Tagraxofusp: First Global Approval.

Tagraxofusp (tagraxofusp-erzs) [Elzonris™] is an intravenously administered CD123-directed cytotoxin (composed of human interleukin-3 and a truncated diphtheria toxin payload) that was developed by Stemline Therapeutics, Inc. for the treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN). In December 2018, tagraxofusp received its first global approval in the USA for the treatment of BPDCN in adults and in paediatric patients aged 2 years and older. A centralized registration application for the use of tagraxofusp in patients with BPDCN is under review in the EU. This article summarizes the milestones in the development of tagraxofusp leading to its first global approval for the treatment of BPDCN.

Pharmacokinetics of recombinant human interleukin 3 administered subcutaneously and by continuous intravenous infusion in patients after chemotherapy for ovarian cancer.

Twenty chemotherapy-naive patients with ovarian carcinoma received 1, 5, 10, or 15 micrograms/kg/day (five patients per dose step) of recombinant human interleukin 3 (rhIL-3) over 7 days after carboplatin/cyclophosphamide in Cycles 1 and 3. Patients received rhIL-3 by continuous i.v. infusion or once daily s.c. injection in Cycle 1 and the alternate route in Cycle 3. Plasma rhIL-3 samples were obtained once daily on Days 1 to 6 and serially over a 24-h period on Day 7 for pharmacokinetic assessment of s.c. and i.v. administered rhIL-3 in 16 and 17 patients, respectively. Concentrations were assayed by a time-resolved fluorescence sandwich immunoassay. Pharmacokinetic parameters were derived by noncompartmental methods. Mean steady-state concentrations during continuous i.v. infusion ranged from 117 pg/ml (1 microgram/kg/day) to 2217 pg/ml (15 micrograms/kg/day) and were linearly related to dose (r = 0.87, P < 0.001). When dose normalized, the mean steady-state concentrations were comparable at all doses. The total-body clearance was approximately 4 to 5 ml/min/kg. Elimination half-life (t1/2 i.v.) could be assessed for the 5- to 15-micrograms/kg/day dose levels and was 53, 41, and 26 min for the 5-, 10-, and 15-micrograms dose levels, respectively (not significant between dose levels). Following s.c. injection, the maximum rhIL-3 plasma concentration ranged from 206 pg/ml (1 microgram/kg/day) to 6930 pg/ml (15 micrograms/kg/day). Both the maximum measured plasma concentration (r = 0.89, P < 0.0001) and the area under the plasma concentration/time curve (r = 0.93, P < 0.0001) were related to dose. Dose-normalized values were comparable over the entire dose range. Elimination t1/2s.c. was 4.8 h at the 1-microgram dose level and roughly half this time for the 5- to 15-micrograms/kg/day dose levels. The systemic clearance of approximately 5 to 6 ml/min/kg was comparable at all dose levels. Based on trough levels of the 7-day s.c. course, no rhIL-3 accumulation occurred. Bioavailability of s.c. administered rhIL-3 was nearly 100%. No correlation between creatinine clearance and pharmacokinetic parameters of rhIL-3 could be demonstrated. Since there was also no difference in hematological efficacy between the two routes of rhIL-3 administration, we conclude that the s.c. route of administration appears to have no disadvantages over the i.v. route and may facilitate its clinical application.

Phase I study of recombinant human interleukin-3 in patients with bone marrow failure.

Interleukin-3 (IL-3) is a T-cell-derived colony-stimulating factor (CSF) whose primary targets include relatively early, multipotential, hematopoietic progenitor cells. In this trial, we treated 24 patients with recombinant human IL-3 given by a daily 4-hour intravenous infusion for 28 days. The dose levels were 30, 60, 125, 250, 500, 750, and 1,000 micrograms/m2/d. At least three patients were entered at every dose level. Each participant suffered from bone marrow failure, with the underlying diagnosis being myelodysplastic syndrome (13 patients), aplastic anemia (eight patients), or aplasia after prolonged high-dose chemotherapy (three patients) for multiple myeloma, breast cancer, or acute myelogenous leukemia. Most patients tolerated therapy well, with the most frequent side effects being low-grade fever and headaches. Hematopoietic changes included modest increases in neutrophil counts (eight patients), eosinophil counts (six patients), platelet counts (three patients), and reticulocyte counts (two patients). An increase in blasts occurred in one patient who had refractory anemia with excess blasts in transformation and was reversible once IL-3 was discontinued. In addition, one patient with chronic myelomonocytic leukemia showed an increase in monocytes (and granulocytes). Progression to acute leukemia did not occur. Pharmacokinetic analyses showed a rapid clearance with a mean half-life of 18.8 minutes at the 60 micrograms/m2/d dose, and 52.9 minutes at the 250 micrograms/m2/d dose. Serum concentrations of 10 to 20 ng/mL of IL-3 were achievable at the 250 micrograms/m2/d dose. Our observations indicate that recombinant human IL-3 can be given safely at doses of 1,000 micrograms/m2/d or less. In addition, on the basis of preclinical data and the biologic activity observed in this study, further trials of this molecule, alone and in combination with other growth factors, are warranted in patients with pancytopenia.

Pharmacokinetic basis for optimal hemopoietic effectiveness of homologous IL-3 administered to rhesus monkeys.

To design an interleukin-3 (IL-3) administration schedule for optimal hemopoietic effectiveness, serum half-life (t1/2) was determined after intravenous (i.v) and subcutaneous (s.c.) bolus injections. The initial t1/2 in serum after i.v. injection was about 10 minutes and the terminal t1/2 close to 2 hours. Subcutaneous administration resulted in plateau levels after 2 to 4 hours, while the apparent terminal t1/2 was similar to that after i.v. infusion. The bioavailability of IL-3 following subcutaneous administration was only about 40% of that following i.v. administration. Hemopoietic effects of continuous i.v. infusion of IL-3 was then compared to s.c. administration in either one, two, or three daily injections. Doses chosen ranged from 1 to 30 micrograms/kg per day. In agreement with the more limited bioavailability of IL-3 following s.c. administration, continuous i.v. infusion was much more effective in stimulating hemopoiesis than s.c. administration. Two or three daily s.c. injections did not improve the hemopoietic response compared to a single s.c. injection, which is in agreement with the apparent terminal t1/2 of 101 min. It is concluded that IL-3 is more effective by continuous i.v. infusion than by subcutaneous administration.

Phase I trial of subcutaneous interleukin 3 in patients with refractory malignancy: hematological, immunological, and pharmacodynamic findings.

We conducted a Phase I trial of s.c. recombinant human interleukin 3 (rhIL-3) to evaluate the toxicity, maximal tolerated dose, pharmacokinetics, and in vivo biological effects of this cytokine. Thirty-one patients with refractory cancer were entered into the study between November 1991 and June 1993. Therapy consisted of s.c. rhIL-3 daily for 15 days administered to cohorts of three to nine patients at dose levels of 60-4000 microgram/m2/day. Cycles were repeated at intervals of 28 days. Seventy-five cycles of rhIL-3 were administered (median, two per patient) and the maximal tolerated dose was 2000 microgram/m2/day. Toxicity was moderate, with most patients developing chills, fever, and myalgia. Dose-limiting toxicity consisted of diarrhea (two patients) and headache (one patient). Hematological effects of rhIL-3 included significant dose-related increases of WBC (P < 0.001), neutrophils (P < 0.001), and eosinophils (P < 0.001). Platelet counts and absolute lymphocyte numbers also increased. Various CD3(+) lymphocyte subsets increased; however, lytic activity (natural killer and lymphokine-activated killer) of peripheral blood lymphocytes was not enhanced. Serum levels of the soluble IL-2 receptor increased in a dose-related fashion, and IL-2-induced lymphocyte proliferation also was increased variably. Pharmacokinetic studies were performed in 13 patients, and area under the curve and maximal concentration values increased with increasing rhIL-3 dose levels (P < 0.001) and correlated with maximal changes from baseline in WBC, neutrophils, and eosinophils. rhIL-3 antibodies were detected in 8% of patients by day 29 of cycle 1 but were not neutralizing. rhIL-3 is well tolerated when administered s.c. and has reproducible hematological and immunological effects. The pleiotropic effects of this cytokine on various in vivo biological parameters were demonstrated clearly. Further studies of its immunoregulatory effects are warranted.

Carbohydrate does not modulate the in vivo effects of injected interleukin-3.

Murine interleukin-3 (IL-3) has extensive N-linked glycosylation. Experiments were performed to determine whether the T cell-derived glycosylated IL-3 differs in its biological activity in vivo when compared with a chemically synthesized form of nonglycosylated IL-3. Groups of mice were treated by intravenous injection with identical units of IL-3 bioactivity as determined in vitro in a cell-proliferation assay. Mice that were treated with seven 5000-unit doses of either form of IL-3, given in 12-hour intervals, showed a small but significant increase in the frequency of mast cell precursor cells in the spleen and of IL-3-responsive colony-forming unit cells (CFU-C). There was no difference in potency of glycosylated and nonglycosylated IL-3. Induction, by IL-3, of histidine decarboxylase in bone marrow and spleen cells was used as a second measure for IL-3 bioactivity. Both IL-3 preparations showed good in vivo histidine decarboxylase inducing activity; however, T cell-derived glycosylated IL-3 was significantly more effective than synthetic IL-3 in inducing the enzyme histidine decarboxylase in bone marrow and in spleen cells. Pharmacokinetic studies showed that chemically synthesized IL-3 was cleared about twice as fast as the T cell-derived IL-3 and that there may be some tissue trapping of glycosylated IL-3. The shorter in vivo half-life of nonglycosylated IL-3 appears to have significant pharmacological consequences on the short-term effect of inducing histidine decarboxylase activity, but not on the effect of the long-term treatment of IL-3 on stimulating the increase of hematopoietic progenitor cells.

Granulocyte-macrophage colony-stimulating factor regulation in murine T cells and its relation to cyclosporin A.

The coordinated expression of the hematopoietic growth factors, interleukin 3 (IL3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in activated T cells suggests common synthetic pathways. However, cyclosporin A (CsA) appears to differentially effect the synthesis of these two lymphokines. When the supernatants from the concanavalin A-stimulated thymoma EL-4 or the T-cell hybrid 2B4 were assayed on the GM-CSF/IL3-dependent PT-18 and on the IL3-dependent DA-1 cell lines, IL3 activity could not be detected following CsA treatment, but substantial growth activity, which was identified as GM-CSF, was observed on the PT-18 cell line. CsA did not affect the kinetics of GM-CSF release, but inhibited the release of IL3 over a period of 40 h after the cells were stimulated and treated with CsA. In addition, CsA could be added up to 1 h after stimulation of the cells without affecting GM-CSF activity but inhibiting completely the IL3 activity. To substantiate the inability of CsA to inhibit GM-CSF activity, GM-CSF gene expression was evaluated. By Northern analysis GM-CSF mRNA was not inhibited by doses of CsA up to 1 microgram/ml. The data reveal that CsA can dissociate between the production of IL3 and GM-CSF, suggesting that these two CSFs are regulated by different mechanisms.

Pharmacodynamics of daily subcutaneous recombinant human interleukin-3 in normal volunteers.

Normal volunteers received subcutaneous injections of recombinant human interleukin-3 (rhIL-3) on 4 consecutive days to characterize toxicity, pharmacokinetics, and hematopoietic effects. Dosages were 2.5, 5.0, and 7.5 micrograms/kg/day (n = 6 subjects per group). Adverse effects consisted predominantly of flu-like symptoms such as fever and headache. Mean area under the serum concentration-time curve and maximum serum concentration were linearly related to dose. Serum clearance was not apparently related to dose. Clearance increased slightly but significantly between days 1 and 4. Rapid but modest elevations in neutrophil and eosinophil counts were observed during treatment. Mean platelet counts rose modestly, peaking on day 10. Increases of CD34+ cell counts were correlated with increases of colony-forming unit-granulocyte macrophage (peak, day 7).

Clinical effects and pharmacokinetics of the fusion protein PIXY321 in children receiving myelosuppressive chemotherapy.

UNLABELLED: A hemopoietin with the ability to accelerate both platelet and granulocyte recovery after intensive chemotherapy would have great clinical utility. The recombinant fusion protein composed of human granulocyte-macrophage colony-stimulating factor and interleukin-3 (PIXY321), showed some promise in early adult trials. However, studies for pediatric patients are limited, and there are no systematic data on the pharmacokinetics of PIXY321 given over prolonged periods at current dosage levels. PURPOSE: To determine the safety, clinical effects and plasma concentrations of increasing doses of PIXY321 in children treated with myelosuppressive chemotherapy. METHODS: A total of 39 children with relapsed or high-risk solid tumors were enrolled in this phase I/II study. PIXY321 was administered once or twice daily by subcutaneous injection in total doses of 500 to 1000 microg/m2 per day for 14 days after each course of chemotherapy with ifosfamide, carboplatin, and etoposide (ICE). Pharmacokinetic studies were performed on day 1 of the first course in 33 patients and repeated on day 14 in 13 patients (once-daily schedule only). RESULTS: Although mild local skin reactions and fever were frequent, no dose-limiting toxicity was identified at the maximum dose studied (1000 microg/m2 per day). There were no statistically significant differences in chemotherapy-induced hematologic toxicity with increasing doses of PIXY321 or with twice-daily vs once-daily dosing. On day 1, the median PIXY321 clearance was 657 ml/min per m2 (range 77 1804 ml/min per m2) and the median half-life was 3.7 h (range 2.1-20.8 h). On day 14, clearance increased in all patients studied (median increase 63%), with a corresponding decrease in the median 12-h concentration (from 1.2 to 0.25 ng/ml). Maximum concentrations were < 1 ng/ml in 81% of patients, and only two patients had maximum plasma concentrations equivalent to those required for consistent activity in vitro. CONCLUSIONS: The recombinant fusion protein PIXY321 proved safe in children treated with myelosuppressive ICE chemotherapy but had no demonstrable clinical benefits. The pharmacokinetic studies suggest that the observed lack of hematologic benefit may be explained by low plasma concentrations resulting from increased clearance with prolonged administration. Moreover, the significant increase in PIXY321 systemic clearance in the absence of increased circulating myeloid cells suggests that the upregulation of either extravascular compartment hematopoietic progenitor cells or nonhematopoietic cells may play an important role in controlling circulating concentrations of this unique cytokine. These findings highlight the importance of a thorough assessment of the systemic disposition of cytokines when determining the dose and schedule necessary to achieve clinical activity in patients.

Toxicology and pharmacokinetics of DT388IL3, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human interleukin 3 (IL3), in cynomolgus monkeys.

The fusion toxin DT388IL3 composed of the catalytic and translocation domains of diphtheria toxin (DT388) linked to interleukin-3 (IL3) was administered to 6 cynomolgus monkeys which possessed cross-reactive IL3 receptors. Groups of 2 animals (1 male and 1 female) received up to 6 every other day slow intravenous infusions of 40, 60, or 100 microg/kg DT388IL3. Monkeys given 40 or 60 microg/kg showed mild or moderate transient malaise and anorexia, respectively, without evidence of organ damage by blood tests or histopathology. Animals treated at 100 microg/kg showed severe malaise and anorexia. The female monkey had moderate to severe vasculitis in multiple tissues. Necropsies were performed on the 40 microg/kg monkeys on day 14 and the 100 microg/kg monkeys on days 6 and 7. DT388IL3 plasma half-life was approximately 30 min with a peak concentration of 0.45 microg/ml or 10,000 pM (IC50 for AML blasts treated in vitro was 6 pM). Immune responses were minimal in 4 animals tested at 12 days and 2 animals tested at 30 days post treatment with anti-DT388IL3 levels < 1 microg/ml. Bone marrow aspirates were obtained on all animals at day 19 or at necropsy and revealed myeloid suppression in the females and myeloid hyperplasia in the males irrespective of dose groups. The maximal tolerated dose of 60 microg/kg for 6 doses is markedly higher than other recombinant diphtheria toxins and provides a dose level sufficient for anti-leukemic activity in vitro and in rodent models. Thus, we propose this agent is a promising drug for AML patients.

Poly(ethylene carbonate)s, part II: degradation mechanisms and parenteral delivery of bioactive agents.

The degradation and drug carrier properties of poly(ethylene carbonate) (PEC) were investigated in vitro and in rats and rabbits. PEC was found to be specifically degraded in vivo and in vitro by superoxide radical anions O2-*, which are, in vivo, mostly produced by inflammatory cells. No degradation of PEC was observed in the presence of hydrolases, serum or blood. PEC is biodegraded by surface erosion without significant change in the molecular weight of the residual polymer mass. The non-hydrolytic biodegradation by cells producing O2-* is unique among the polymers used as biodegradable drug carriers. The main degradation product of PEC in aqueous systems is ethylene glycol, formed presumably by hydrolysis of ethylene carbonate. The splitting off of a five-membered ring structure from the polymer chain indicates a chain reaction mechanism for the biodegradation. PEC is a suitable drug carrier, particularly for labile drugs. Using human interleukin-3 and octreotide as model drugs, surface erosion of the PEC formulations was indicated by a 1:1 correlation between drug release and polymer mass loss.

Modulation of the sustained delivery of myelopoietin (Leridistim) encapsulated in multivesicular liposomes (DepoFoam).

Myelopoietins (MPO) are novel chimeric growth factors containing IL-3 and G-CSF receptor agonists that enhance the biological properties of both cytokines. These cytokines, like many therapeutic proteins, clear rapidly from circulation and must be administered daily to provide efficacy. Therefore, a controlled and sustained delivery system comprised of a biocompatible and biodegradable matrix, would offer important therapeutic advantages in the clinic, such as significantly reducing dose frequency and providing efficacy without toxicity. We report here the encapsulation of Leridistim (a protein from the MPO family) in multivesicular liposomes (DepoFoam) for sustained delivery, and demonstrate that a single injection of DepoFoam-encapsulated Leridistim results in elevated neutrophil counts for 10 days, in contrast to only 2 days for un-encapsulated Leridistim. Moreover, varying the lipid content of the DepoFoam matrix modulated the duration of elevated neutrophils from 2-3 to 9-10 days. The encapsulated Leridistim was released in vivo from the multivesicular liposomes in a uniform manner, consistent with its pharmacodynamic duration. Finally, a reproducible pharmacodynamic effect was observed with several batches of a DepoLeridistim formulation, indicating consistency of the manufacturing process of the DepoFoam delivery system. The capability of altering the release rates by varying the lipid composition provides maximum flexibility for controlled delivery of cytokine therapeutics.

Effect of dexamethasone on IL-1 and IL-3-LA release by unstimulated human mononuclear cells.

The effect of dexamethasone on the in vitro release of IL-1 and IL-3-LA by human unstimulated mononuclear cells was studied. The drug elicited a dose-dependent effect on the production of both interleukins. In addition, a time course effect of dexamethasone on IL-3-LA was demonstrated. It appeared after 5 min of incubation, reached a maximal level after 15 min, and remained at that level after 24 h. The drug also caused a dose-dependent inhibitory effect on the ability of human mononuclear cells to incorporate [3H] uridine. The inhibitory effect of dexamethasone on these interleukins may serve as a partial explanation of the susceptibility to infection in patients treated with corticosteroids.

Delivery to macrophages of interleukin 3 loaded in mouse erythrocytes.

Mouse carrier erythrocytes containing 125I-interleukin 3 have been prepared and treated with band 3 crosslinking reagents. The incorporation of interleukin 3 by hypotonic treatment into mouse erythrocytes reached levels of about 15% of the interleukin 3 added to the medium being predominantly present in the cytosolic fraction (73%). Uptake fell to about 7.4% when using the same conditions but omitting hypotonic shock. The interaction of band 3 crosslinked interleukin 3 loaded erythrocytes with macrophages was also studied. A high level of incorporation of interleukin 3 into macrophages was observed either from band 3 crosslinked, interleukin 3-loaded erythrocytes or from interleukin 3 loaded erythrocytes. The observations encourage the view that the system may be able to deliver and target cytokines and other growth factors to macrophages.

Cytokines in tumor therapy. Interleukin-3.

IL-3 is a T-cell-derived hematopoietin. This cytokine stimulates the growth of multipotential progenitor cells and is therefore of clinical interest for the therapy of pancytopenic processes. Both in vitro and animal studies suggest that the development and maturation of multiple lineages of blood cells can best be achieved by combining IL-3 with other growth factors. IL-3 may therefore act to expand a primitive pool of precursor cells, and the presence of additional factors may aid in the differentiation of these cells to functional status. Studies of IL-3 in human beings are in early phase I stages. These trials indicate that this molecule has a short half-life, and hence should be given by routes that permit prolonged serum levels. IL-3 is generally well tolerated, and stimulation of progression to acute leukemia was not observed in our subjects with preleukemic states. This agent increased neutrophil, eosinophil, platelet, and reticulocyte counts in individual patients. However, responses varied from patient to patient, and the IL-3-induced changes in neutrophil counts were modest as opposed to those previously described after GM-CSF therapy. Comparison of the in vitro effects of IL-3 in the colony/culture assay to subsequent in vivo effects in a small number of patients indicates that increases in CFU-G, CFU-M, and CFU-GM can, at times, predict for changes in granulocyte and monocyte counts. A synthesis of currently available data indicates that future trials should be designed to treat additional patients with IL-3 alone and to combine IL-3 with GM-CSF, G-CSF, erythropoietin, and other growth factors.

Clinical effects of recombinant human interleukin-3.

Interleukin-3 (IL-3) is a glycoprotein belonging to the hematopoietic growth factor family that in preclinical in vitro and in vivo studies has exhibited a multilineage activity. Phase I/II trials with recombinant human IL-3 (rhIL-3) expressed in yeast are being done in patients with advanced malignancies as well as in patients with bone marrow failure states. Subcutaneous administration of rhIL-3 at dosages between 30 and 500 micrograms/m2 for 15 consecutive days has resulted in a dose-dependent increase in platelet counts as well as in a substantial increase in the number of circulating neutrophils, eosinophils, monocytes, and lymphocytes in patients with advanced malignancies but normal hematopoiesis. Erythropoiesis is less stimulated with an increase in hemoglobin concentration only in a minority of patients. In patients with secondary hematopoietic failure due to prolonged chemo-/radiotherapy or bone marrow infiltration by tumor cells, treatment with rhIL-3 leads to a clinically significant restoration of hematopoiesis, especially of thrombopoiesis and granulopoiesis. rhIL-3 has also been shown to improve neutrophil and platelet counts in patients with myelodysplastic syndromes, while improvement of hematopoiesis is rarely observed in patients with severe aplastic anemia with the presently used treatment schedules. Adverse effects of rhIL-3 are minor at the clinically used dosages and include fever, bone pain, headache, and stiffness of the neck. Transient thrombocytopenia has been observed in a few patients with myelodysplastic syndrome or aplastic anemia treated at dosages of 250-500 micrograms/m2. rhIL-3 is a multilineage hematopoietic cytokine with promising effects on platelet and neutrophil counts and special usefulness in patients with secondary hematopoietic failure.

Iontophoresis of a 13 kDa protein monitored by subcutaneous microdialysis in vivo.

BACKGROUND: The purpose of this study was to optimize parameters pertaining to microdialysis technique so as to make this method feasible for evaluating transdermal transport of macromolecules. RESULTS: Microdialysis experiments were performed in vivo using hairless rats with daniplestim as the model protein. Two perfusion fluids - phosphate-buffered saline (PBS) and 3% dextran in PBS - were evaluated with respect to their effect on sample volume retrieval and recovery of the target protein from the microdialysis probe. Incorporation of dextran-60 in the perfusion fluid reduced fluid loss to 10% as opposed to 34% in the absence of dextran-60. Improvement in daniplestim recovery was also seen with dextran-PBS (56.5 ± 10.3%) as the perfusion fluid than with PBS alone (26.7±4.5%). CONCLUSION: Subcutaneous levels of daniplestim were measured following iontophoresis after improving recovery and minimizing fluid loss from the microdialysis probe.

Transdermal delivery of a approximately 13 kDa protein--an in vivo comparison of physical enhancement methods.

The availability of several enhancement techniques has made it possible to study delivery of macromolecules through skin. This study was conducted to evaluate the transdermal delivery of a ~13 kDa protein using iontophoresis, sonophoresis, and microneedles alone or in combination. In vivo delivery experiments were carried out using hairless rats with daniplestim (DP) as the model protein (molecular weight: 12.760 kDa; isoelectric point, 6.2). Delivery enhancement abilities of the above techniques were evaluated at two different drug concentrations in the patch: 2 mg/mL and 5 mg/mL. At a drug loading concentration of 2 mg/mL maximum delivery was seen with the combination of microneedles and iontophoresis. At 5 mg/mL, sonophoresis alone gave a C(max) of 8.22 +/- 5.9 ng/mL and a combination of sonophoresis and iontophoresis gave a C(max) of 4.9 +/- 1.8 ng/mL. The results of this study suggest that combination of microneedles and iontophoresis was the most effective approach in delivering a 13 kDa protein through the skin.

Molecular charge mediated transport of a 13 kD protein across microporated skin.

Transport of proteins across the skin is highly limited owing to their hydrophilic nature and large molecular size. This study was conducted to assess the skin transport abilities of a model protein across hairless rat skin during iontophoresis alone and in combination with microneedles as a function of molecular charge. The effect of microneedle pretreatment on electroosmotic flow was also investigated. Skin permeation experiments were carried out in vitro using daniplestim (DP) (MW, 12.76 kD; isoelectric point, 6.2) as a model protein molecule. The effect of molecular charge on protein transport was evaluated by performing studies in two different buffers--TRIS (pH 7.5) and acetate (pH 4.0). Iontophoretic transport mechanisms of DP varied with respect to molecular charge on the protein. The combination approach (iontophoresis and microneedles) gave much higher flux values compared to iontophoresis alone at both pH 4.0 and pH 7.5, however, the delivery in this case was also found to be charge dependent. The findings of this study indicate that electroosmosis persisted upon microporation, thus retaining skin's permselective properties. This enables us to explore the combination of microneedles and iontophoresis as a potential approach for delivery of proteins.

Specific binding, internalization, and degradation of human recombinant interleukin-3 by cells of the acute myelogenous, leukemia line, KG-1.

We have studied the interaction of 35S-labeled recombinant IL-3 with the acute myelogenous leukemia cell line, KG-1. 35S-IL-3 bound to these cells in a time dependent, saturable, and specific manner at 4 degrees C. Scatchard transformation of binding isotherms demonstrated the existence of a small number (200) of binding sites, with an apparent dissociation constant of 70-105 pM. After a temperature shift from 4 degrees C to 37 degrees C, surface-bound 35S-IL-3 was rapidly internalized and processed into a trichloroacetic acid soluble form that was released into the medium. Experiments to address the specificity of the IL-3 binding site revealed that neither human IL-2, M-CSF, erythropoietin, transferrin, bovine insulin, nor murine nerve growth factor compete with IL-3 for binding to KG-1 cells. Both human and gibbon recombinant IL-3 and, surprisingly, human recombinant GM-CSF effectively competed the binding of the labeled IL-3 to these cells at 4 degrees C. The competition by GM-CSF was found to be concentration dependent, but much higher concentrations were required to achieve the levels obtained with IL-3. These results suggest that GM-CSF may also interact with the high-affinity IL-3 binding site on KG-1 cells or, alternatively, that GM-CSF binding to its own receptor may decrease the affinity of the IL-3 receptor for its ligand.