Gaucher disease has become a prototype genetic disease for the development of prenatal diagnosis [6], genotype-phenotype correlations [2, 7, 8], and effective therapy [9-13]. On the basis of the clear efficacy of targeted enzyme therapy [10], recent studies [9, 12, 14, 15] in more than 90 patients have established that regular infusions of enzyme purified from placenta (alglucerase [Ceredase; Genzyme Corp., Cambridge, Massachusetts]) lead to regression of the clinical manifestations of Gaucher disease. In addition, antibody-mediated and non-antibody-mediated adverse events have occurred in only 5% to 7% of treated patients [15, 16]. Ceredase is a commercial form of placenta glucocerebrosidase that has been modified for targeting mannose receptor sites on macrophages and other cells [17, 18]. A theoretical limitation to the use of Ceredase is the remote possibility of infective contaminants in the preparation from human placenta. A practical limitation is the finite availability of acceptable placentae. For each patient, approximately 10 to 12 tons of placentae or about 50 000 per year are needed as source material for Ceredase.

Enzyme produced by heterologous expression of human complementary DNA (cDNA) for glucocerebrosidase in eukaryotic cells could eliminate both of these limitations. To determine the efficacy of recombinant glucocerebrosidase, we did a randomized, double-blind, parallel trial with mannose-terminated glucocerebrosidase (alglucerase, Ceredase) from human placenta and the human enzyme that is produced in Chinese hamster ovary cells and deglycosylated to expose mannose residues in the oligosaccharide chains (imiglucerase, Cerezyme [Genzyme Corp.]).

Methods

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Thirty patients with non-neuronopathic type 1 Gaucher disease were entered into the trial after consent was obtained. Deficiency of glucocerebrosidase (5% to 15% of mean normal activities) was shown by natural or artificial substrates in peripheral blood mononuclear cells, cultured skill fibroblasts, or lymphoblastoid cell lines obtained from each patient [19, 20]. Inclusion criteria for the study were as follows: 1) enzymatically confirmed glucocerebrosidase deficiency; 2) patient age between 2 and 75 years; 3) an intact, enlarged spleen; 4) a hemoglobin level at least 10 g/L less than the lower limit of normal; and 5) in women, a willingness to avoid pregnancy during the trial. Exclusion criteria were as follows: 1) inability to comply with study requirements; 2) previous receipt of any form of glucocerebrosidase; 3) total splenectomy; 4) a concurrent major medical disorder, such as active infectious disease or substance abuse; and 5) positive serologic response to hepatitis B surface antigen or human immunodeficiency virus (HIV) type 1, or both. The patients had moderate to severe Gaucher disease.

The study was a double-blind, parallel trial with random assignment to Ceredase or Cerezyme. We categorized randomization into three groups according to patient age: 1) younger than 12 years; 2) 12 to 17 years; and 3) older than 17 years. In each study center, patients were independently randomly assigned in blocks by age and were assigned study numbers. All study personnel except the pharmacist at each institution were blinded to the allocated treatment. No children younger than 12 years were enrolled in the study. Of the 30 patients enrolled, 17 were male and 13 were female (age range, 12 to 69 years). Of the 7 patients between 12 and 17 years of age, 4 received Ceredase and 3 received Cerezyme. Twenty-three patients older than 17 years were enrolled; 11 of them received Ceredase and 12 received Cerezyme. The groups of patients treated with Ceredase or Cerezyme did not differ in sex distribution, age, weight, or height. All patients received Cerezyme or Ceredase at a dose of 60 U/kg body weight once every 2 weeks for 9 months. Complete hematologic and clinical chemistry data were available for this period. At this dose, complete data for hepatic and splenic volumes were available for all patients for the first 6 months. Hepatic and splenic volumes were evaluated in the 16 patients from the Mt. Sinai School of Medicine at 9 months, just before a dose-reduction study began. The hepatic and splenic volumes of the patients from the National Institutes of Health (NIH) were evaluated at 12 months. We did not include these data in our report. We did analyses of variance and other statistical analyses using Systat software (Systat Inc., Evanston, Illinois).

In addition to physical examinations, clinical laboratory studies were done to monitor both therapeutic efficacy and potential toxic effects, including total serum acid phosphatase levels, angiotensin-converting enzyme levels, serum bilirubin levels, hemoglobin levels, platelet counts, peripheral blood leukocyte counts, and serum iron and clotting studies. At baseline and at study completion, hepatitis B surface antigen assay; HIV-1 serologic testing; serum protein electrophoresis; and complement C3, C4, and CH-50 studies were done. Hepatic and splenic volumes were estimated by computed tomography (at Mt. Sinai School of Medicine) [12, 15] or magnetic resonance imaging (at NIH) [21]. We calculated the increases over normal volumes by assuming that the hepatic and splenic masses (1 g/mL density) were 2.5% and 0.2% of body weight, respectively [22]. To avoid biasing the results because of the nearly universal weight gain in treated patients, we averaged the body weight of adults over the study period. In children (patients younger than 18 years), we calculated the hepatic and splenic volumes on the basis of body weight at each time point to allow for growth.

We monitored the formation of antibodies to natural or recombinant glucocerebrosidases every 3 months by radioimmunoprecipitation assay [16]. Adverse events were monitored at each infusion. Before each infusion, we also obtained a history of intra-infusion adverse events.

Ceredase was supplied as a clear liquid solution stored at 4 °C, solubilized in the presence of albumin. Cerezyme was purified from culture media of Chinese hamster ovary cell clones that contained numerous copies of the human glucocerebrosidase cDNA. Carbohydrate removal to expose core mannose moieties was done by the sequential exoglycosidase treatment used to produce Ceredase. The amino acid sequences of glucocerebrosidase in Ceredase and Cerezyme were identical except for a single amino acid substitution of a histidine for a natural arginine at position 495 in the latter. The lack of effect of this amino acid change on the catalytic function of glucocerebrosidase has been documented [23]. Cerezyme was supplied as a lyophilized powder containing mannitol, sodium citrate, and polysorbate 80. Cerezyme as lyophilized powder was stored at 4 °C until use. Immediately before administration, Cerezyme was reconstituted with sterile water to a concentration of 40 U/mL. Ceredase or Cerezyme stocks were diluted in 0.9% NaCl just before infusion. During the study, the patients were weighed at each infusion, and the total dose to be administered was adjusted to 60 U/kg on the basis of the current weight. A unit of enzyme activity (U) is defined as the amount of enzyme required to cleave 1 µmol of p-nitrophenol-ß-d-glucopyranoside per minute.

Results

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Hematologic Findings

The effects of Ceredase and Cerezyme infusions on hemoglobin levels by 6 and 9 months are shown in Tables 1 and 2. Neither the mean initial hemoglobin level (approximately 107 g/L) nor changes in hemoglobin levels differed between the two treatment groups. During the first 6 months of therapy, hemoglobin levels increased by a mean of 17.1 g/L. In patients with hemoglobin levels less than 120 g/L, 54% and 30% of the patients receiving Ceredase and Cerezyme, respectively, achieved this or a greater level by 6 months. Sixty-nine percent and 40%, respectively, of patients receiving Ceredase and Cerezyme achieved these levels by 9 months. The rates at which hemoglobin levels increased by 10 and 15 g/L were also similar. An average of 92 days was required for the hemoglobin level to increase 10 g/L in the Ceredase group, whereas an average of 77 days was needed in the Cerezyme group. For the Ceredase and Cerezyme groups, hemoglobin levels increased 15 g/L in 113 and 125 days, respectively. None of the above differences was significant (P > 0.2). In both treatment groups, we observed lesser degrees of response in patients with higher initial hemoglobin levels. However, this conclusion was strongly influenced by two patients who had initial hemoglobin levels less than 90 g/L and large responses to treatment, that is, an increase of more than 30 g/L during the first 6 months of therapy.

All patients in the study had thrombocytopenia (mean platelet count, approximately 71.5 x 10⁹/L). About half (7 of 15) of the patients in each group had increases in platelet counts of 20% and 40% or more during the 6- and 9-month treatment periods, respectively (Table 2). These responses to therapy did not differ between the Ceredase and Cerezyme groups. In each group and in the entire study group, the platelet responses to therapy were unrelated to the initial level of thrombocytopenia.

Influence of Visceral Organ Volumes on Hemoglobin and Platelet Responses

The influences of the initial hepatic and splenic volumes on hematologic responses are summarized in Table 3. In these analyses, we noted no significant differences between the distributions of initial organ volumes or hematologic responses with either Ceredase or Cerezyme [data not shown]. On the basis of initial hepatic and splenic volumes, we could discern three types of patients: 1) those with hepatic volume less than 1.4-fold and splenic volume less than 19-fold the normal volume; 2) those with hepatic volume greater than 1.4-fold and splenic volume less than 19-fold the normal volume; and 3) those with hepatic volume greater than 1.4-fold and splenic volume greater than 19-fold the normal volume. No patients had a hepatic volume less than 1.4 times the normal volume or splenic volume greater than 19 times the normal volume. As shown in Table 3, the splenic volume partially influenced hemoglobin and platelet responses to therapy. Because of the large variances, these differences were trends suggesting that hemoglobin response was better and platelet response was poorer in patients with larger spleens.

The results of hepatic and splenic volumes changes in patients receiving Ceredase and Cerezyme at 6 and 9 months are summarized in Table 4 and Figure 1. Initial hepatic and splenic volumes in either treatment group or between centers did not differ (P > 0.2). The overall reductions in hepatic volume at 6 and 9 months were approximately 12.4% and 19%, respectively. The overall splenic volume decreases at 6 and 9 months were 34.7% and 44.6%, respectively. In both groups, the rate of decrease in hepatic and splenic volumes during the 6- to 9-month period was less than the rates obtained during the first 6 months of therapy. The mean decrease in splenic volume during this 3-month period was about 13.5% in either group (P > 0.2). We did not include 14 patients from the NIH study group in the 6- to 9-month interim analyses, but the trends at 12 months were similar.

View larger version (16K): [in this window] [in a new window]   Figure 1. Decreases in splenic volume from initial volume measured at 6 months in the Cerezyme (Group 1) and Ceredase (Group 2) treatment groups. The values are presented as the percentage change from initial volume. The distribution of individual values, median value (horizontal line in boxes), and interquartile range (boxes), are shown. The outer quartile range is indicated by the vertical capped lines.

The initial hepatic and splenic volumes influenced the observed degree of decrease (Table 5). When we used the three groups of patients defined by hepatic and splenic volumes, the differences in decreases in hepatic volume between those with an initial hepatic volume less than 1.4-fold the normal volume and those with an initial hepatic volume greater than 1.4-fold the normal volume were significant at both 6 and 9 months (P = 0.03). For these studies, the greatest changes were in patients with larger initial hepatic volumes (>1.4-fold normal volumes). In patients who had a hepatic volume greater than 1.4-fold and a splenic volume less than 19-fold the normal volume, the influence of organ volume was highly significant (P = 0.007). In patients with the largest organs (hepatic volume >1.4-fold and splenic volume >19-fold the normal volume), the differences were somewhat less significant (P = 0.069). We did not see similar levels of statistical significance with changes in splenic volume. In patients with a hepatic volume less than or greater than 1.4-fold normal volumes, a P value of greater than 0.3 was obtained for all paired groups. However, the trend was still apparent for the splenic volumes, and patients with the largest spleens had the largest decreases.

Changes in Other Indicators of Gaucher Disease

Eleven patients receiving Ceredase and 13 patients receiving Cerezyme had elevated levels of angiotensin-converting enzyme when therapy began. During the 9-month study, 93% of patients receiving Cerezyme and 73% of patients receiving Ceredase had decreases of greater than 30% in serum angiotensin-converting enzyme levels. Fourteen of 15 patients receiving Cerezyme and 12 of 15 patients receiving Ceredase had reductions in total acid phosphatase levels of more than 30%. Other serum enzyme levels, such as those of serum aspartate aminotransferase, serum alanine aminotransferase, and -glutamyl transpeptidase, decreased during therapy. However, none of these values was initially in the abnormal range, which probably indicates minor degrees of fluctuation around the normal level or reflects a reduction in hepatic volume.

Adverse Events

Antiglucocerebrosidase antibodies developed in six patients receiving Ceredase and in three patients receiving Cerezyme during the 9-month study period. Patients in the Cerezyme group developed antibodies by 3 to 6 months. Three patients receiving Ceredase developed antibodies during the first 6 months, and three additional patients became seropositive by 9 months. Three patients positive for antibodies reported dizziness, pruritus, or rash that developed after the enzyme was administered. In no case were symptoms severe enough to discontinue therapy. Diminished therapeutic response was not apparent in patients positive for antibodies.

Discussion

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Until recently, enzyme or protein therapies have been limited to agents that are retained in the plasma space (that is, factor VIII) or have actions at the plasma membrane (that is, insulin). In addition, enzyme therapy for inherited metabolic diseases has been limited by the lack of sufficient amounts from natural sources for evaluation of biochemical or clinical efficacy [24]. The demonstration that intravenous glucocerebrosidase could reverse disease symptoms and signs in patients with Gaucher disease indicates that functional enzymes can be delivered to intracellular sites [9-12, 15]. However, the extraction and purification of glucocerebrosidase from tons of placenta per year per patient indicates practical and theoretical problems associated with this approach to enzyme therapy. These problems include the following: The availability of natural glucocerebrosidase is limited by the number of placentae; a massive number of placentae must be screened for known infectious agents; and purification methods must be developed to ensure freedom from such agents. This latter requirement adds several organic solvent extraction steps to the purification procedure for Ceredase. Each of these steps increases the potential for partial denaturation or conformational changes of the enzyme. Although the potential for contamination of Ceredase by viruses is low, the possibility of abnormal, immunogenic forms of the enzyme is real. In comparison, recombinant glucocerebrosidase is less likely to contain infectious pathogens, thereby permitting more direct and less denaturing purification.

Glucocerebrosidase requires glycosylation to assume an active conformation and consequently cannot be produced in an active state in bacterial systems [25, 26]. In addition, glucocerebrosidase is tightly associated with the lysosomal membrane and is not normally secreted in significant amounts. However, as with other lysosomal proteins [27], massive overexpression of the glucocerebrosidase in Chinese hamster ovary cells can lead to substantial secretion of an active enzyme into the media of the selected clones. These cells retain all the necessary post-translation processing systems for complex carbohydrate modification. The glucocerebrosidase of these cells, which is isolated from spent serum-free media, requires sequential exoglycosidase treatment for the exposure of mannose residues for targeting to the macrophages. Our data show the similar therapeutic efficacy of the enzymes isolated from placenta or from recombinant DNA sources. These findings show that an intracellular protein produced by recombinant methods can be delivered to a subcellular compartment (the lysosomes) and affect clinical reversal of the signs and symptoms of a human-inherited metabolic disease.

Direct comparison of the therapeutic results of Ceredase and Cerezyme shows no significant difference between the enzyme preparations or within the study groups at each institution. The sample studied was highly varied and typical of many patients with moderate to severe Gaucher disease. Such wide variation in clinical symptoms and signs is typical of these patients, but patient involvement in each group was similar. The rates of increases in hemoglobin levels or platelet counts and decreases in visceral organ volumes were essentially identical between the two study groups. As in several other studies, increases in hemoglobin levels have been more rapid than those of platelet counts [9, 10, 15]. In general, the percentage responses in hemoglobin levels were less than the percentage increases in platelet counts, particularly during the 6- to 9-month interval. This probably reflects the fact that approximately 50% of the patients attained near-normal hemoglobin levels during the first 6 months of therapy. In comparison, most patients remained significantly thrombocytopenic throughout the entire study period. Curiously, the volumes of the liver and spleen differentially influenced the responses of hemoglobin levels and platelet counts to enzyme therapy (Table 2). Because of the large variances in responses within each group, statistical significance was not achieved for either hemoglobin or platelet responses as a function of hepatic and splenic volume, but a trend of direct relation was evident at 6 and at 9 months. We saw the largest hemoglobin responses in patients with hepatic volumes greater than 1.4 times the normal volume and those with splenic volumes greater than 19 times the normal volume. Patients with the largest livers and spleens had nearly twice the response in hemoglobin as patients with livers less than 1.4 times the normal volume and splenic volumes less than 19 times the normal volume. We observed the opposite result with platelets during the first 6 months of therapy (Table 2). Patients with the largest livers and spleens had about half the initial platelet response as did those with smaller livers and spleens. This difference nearly disappeared during the subsequent 3 months of therapy.

We noted no differences in the decreases in hepatic volume or splenic volume between the two study groups. The mean decrease of hepatic volume was approximately 12.5% and 20% by 6 and 9 months, respectively. Splenic volume had decreased by about 35% at 6 months and 46% by 9 months. The fact that the initial hepatic volumes did not differ between the two study groups is important in evaluating the results. If the smallest or largest livers had been differentially distributed to one of the two study groups, we could have observed a clear influence on the rate of response. As shown in Table 4, the initial hepatic volume clearly influenced the degree of hepatic volume decrease observed with enzyme therapy. In patients with hepatic volumes less than 1.4 times the normal volume, volume decreased by about 4% and 11% during the first 6 and 9 months of therapy. Patients with hepatic volumes greater than 1.4 times the normal size had responses 2 to 5 times those observed in patients with the smaller livers. This seemed to be independent of splenic volume. Patients with hepatic volumes greater than 1.4 times the normal volume and splenic volumes less than 19 times the normal volume had a response 2 to 5 times greater than those with hepatic volumes less than 1.4 times the normal volume and splenic volumes less than 19 times the normal volume. Although these differences were not highly significant (P < 0.01), the trend was convincing (P = 0.03). Consequently, patients with the smallest livers seem to have the smallest responses. These trends were less convincing with splenic volumes in which similar responses were obtained in all groups. After 9 months of therapy, patients with the smallest livers and spleens had a mean decrease in splenic volume that was nearly 10% less than that of patients with the largest livers and spleens ( –42% compared with –52%)(P = 0.09). These results indicate that the responses to enzyme therapy in Gaucher disease are multivariate and that comparisons between groups of patients should be done in persons with similar degrees of involvement on the basis of quantitative assessment of their clinical status. Our findings provide guidelines for monitoring therapeutic responses in patients with moderate to severe disease.

The greatest difference between the two study groups was the incidence of the formation of antibodies to the administered enzyme. During the 9-month study period, 40% of patients receiving Ceredase developed antibodies to glucocerebrosidase compared with 20% of patients receiving Cerezyme. These are both higher rates of antibody conversion than the previously reported rates of approximately 12% to 15% [15, 16]. In no case did antibody formation interfere with therapeutic efficacy, nor did it result in any major immune-mediated reactions. The conversion to antibody positivity in the two cohorts was the same during the first 6 months of therapy, whereas none of the patients receiving Cerezyme became positive for antibodies during the second 3 months of therapy. These results suggest that the product purified from natural sources might be more antigenic than the recombinant enzyme form because of trace impurities, denaturation, or abnormal conformations of the administered enzyme. The results also suggest that patients who convert to antibody positivity when receiving Cerezyme may convert sooner than when receiving Ceredase. If confirmed in larger studies, the period of direct medical observation for administration of Cerezyme may be shorter than that suggested for Ceredase.

Overall, our results show the general similarity of patient responses to natural and recombinant glucocerebrosidase for enzyme therapy in Gaucher disease. Currently, both forms of the drug have the same unit price. We show that the first generation of recombinant enzymes for delivery to intracellular and subcellular compartments can result in efficacious treatment of an inherited inborn metabolism error. Clearly, these approaches are directly applicable to other lysosomal storage diseases and disorders requiring intracellular replacement of proteins. Second- and third-generation recombinant drugs could be produced in different cell lines to synthetically incorporate specific targeting signals for proteins and to reduce the number of manufacturing steps and the cost in producing these therapeutic biological agents.