Development and characterization of novel erythropoiesis stimulating protein (NESP).

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP), which was engineered to contain five N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximately 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.

Development and characterization of novel erythropoiesis stimulating protein (NESP).

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP, ARANESP, Amgen Inc, Thousand Oaks, CA), which was engineered to contain 5 N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximate 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.

The effects of normal as compared with low hematocrit values in patients with cardiac disease who are receiving hemodialysis and epoetin.

BACKGROUND: In patients with end-stage renal disease, anemia develops as a result of erythropoietin deficiency, and recombinant human erythropoietin (epoetin) is prescribed to correct the anemia partially. We examined the risks and benefits of normalizing the hematocrit in patients with cardiac disease who were undergoing hemodialysis. METHODS: We studied 1233 patients with clinical evidence of congestive heart failure or ischemic heart disease who were undergoing hemodialysis: 618 patients were assigned to receive increasing doses of epoetin to achieve and maintain a hematocrit of 42 percent, and 615 were assigned to receive doses of epoetin sufficient to maintain a hematocrit of 30 percent throughout the study. The median duration of treatment was 14 months. The primary end point was the length of time to death or a first nonfatal myocardial infarction. RESULTS: After 29 months, there were 183 deaths and 19 first nonfatal myocardial infarctions among the patients in the normal-hematocrit group and 150 deaths and 14 nonfatal myocardial infarctions among those in the low-hematocrit group (risk ratio for the normal-hematocrit group as compared with the low-hematocrit group, 1.3; 95 percent confidence interval, 0.9 to 1.9). Although the difference in event-free survival between the two groups did not reach the prespecified statistical stopping boundary, the study was halted. The causes of death in the two groups were similar. The mortality rates decreased with increasing hematocrit values in both groups. The patients in the normal-hematocrit group had a decline in the adequacy of dialysis and received intravenous iron dextran more often than those in the low-hematocrit group. CONCLUSIONS: In patients with clinically evident congestive heart failure or ischemic heart disease who are receiving hemodialysis, administration of epoetin to raise their hematocrit to 42 percent is not recommended.

Use of recombinant human erythropoietin for management of anemia in dogs and cats with renal failure.

OBJECTIVE: To test efficacy and safety of recombinant human erythropoietin (r-HuEPO) administration in dogs and cats with naturally developing chronic renal failure. DESIGN: Case series. ANIMALS: 6 client-owned dogs and 11 client-owned cats with chronic renal failure. PROCEDURES: r-HuEPO was administered intravenously or subcutaneously. Erythropoietic effects were monitored by determining CBC, performing cytologic examination of bone marrow aspirates, and measuring serum iron concentration before and during treatment. Development of adverse effects was monitored by performing sequential clinical assessments, CBC, and serum biochemical tests and by measuring indirect blood pressure and anti-r-HuEPO antibody titers. RESULTS: Administration of r-HuEPO increased RBC and reticulocyte counts, hemoglobin concentration, and Hct comparably in dogs and cats. Assessments of clinical well-being, including appetite, energy, weight gain, alertness, strength, and playfulness, were improved variably. Adverse effects, including anemia, anti-r-HuEPO antibody production, seizures, systemic hypertension, and iron deficiency, were demonstrated inconsistently in dogs and cats. CLINICAL IMPLICATIONS: Anemia contributes to clinical manifestations of chronic renal failure in dogs and cats. Administration of r-HuEPO has the potential to resolve anemia and improve clinical well-being. However, its administration poses risks of antibody production and adverse effects associated with correction of RBC mass. Use of r-HuEPO in dogs and cats requires conscientious assessment of risks and benefits until homologous forms of erythropoietin are available.

Isolation, characterization, and expression of a cDNA encoding N-acetylglucosaminyltransferase V.

A cDNA clone for the complete coding sequence for alpha-1,3(6)-mannosylglycoprotein beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155) was isolated and expressed in COS-7 cells. Degenerate oligonucleotide primers for polymerase chain reaction were synthesized based on the amino acid sequence of three tryptic peptides isolated from affinity-purified GlcNAc-T V. Polymerase chain reaction amplimers were isolated from rat and mouse mRNA. A cDNA-encoding full-length enzyme was isolated from a rat 1 cell (EJ-ras-transformed) library and sequenced. Transient expression of this clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes GlcNAc-T V. Northern analysis of rat kidney mRNA revealed a single band corresponding to a length of about 7 kilobases. Sequence analysis of the cDNA clone demonstrated an open reading frame that encoded a type II membrane protein of 740 amino acids.

Enhancement of the amplitude of somatosensory evoked potentials following magnetic pulse stimulation of the human brain.

In this study we have demonstrated an enhancement of cortically generated wave forms of the somatosensory evoked potential (SEP) following magnetic pulse stimulation of the human brain. Subcortically generated activity was unaltered. The enhancement of SEP amplitude was greatest when the median nerve was stimulated 30-70 msec following magnetic pulse stimulation over the contralateral parietal scalp. We posit that the enhancement of the SEP is the result of synchronization of pyramidal cells in the sensorimotor cortex resulting from the magnetic pulse.

Suppression of cutaneous perception by magnetic pulse stimulation of the human brain.

We have demonstrated that magnetic pulse stimulation of the sensorimotor cortex suppresses perception of threshold electrical stimuli to the fingers of the contralateral hand. Maximum suppression of perception occurs when the fingers are stimulated 30-90 msec after the magnetic pulse. Thereafter, errors in perception of the cutaneous stimulus decrease to control levels by 300-400 msec after the magnetic pulse. The period of maximum suppression of perception coincides with the period during which cortically generated somatosensory evoked potentials (SEPs) are enhanced following magnetic pulse stimulation of the brain. The duration of suppression of perception, however, outlasts the duration of SEP enhancement. When the magnetic pulse is delivered after finger stimulation there is also suppression of perception. The suppression of perception is maximal when the magnetic pulse occurs 20-30 msec after finger stimulation. This interval coincides with the arrival of the afferent volley at the primary sensory cortex.

Recombinant human erythropoietin in anemic patients with end-stage renal disease. Results of a phase III multicenter clinical trial.

STUDY OBJECTIVE: To determine the effectiveness and safety of recombinant human erythropoietin (rHuEpo). PATIENTS: Hemodialysis patients (333) with uncomplicated anemia (hematocrit less than 0.30). All received rHuEpo intravenously, three times per week at 300 or 150 U/kg body weight, which was then reduced to 75 U/kg and adjusted to maintain the hematocrit at 0.35 +/- 0.03 (SD). RESULTS: The baseline hematocrit (0.223 +/- 0.002) increased to 0.35, more than 0.06 over baseline within 12 weeks in 97.4% of patients. Erythrocyte transfusions (1030 within the 6 months before rHuEpo therapy) were eliminated in all patients within 2 months of therapy. Sixty-eight patients with iron overload had a 39% reduction in serum ferritin levels after 6 months of therapy. The median maintenance dose of rHuEpo was 75 U/kg, three times per week (range, 12.5 to 525 U/kg). Nonresponders had complicating causes for anemia, myelofibrosis, osteitis fibrosa, osteomyelitis, and acute or chronic blood loss. Adverse effects included myalgias, 5%; iron deficiency, 43%; increased blood pressure, 35%; and seizures, 5.4%. The creatinine, potassium, and phosphate levels increased slightly but significantly. The platelet count increased slightly but there was no increase in clotting of vascular accesses. CONCLUSIONS: The anemia of hemodialysis patients is corrected by rHuEpo resulting in the elimination of transfusions, reduction in iron overload, and improved quality of life. Iron stores and blood pressure must be monitored and treated to maintain the effectiveness of rHuEpo and to minimize the threat of hypertensive encephalopathy.

Conversion of a Golgi apparatus sialyltransferase to a secretory protein by replacement of the NH2-terminal signal anchor with a signal peptide.

The beta-galactoside alpha 2,6 sialyltransferase, an integral membrane protein localized to the trans-region of the Golgi apparatus, has been converted into a catalytically active secreted protein by the replacement of the NH2-terminal signal-anchor domain with the cleavable signal peptide of human gamma-interferon. Pulse-chase analysis of the wild type and recombinant proteins expressed in stably transfected Chinese hamster ovary cells showed that the wild type sialyltransferase (47 kDa) remained cell-associated. In contrast, the signal peptide-sialyltransferase fusion protein yielded an enzymatically active 41-kDa polypeptide which was secreted with a half-time of 2-3 h, consistent with cleavage of the signal peptide. The data indicate that the catalytic domain does not contain sufficient information for retention in the Golgi apparatus and that retention signals are likely to be found in the NH2-terminal 57 amino acids of the wild type enzyme.

Short latency somatosensory evoked potentials following mechanical taps to the face. Scalp recordings with a non-cephalic reference.

Trigeminal somatosensory evoked potentials were recorded over the scalp using non-cephalic reference sites following mechanical taps to the face. A negative wave form, Nf17, was recorded bilaterally with its highest amplitude over the frontal scalp contralateral to the side of stimulation. A localized negative wave form, Np25, was recorded over the centro-parietal scalp contralateral to the side of stimulation. Np25 had an onset latency of 16.46 msec. The location and restricted spatial distribution of Np25 suggest that it represents the initial activation of the face area of the primary sensory cortex. The widespread bilateral nature of Nf17 and its latency of onset preceding that of Np25 suggest that Nf17 may be a 'far-field' potential reflecting activity in subcortical sensory pathways subserving the face.

The use of recombinant human erythropoietin (EPO) to correct the anemia of end-stage renal disease: a progress report.

End-stage renal disease (ESRD) typically is associated with severe anemia. The major contributor to the anemia appears to be the absolute or relative deficiency of erythropoietin (EPO) production by the kidney. A series of clinical trials have been conducted in the United States using recombinant human EPO (rh EPO) to treat anemic patients with ESRD. The encouraging results of the Phase I-II clinical trials have been confirmed in a multicenter trial in which over 250 patients have been treated. The results indicate that rh EPO can effectively correct the anemia of ESRD and the rate of correction is dependent upon the initial dose given. The rHuEpo was well tolerated, produced few or no direct side effects, and was effective in greater than 95 percent of the patients. rh EPO should have a major role in the correction of the anemia of ESRD and contribute significantly to the rehabilitation of such patients.

Recombinant human erythropoietin and autologous blood donation.

Risks of transfusion are minimized with autologous blood. However, autologous donation programs require 2 to 5 weeks to yield only 2.2 units per patient. Recombinant human erythropoietin (r-HuEPO) has been shown to increase erythropoiesis. This study evaluated the effects of r-HuEPO on an aggressive autologous donation program. Twelve adult male baboons were randomized into two groups of six. All animals were studied three times per week for 5 weeks. A unit of blood was donated when on any study day the hematocrit was greater than 30%. Animals received intravenously either 750 units/kg of r-HuEPO (n = 6) or placebo (n = 6) on each study day. Iron dextran was given intravenously to replace 150% of shed iron. The r-HuEPO group had an earlier onset of reticulocytosis (2.7 vs 5.5 days, p less than 0.01) and donated 35% more blood (13.5 vs 10.0 units, p = 0.01) than the control group. No adverse reactions to r-HuEPO were observed. The data show that an aggressive autologous donation program can yield 10 units of blood over a 5-week period. Further, r-HuEPO increases that yield by an additional 35%. This aggressive autologous donation program with r-HuEPO may significantly reduce the need for homologous transfusion and its attendant risks.

Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial.

We administered recombinant human erythropoietin to 25 anemic patients with end-stage renal disease who were undergoing hemodialysis. The recombinant human erythropoietin was given intravenously three times weekly after dialysis, and transfusion requirements, hematocrit, ferrokinetics, and reticulocyte responses were monitored. Over a range of doses between 15 and 500 units per kilogram of body weight, dose-dependent increases in effective erythropoiesis were noted. At 500 units per kilogram, changes in the hematocrit of as much as 10 percentage points were seen within three weeks, and increases in ferrokinetics of three to four times basal values, as measured by erythron transferrin uptake, were observed. Of 18 patients receiving effective doses of recombinant human erythropoietin, 12 who had required transfusions no longer needed them, and in 11 the hematocrit increased to 35 percent or more. Along with the rise in hematocrit, four patients had an increase in blood pressure, and a majority had increases in serum creatinine and potassium levels. No organ dysfunction or other toxic effects were observed, and no antibodies to the recombinant hormone were formed. These results demonstrate that recombinant human erythropoietin is effective, can eliminate the need for transfusions with their risks of immunologic sensitization, infection, and iron overload, and can restore the hematocrit to normal in many patients with the anemia of end-stage renal disease.

Characterization and biological effects of recombinant human erythropoietin.

Human recombinant erythropoietin (rHuEPO) has been purified to apparent homogeneity and compared to purified human urinary erythropoietin (EPO). Both the purified natural and recombinant EPO preparations were characterized in a competition radioimmunoassay (RIA), the exhypoxic polycythemic mouse bioassay, in vitro tissue culture bioassays using bone marrow cells, and by Western analysis. In the immunological and biological activity assays, the rHuEPO shows a dose response which parallels that of the natural hormone. By Western analysis, the recombinant and human urinary EPO migrate identically. Administration of rHuEPO increases the hematocrit of normal mice in a dose-dependent manner. Additionally, the rHuEPO is able to increase the hematocrit of rats made uremic as a result of subtotal nephrectomy. In summary, by all criteria examined, the rHuEPO is biologically active and equivalent to the natural hormone.

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene.

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.

Expression of biologically active bovine luteinizing hormone in Chinese hamster ovary cells.

Biologically active bovine luteinizing hormone (LH) has been obtained through expression of the alpha- and LH beta-subunit genes in stably transformed clones of DUXB11, a Chinese hamster ovary cell line deficient in dihydrofolate reductase (DHFR). Expression of alpha-and LH beta-subunit mRNAs of the expected sizes (approximately 910 and 770 nucleotides, respectively) were revealed by blot analysis after electrophoresis of total cellular RNA. Furthermore, presence or absence of the gonadotropin mRNAs in several clonal lines was directly correlated with the appearance of one or both bovine LH subunits in the culture medium. Media from three clones secreting significant immunoreactive levels of both subunits also stimulated the release of progesterone in ovine luteal cells, suggesting that the secreted LH was assembled into a biologically active and glycosylated dimer. Immunoprecipitation and NaDodSO4/PAGE of [35S]methionine-labeled proteins secreted from one of the clones, CHODLH20, further confirmed the presence of an alpha/beta dimer with apparent subunit molecular weights of 20,500 and 16,000, only slightly higher than those of pituitary alpha and LH beta subunits.