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PHARMACOKINETICS/SPECIAL POPULATIONS |
From Ohnishi Hospital, Saitama, Japan (Dr Ohnishi) and Mitsubishi Tanabe Pharma Corporation, Osaka, Japan (Dr Kawaguchi, Mr Nakajima, Mr Mori, Mr Ueshima).
Address for correspondence: Atsuhiro Kawaguchi, PhD, Mitsubishi Tanabe Pharma Corporation, 2-2-6 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8405, Japan; e-mail: Kawaguchi.Atsuhiro{at}mf.mt-pharma.co.jp.
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ABSTRACT |
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 TOP ABSTRACT STUDY DESIGNMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: Recombinant human serum albumin • Pichia pastoris • pharmacokinetics • bioequivalence • liver cirrhosis
Therapeutic use of plasma-derived human serum albumin (pHSA) has long been carried out, employing improved purification techniques, including inactivation and elimination of known viruses.5 Plasma-derived human serum albumin sourced from human blood still poses the risk of infection from contaminated unknown viruses, with another issue being extremely limited supplies. Extensive research on substitutes for pHSA, using Bacillus subtilis,6 Saccharomyces cerevisiae,7,8 and Pichia pastoris9 have yet to produce any clinical results with commercial applications, leaving a safe and mass-producible product much in demand.
Recombinant human serum albumin (rHSA) is a high-purity, mass-produced product derived from methylotrophic Pichia pastoris, developed using recombinant DNA technology, employing a successfully established high-producer strain and an originally developed high-level purification system.10-12 Structural and physicochemical properties revealed rHSA identical to pHSA,13-16 and pharmacokinetic profiles were deemed similar in animal studies.17,18 Clinically, rHSA demonstrated efficacy and safety equivalent to pHSA during a phase III study in patients with ascites due to liver cirrhosis.19 Other clinical studies indicated efficacy and safety in hemorrhagic shock, nephrosis, burns, acute abdomen, poor-risk before surgery, and post-open heart surgery patients.20
In this study, we evaluated the pharmacokinetic profiles of rHSA in liver cirrhosis patients in comparison to pHSA using serum albumin concentrations.
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STUDY DESIGN |
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TOPABSTRACTSTUDY DESIGNMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Patients
Prior written informed consent was obtained from 22 patients with liver cirrhosis, who fulfilled the inclusion and exclusion criteria and enrolled into the study, which was conducted from September 2002 to March 2003.
Liver cirrhosis patients without ascites or edema, aged 20 to 75 years inclusive, who could be admitted for the study period were eligible for participation.
The following exclusion criteria were applied: patients who received blood transfusions or plasma protein products within 3 months before screening; a total bilirubin of 3.0 mg/dL or more; renal dys-function manifested by serum creatinine levels of 4.0 mg/dL or more; prothrombin time (PT) of less than 50%; hepatocellular carcinoma (HCC) verified by tumor embolism in the portal vein (main trunk or primary/secondary branch), inferior vena cava, or main trunk of the hepatic vein; cardiac functions rated class III or IV according to the New York Heart Association (NYHA) classification; history of shock or hypersensitivity to any ingredient of pHSA products; patients scheduled to undergo invasive testing or treatment for underlying diseases or complications (eg, HCC, gastric/esophageal varices) during the study period; hepatic encephalopathy (coma scale class II or more severe) at receipt of informed consent; a positive prick test result for rHSA; female patients who were pregnant, nursing, could be pregnant, or intended to become pregnant during the study period; and other patients deemed ineligible for participation by the investigators.
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MATERIALS AND METHODS |
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TOPABSTRACTSTUDY DESIGNMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Assignment and Sample Size
To minimize intergroup bias in baseline values of serum albumin concentrations, assignment was dynamically conducted, using only this factor, by the minimization method according to concentrations measured 1 day prior to administration (day-1) by a third party (Bellsystem24 Inc., Tokyo, Japan). Multiple factors were not incorporated, as the study population was small. Because serum albumin concentration is the only objective parameter used to assess the primary endpoint, an open-label design was selected.
Half-life (t
) of albumin is known to be long, 15 to 19 days,21-23 thus parallel-group was chosen versus crossover as the study design. Sample size was estimated at 10 patients per group according to the Guidelines for Bioequivalence Testing of Generic Drugs24 and set at 11 patients, taking dropouts into consideration. Serum albumin concentrations observed in a previous phase III study in patients with ascites due to liver cirrhosis and the data collected until day 7 were used to calculate the area under the curve (AUC) in each group. Based on the AUC (g·hr/dL) (497.5 ± 62.1, 47 [mean ± SD, number of subjects] in the rHSA group, and 514.4 ± 69.3, 45 in the pHSA group), power was calculated when 90% confidence interval (CI) fell within 80% to 125% of the AUC, and indicated 10 patients as sufficient to provide 90% power to detect bioequivalence.
Prohibited Concomitant Medications and Therapies
Concomitant use of the following medications and therapies was prohibited: blood transfusion or administration of plasma protein products, other investigational products, invasive testing, or treatment for HCC (excision, percutaneous ethanol injection therapy [PEIT], transcatheter arterial embolization [TAE] therapy, transcatheter arterial injection [TAI] of carcinostatics, microwave coagulation therapy [MCT], high frequency coagulation therapy [radiofrequency wave], transcatheter contrast imaging, etc), sclerotherapy for gastric or esophageal varices, ligation of varices, or endoscopy from receipt of consent to day 8. Concomitant use of diuretics was permitted only at a stable dosage and dose regimen during the study period.
Observation, Examination, and Assessment Items
Patients were admitted on day -1, treated with rHSA or pHSA from days 1 to 3, and observed until discharge on day 8. Serum albumin concentrations were measured using the bromcresol green (BCG) method at Mitsubishi Chemical Medience Corporation (Tokyo, Japan), and concentrations monitored on day -1, immediately predose and postdose during the treatment phase (days 1-3), and daily for the observation period (days 4-8). To assess safety, patients were observed for any clinical signs and symptoms from days 1 to 8. In addition, laboratory tests (hematology and urinalysis) were conducted on days -1, 4, and 8, and vital signs recorded predose and postdose during the treatment phase and once daily during the observation period.
Statistical Analysis
Primary analysis was performed on AUC using the trapezoidal method from day 1 predose to day 8 (168 hr). Since Cmax of intravenous HSA administrations may not be evaluated, as with oral agents, AUC was the other major parameter that could be considered a primary endpoint for bioequivalence and Cmax another major parameter calculated as reference data. If the 90% CI for the difference in mean log-transformed AUC fell within log (0.8) and log (1.25), both products were determined as bioequivalent.24 Analyses were performed using the statistical software SAS Version 8.2 (SAS Institute Inc., Cary, North Carolina).
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RESULTS |
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TOPABSTRACTSTUDY DESIGNMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Serum albumin concentrations on day -1 were 3.34 ± 0.43 g/dL (mean ± SD) in the rHSA group, and 3.31 ± 0.43 g/dL in the pHSA group, suggesting patients were equally assigned to treatment groups by the minimization method (Table I). Background characteristics, such as age and weight, excluding gender, were comparable between the 2 groups. An intergroup gender difference was found between the all-male rHSA group and the 7-male/4-female pHSA group.
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Analyses of Serum Albumin Concentrations and Bioequivalence
In both groups, changes in plasma concentrations showed a similar pattern after each intravenous dose (repeated for 3 days) and after termination of administration, with similar serum albumin concentrationtime profiles (Figure 1).
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Geometric mean AUC0-168hr (g·hr/dL) was 637.12 in the rHSA group, and 635.93 in the pHSA group. The difference in AUC0-168hr was 0.0008 ± 0.0191, and 90% CI ranged from -0.0321 to 0.0337 (92.9%-108.1%), falling within the acceptable bioequivalence range, -0.0969 to 0.0969 (log [0.8] to log [1.25]), indicating bioequivalence between rHSA and pHSA (Table II).
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Geometric mean Cmax (g/dL) was 4.16 in the rHSA group, and 4.19 in the pHSA group. The difference in Cmax was -0.0030 ± 0.0174, and 90% CI ranged from -0.0330 to 0.0269 (92.7%-106.4%) (Table II).
Safety
Three adverse events were observed in 2 patients from the pHSA group among the 22 patients examined. No serious or allergic adverse events were observed in either group. A single event of insomnia was reported in a male patient from the pHSA group and was considered moderate, resolved by day 8 with treatment using an antianxiety drug; it was regarded as being unrelated to the investigational product and caused by the environmental change from hospitalization. Two abnormal laboratory changes observed in 1 male patient from the pHSA group (increase in BUN and serum creatinine levels: BUN values on days -1, 4, and 8 were 19, 43, and 52 mg/dL, respectively, and serum creatinine levels were 1.0, 2.4, and 1.9 mg/dL, respectively) being treated concomitantly with 2 diuretic agents were possibly related to the investigational product. Exogenous albumin increased total serum albumin concentrations that possibly enhanced the effect of the diuretic agents, causing transient intravascular dehydration. These abnormalities almost returned to baseline levels during the follow-up period.
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DISCUSSION |
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TOPABSTRACTSTUDY DESIGNMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Human serum albumin doses are decided according to patient status. Patients presenting with hypoalbu-minemia accompanied by recurring ascites are generally administered albumin at 8 to 10 g/L of abdominal fluid removed after paracentesis of more than 4 to 5 L.25 In this study, bioequivalence of rHSA and pHSA was evaluated using a clinically accepted dose of 25 g/day. Multiple dosing (3 days) was chosen to facilitate calculation of AUC to compare pharmacokinetic profiles between the groups. Repeated administration is a clinically accepted form of albumin usage. According to the Japanese Guidelines for Bioequivalence,24 repeated administration may be used when comparing bioequivalence if clinically practiced.
The number of pharmacokinetic samples collected and sampling times were projected based on simulation results of research conducted by Kato et al26 who analyzed metabolic movement of albumin in the liver of cirrhosis patients. In addition, the number of samples collected was reduced and limited to a bare minimum, as these subjects were already presenting with liver cirrhosis. As t for serum albumin is long, daily sampling was considered sufficient to calculate AUC. Hence, serum albumin concentrations monitored immediately predose and postdose during the treatment phase (days 1-3) and every 24 hours during the observation period (days 4-8) were used to calculate AUC.
Minimization was aimed at ensuring treatment groups were allocated with respect to predefined serum albumin concentrations on day -1 and achieved this balance from results obtained. Stepped homologous increases in serum albumin concentration-time profiles were observed in both the rHSA and pHSA groups after administration from days 1 to 3, and parallel concentration-time profiles were maintained till day 8 with a small linear decrease (Figure 1). The difference in mean log-transformed serum albumin AUC0-168hr was 0.0008 ± 0.0191 (90% CI: 92.9%-108.1%), indicating bioequivalence between rHSA and pHSA (Table II). Cmax was the other major parameter used in the investigation of bioequivalence. In this study, Cmax was calculated as reference data since HSA was administered intravenously. The 90% CI for the difference, from 92.7% to 106.4%, lies within the bioequivalence range.
On the other hand, it is difficult to estimate other pharmacokinetic parameters such as t accurately in this study, as albumin is an endogenous protein with a long t, and a small rise of approximately 1.0 g/dL in concentration after multiple dosing is also insufficient. As reference data, we calculated the t using the least square method from day 3 postdose to day 8. Since t of 2 subjects (77.5 and 209.2 days) was 3 times larger than the reference data (15 to 19 days21-23), this data was excluded from this analysis. The t was 31.1 ± 7.0 day (n = 10) in the nHSA group, and 29.6 ± 6.2 (n = 10) in the rHSA group. We also estimated t from day 3 postdose to day 7. It is better to calculate t excluding data on day 8 because serum albumin concentration between day 7 and day 8 are close and plateaued (Figure 1). The t was 23.8 ± 8.3 day (n = 11) in the nHSA group, and 23.9 ± 7.2 (n = 10) in the rHSA group. One t value in the rHSA group was excluded because the calculated t was negative. The differences between these values were not remarkable. To evaluate the t of albumin accurately, a long-term radioisotope study would be needed to assess the elimination phase of albumin.
Patient demographics and baseline characteristics generally showed no remarkable bias, including baseline serum albumin concentrations, except for gender. Although this difference existed between the rHSA (11 male) and pHSA (7 male; 4 female) groups, in post-hoc analysis (P = .0902, Fisher), intergroup analysis exclusively among male patients provided a similar 90% CI (93.3%-111.9%) compared to primary analysis results. Since weight differs by gender, exploratory bioequivalence analysis using adjusted weight as a covariate showed no significant differences between the groups (90% CI: 92.7%-108.4%).
In addition, baseline adjustments were made for the AUC0-168hr and Cmax as serum albumin is an endogenous protein. The following parameters were calculated after subtracting immediately predose (baseline) values. Baseline-adjusted geometric mean AUC0-168hr (g·hr/dL) was 99.96 and 106.74 in the rHSA and pHSA groups, respectively, while difference in mean AUC0-168hr was -0.0285 ± 0.0333 (90% CI: 82.0%-106.9%). Baseline adjusted geometric mean Cmax (g/dL) was 0.96 and 1.04 in the rHSA and pHSA groups, respectively, and difference in mean Cmax was -0.0383 ± 0.0276 (90% CI: 82.0%-102.2%). These differences do not contradict bioequivalence. Overall, both rHSA and pHSA were safe and well tolerated in the study. In the pHSA group, insomnia (unrelated) was reported in 1 patient, and increases in BUN and serum creatinine levels (possibly related) were observed in 1 patient. No adverse events of clinical concern were observed in either treatment group. Examination of safety data obtained from a previous phase III study resulted in little adverse reaction.19
Recombinant human serum albumin is a high purity, virus-free albumin product based on recombinant DNA technology without using blood-derived components in the manufacturing process.10-12 It is successfully being mass produced on a commercial basis, suggesting a stable supply can be realized without using finite human blood, and that limitation on plasma procurement can be overcome.
These results indicate bioequivalence between rHSA and pHSA, and can be considered a possible alternative in the clinical environment.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTSTUDY DESIGNMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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