analysis including bowel length, height, and weight as covariates demonstrated a significant dependence of the oral dose on these factors (multiple r = 0.77, P = 0.0001), but as shown in Table 2, only bowel length had an independent effect on the required dose. These findings confirm that bowel length is the principal determinant of the requirement for orally administered cyclosporine in children.

Figure 2. Figure 2. Log(Oral Cyclosporine Dose) Plotted against the Terminal Plasma Clearance Rate in 17 Children.

There was a linear correlation between the two variables (r = 0.57, P<0.02).

The pharmacokinetic studies demonstrated that the rate of drug clearance is another determinant of the dose requirement. As expected, there was a close relation between the oral cyclosporine dose and the fractional availability of oral cyclosporine in the children (r = −0.80, P = 0.0001). The log(oral dose) was directly related to the rate of terminal clearance (r = 0.57, P = 0.017), as shown in Figure 2. There were no age-related effects that could explain the large doses of orally administered cyclosporine required by small children. The terminal clearance rate was not related to age, bowel length, height, or weight (P not significant for all analyses). The rate of terminal clearanee is therefore an individual, but not group-related, determinant of the dose requirement.

Multiple regression analysis was performed to assess the contributions of the independent variables (bowel length, terminal clearance rate, height, and weight) to the oral cyclosporine dose. This analysis involved the 14 children for whom estimates of both bowel length and pharmacokinetics were available. The simple linear relation between the log(dose) and bowel length (r = −0.79, P = 0.0007) and the log(dose) and the terminal clearance rate (r = 0.55, P = 0.04) was maintained in this group. Simple linear regression also demonstrated that the log(dose) was related to height (r = −0.71) and weight (r = − 0.57), but these variables, of course, are related to bowel length. The results of the multiple regression analysis are given in Table 2. Only bowel length and the terminal clearance rate were independently related to the oral dose. The equation for the relation between dose and these two independent variables was log(dose in milligrams per square meter per day) = 3.429 − 0.002 (bowel length in centimeters) + 1.264 (clearance rate in nanograms per milliliter per minute) (multiple r = 0.69, P = 0.001).

Two factors determine the functional bowel length: body size and the amount of bowel excluded or removed at surgery. Among the 46 patients who had cholangioenterostomies, the median fraction of bowel removed and excluded was 12 percent. The seven children in whom primary-duct anastomoses were performed required significantly lower doses of cyclosporine than those who had Roux-en-Y anastomoses (408±115 vs. 1012±683 mg per square meter per day, P<0.04), although they were somewhat older (7.51±4.29 vs. 4.26±4.53 years, P not significant). The five infants who had had the most bowel removed (averaging 27 percent of the estimated bowel length) required 1682±646 mg of cyclosporine per square meter per day. Multiple regression analysis demonstrated that the amount of intestine not available for cyclosporine absorption (expressed as a percentage of the expected length of intestine removed or excluded) was an independent factor contributing to the log(dose) of cyclosporine, with height and weight also included as independent variables to control for body size (multiple r = 0.763, P = 0.001, df = 52; for percentage of bowel excluded: slope = 0.023; 95 percent confidence interval, 0.001 to 0.045; t value = 2.124; P<0.04).

Children, particularly infants, require much higher doses of orally administered cyclosporine than adults after liver transplantation. Our data demonstrate that bowel length and the rate of terminal clearance of the drug are important variables in determining the dose required to maintain therapeutic blood levels and that, of the two factors, bowel length is probably the main determinant of the large difference between the two populations.

A major problem in analyzing the data was the way the drug requirement was expressed. The expression of the required cyclosporine dose as milligrams per square meter per day, although widely accepted as a method of correcting drug doses for body size, produced statistical complexity, in that the denominator included both height and weight and was directly related to age. Therefore, all variables related to body size might have been inversely related to dosage simply because of the method of expression. The first step in assessing the factors contributing to the dose of cyclosporine needed to maintain therapeutic blood levels was to perform linear regression analysis with the total daily dose (uncorrected) as the dependent variable plotted against each of the independent variables (height, weight, age, and functional bowel length). Empirically, the dose, whether administered intravenously or orally, would be expected to increase with increasing body size, a relation that was found to be true only for the intravenous dose.

Since the principal factor contributing to increasing bowel length up to about four years of age is increasing body size,¹⁴ growth in general would result in conflicting effects on the amount of orally administered cyclosporine required; increasing body size would cause the dose to increase, and increasing bowel length, by improving absorption, would cause it to decrease. The relative magnitudes of these effects would determine the overall effect of growth on the cyclosporine dose. The finding that the uncorrected cyclosporine dose decreased with increasing body size suggested that bowel length was the predominant variable related to body size that affected the required oral dose of cyclosporine. It was valuable to perform similar analyses with corrected doses, because the daily dose per square meter, not the total daily dose, is what is so much larger in small children after liver transplantation. Furthermore, there is no a priori reason that the dose should decrease with increasing body size or age, if not by improved absorption or reduced clearance. The argument for the dependence of the required dose of orally administered cyclosporine on the functional gut length was strengthened by the finding of a significant inverse correlation between the oral dose and gut length, whereas the intravenous dose was unrelated to factors that change with body size. Furthermore, expressing the oral requirement as a ratio of the required intravenous dose, which should have corrected for all effects on drug distribution and metabolism that were related to body size, produced similar results. Therefore, it seems that the large oral dose required by small children is mainly related to poor absorption, which in turn is probably related to bowel length and absorptive surface area.

Another difficulty in completing these studies was the estimation of bowel length, which must be considered to be at best a rough estimate. Measuring intestinal length is difficult, and in situ measurements cannot readily be compared with those obtained at autopsy. In addition, in our calculations we accepted some measurements made by other surgeons during previous operations for biliary atresia. Although every effort was made to be accurate in the estimate, there were some errors in measurement. Despite this imprecision, a very strong correlation between residual bowel length and cyclosporine dose cannot be denied.

Although mostly the result of the relation between bowel length and age, the required oral dose can also be affected by surgical bowel removal and exclusion. During liver transplantation in children, biliary reconstruction usually involves the formation of a Roux-en-Y cholangioenterostomy. Surgical teaching suggests that biliary and afferent limbs measuring between 30 and 40 cm in length are necessary to prevent ascending biliary infection. Age has not been a factor in these considerations. Our studies in rats have shown that neither limb of the Roux-en-Y is available for cyclosporine absorption because bile is critical for its absorption.^{3 4 5} Standard construction of the Roux-en-Y loop may exclude 20 to 40 percent of a small infant's intestine from cyclosporine absorption. Superimposed on this may be the effect of a previous operation for biliary atresia, the most common indication for liver transplantation in infants.¹⁸ Recent modifications of the Kasai portoenterostomy include long and complex enterostomy loops,¹⁹ which often cannot be dissected for use at the time of liver transplantation. As a result of these surgical procedures, many young infants lose the use of large portions of what are already short intestines, further restricting cyclosporine absorption. For example, one infant had 133 cm of bowel (38 percent of the expected bowel length) removed or excluded, mostly during surgical care before transplantation. His cyclosporine dose was 2350 mg per square meter per day. Because of this huge requirement, surgical revision of his intestinal anatomy was undertaken, and the amount of bowel excluded was reduced to 60 cm. As a result, the cyclosporine dose dropped to 733 mg per square meter per day. Therefore, careful conservation of intestinal length before and during liver transplantation should be a surgical goal in the treatment of infants with biliary atresia. This detail is much less important in the treatment of larger children and adults.

The rate of drug clearance is also related to the dose, but our studies failed to show any statistically significant difference in clearance between children and adults. There is some potential for a beta statistical error in our study, considering the limited number of adult subjects. However, it is unlikely that the small differences that could be detected with a larger sample would account for the large difference in the dose between groups. We conclude that the rate of cyclosporine clearance is an important variable that contributes to individual differences in the required dose, but it does not represent a particular risk for children.

Our observations have important implications for the performance of liver transplantation. First, it is difficult to administer enough drug to some infants to achieve a therapeutic level, placing these patients at risk for rejection. Second, the transition from intravenous to oral administration can be a problem because of the difficulty in estimating the oral dose for children. For adults, multiplying the intravenous dose by 3 provides a close approximation of the oral dose.^{6 7 8} Our results can be used to estimate the oral dose for infants when height and the length of bowel that was excluded are known. The required doses ranged from 1.7 to 50 times the intravenous dose. Finally, minimizing the loss of intestine before and during liver transplantation in infants can lessen the need for cyclosporine, which is an important part of the cost of liver transplantation.