Pharmacokinetics of glycosylated recombinant human granulocyte colony-stimulating factor (lenograstim) in healthy male volunteers

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1 Pharmacokinetics of glycosylated recombinant human granulocyte colony-stimulating factor (lenograstim) in healthy male volunteers A. C. Houston, 1 L. A. Stevens 2 & V. Cour 3 1 Quintiles (UK) Ltd, Bracknell, UK, 2 Clin Phone Ltd, Nottingham, UK and 3 Rhône-Poulenc-Rorer, Antony, France Aims The aim of this open, randomised, crossover, parallel-group study was to compare the pharmacokinetics and neutrophil responses of lenograstim when administered subcutaneously (s.c.) and intravenously (i.v.). Methods A total of 27 healthy male volunteers was recruited. Lenograstim doses (0.5, 2, 5, or 10 mg kg 1 ) were administered s.c. or i.v. once-daily for 5 days, and then, after a 10-day washout period, vice versa for a further 5 days. Lenograstim concentrations and absolute neutrophil counts (ANCs) were measured predosing and postdosing on days 1 and 5. Results Maximum serum concentrations of lenograstim were higher following i.v. dosing (mean vs ng ml 1 after s.c. dosing on day 1) and attained sooner (median vs h on day 1). However, apparent elimination halflives of lenograstim were longer following s.c. dosing (mean vs h after i.v. dosing on days 1 and 5). ANCs increased in a dose-dependent manner with both routes of lenograstim, but more prolonged rises and higher ANC peaks were attained following s.c. doses. ANCs peaked on day 6 following 5 mg kg 1 s.c. doses (mean peak= cells l 1 ), but on day 2 after 5 mg kg 1 i.v. doses (mean peak= cells l 1 ). Irrespective of route, the most common adverse events were headaches and back/spine pain; at doses of up to 5 mg kg 1 these were mild and generally well tolerated. Conclusions While supporting the use of both s.c. and i.v. administered lenograstim to treat neutropenia, these results demonstrate that neutrophil responses are more sustained and prolonged with the s.c. route. Keywords: absolute neutrophil count, granulocyte colony-stimulating factor, healthy volunteers, lenograstim, pharmacokinetics, rhug-csf Introduction Neutropenia increases risk of infection in patients undergoing cancer chemotherapy, and is frequently a dose-limiting side-effect. Agents shortening the period of chemotherapy-associated neutropenia may reduce incidence or duration of serious infections and enable greater dose-intensification. Production of neutrophils is controlled naturally by granulocyte colony-stimulating factor (G-CSF). G-CSF stimulates specifically the differentiation and activation of neutrophils from bone marrow-derived progenitor cells [1]. The recent development of recombinant DNA Correspondence: Dr Alan C. Houston, Quintiles (UK) Limited, Ringside, 79 High Street, Bracknell RG12 1DZ. Received 25 January 1997, accepted 1 October technology has permitted the large-scale expression, and introduction into clinical practice, of recombinant human G-CSF (rhug-csf). RHuG-CSF can be expressed either in glycosylated form by Chinese Hamster Ovary cells (lenograstim) [2] or as a nonglycosylated molecule by Escherichia coli (filgrastim) [3]. Lenograstim is identical in amino acid sequence and structure to endogenously produced G-CSF, while filgrastim has an extra methionine residue and is not glycosylated. Both rhug-csfs elevate the number of circulating neutrophils. Characteristically an initial decrease (nadir) in neutrophil counts is followed by a dose-dependent increase [4]. When given as prophylaxis or treatment to patients receiving cytotoxic cancer therapy, rhug-csfs reduce the duration of neutropenia, the incidence of infections and the necessity for antibiotics [2, 3]. Lenograstim can be administered either subcutaneously (s.c.) or intravenously (i.v.). This report describes an open, 1999 Blackwell Science Ltd Br J Clin Pharmacol, 47,

2 A. C. Houston et al. randomised, crossover, parallel-group study designed to lated using the linear trapezoidal rule, and the absolute compare the pharmacokinetics and neutrophil responses bioavailability (F) of each s.c. dose level as F=AUC(0, of lenograstim doses when given s.c. and i.v. 24 h) (day 5, s.c.)/auc(0, 24 h) (day 5, i.v.) dose(i.v.)/dose(s.c.) 100. Methods Total clearance (CL) was calculated for i.v. doses as CL=dose/AUC. The apparent elimination half-life (t 1/2 ) Subjects and treatment of s.c. and i.v. doses was determined following administration on days 1 and 5 by log-linear regression of data The study population consisted of 27 healthy male volunteers (age years; weight kg). All points describing the subsequent 24 h phase of the serum provided written consent. The study was approved by concentration-time curve. The terminal t 1/2 of s.c. and the Besselaar UK Independent Review Board. Volunteers i.v. doses was calculated by log-linear regression of were assigned to receive lenograstim at a once-daily dose subjectively identified data points describing the terminal of 0.5, 2, 5 or 10 mg kg 1 either by s.c. injection or i.v. phase of the serum concentration-time curve, using the infusion (over 30 min) for 5 days and then after a 10-day formula t 1/2 =(ln 2)/l z, where l z is the terminal wash-out period, vice versa for a further 5 days. elimination rate constant. The amount of lenograstim excreted in the urine (Ae) after s.c. and i.v. doses was Collection of blood and urine samples obtained by multiplying the collected urine volume by the concentration of lenograstim. Renal clearance (CL r, For each lenograstim s.c. dose on days 1 and 5, venous (0, 24 h)) was determined from CL r, (0, 24 h)=ae (0, blood samples were taken 30 min prior to dosing, and at 24 h)/auc(0, 24 h). 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16 and 20 h postdose; To test for period effects, comparisons of day 5 AUC(0, additional samples were taken 24, 36, 48 and 72 h after 24 h) and C max values for 2 and 5 mg kg 1 i.v. and s.c. the day 5 dose. For lenograstim i.v. doses on days 1 and groups were made by analysis of variance (anova) fora 5, venous blood samples were taken from the contralateral two-way crossover design. A test for sequence effect was arm 30 min before and 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, performed to assess the likelihood of the occurrence of a 4, 6, 8, 10, 12 and 16 h after the start of infusion. carry-over effect. Additional samples were taken 24 and 36 h after the start of the i.v. infusion on day 5. Absolute neutrophil counts For each s.c. and i.v. dose on days 1 and 5, urine was collected predose and during the entire 24 h postdose Absolute neutrophil counts (ANCs) in venous blood period. samples were measured using a Technicon TM H1 Automate. Detection of lenograstim Pharmacokinetic data analysis A noncompartmental approach was used to calculate pharmacokinetic parameters for lenograstim. Actual times of blood sampling were used for each serum lenograstim concentration vs time profile. Serum and urine concentrations below the lower limit of detection were taken as zero. Maximum serum concentrations (C max ) and the time to C max (t max ) were noted directly. The area under the serum concentration-time curve (AUC) was calcu- Safety measurements Serum and urine samples were analysed for lenograstim using an enzyme-immunoassay (EIA) procedure devel- oped by Chugai Pharmaceutical Co. Ltd [5]. The lower limit of detection of lenograstim in this procedure was 32 pg ml 1 and the upper limit was 1000 pg ml 1. Intra-day precision (determined using control samples) was 17.0% at 62.5 pg ml 1, 10.0% at 250 pg ml 1 and 10.1% at 750 pg ml 1. The corresponding accuracy figures were 104.8%, 93.7% and 88.7%. Routine laboratory tests were performed prior to the start of treatment, on days 1, 2, 4 and 5 of each treatment period, and on days 1, 7 and 10 of the washout period. Supine blood pressure, pulse rate and temperature were recorded, and an electrocardiogram was performed, before and at time points between 4 and 24 h after each dose. Investigators monitored adverse events throughout the study and assessed the likelihood of their relationship to lenograstim treatment (none, unlikely, possibly, probably, highly probably). Results A total of 27 volunteers was recruited. Groups of six volunteers were scheduled to receive lenograstim at doses of 0.5, 2, 5 and 10 mg kg 1. Two volunteers (assigned to 2 and 5 mg kg 1 groups) withdrew voluntarily prior to dosing and were replaced. One volunteer given 2 mg kg 1 s.c. withdrew due to vomiting and leg pain Blackwell Science Ltd Br J Clin Pharmacol, 47,

3 Pharmacokinetics of lenograstim after the first dose and was replaced. Volunteers given Table 2 Bioavailability of lenograstim following subcutaneous 10 mg kg 1 s.c. or i.v. did not enter the second treatment dosing on day 5. phase as all experienced intolerable bone pain and changes Bioavailability (%) in alkaline phosphatase (AP) levels during the first Dose 1 (mg kg 1 ) Mean 2 s.d. treatment phase Pharmacokinetic measurements No period or sequence effect was seen for any pharmacokinetic n.c. parameter mg kg 1 doses, n=3; all other doses, n=6. The main pharmacokinetic parameters (C max, t max, 2 For 0.5, 2 and 5 mg kg 1 doses, the mean bioavailability was AUC(0, 24 h) on days 1 and 5 of the s.c. and i.v. calculated for a crossover design; for 10 mg kg 1 doses, the mean formulations are presented in Table 1. At all doses of bioavailability was calculated for a parallel groups design. lenograstim, mean C max and AUC(0, 24 h)were higher s.d.=standard deviation; n.c.=not calculable. and median t max was shorter following i.v. compared with s.c. administration. Mean C max and AUC(0,24 h) 10 mg kg 1 to 94.1 h [range ] at 0.5 mg kg 1 ) values were dose-dependent with both routes. On day 5, was longer than that of its corresponding i.v. dose (15.9 h the mean absolute bioavailabilities of the s.c. doses were [range ] at 10 mg kg 1 to 55.1 h [range 60.8% at 0.5 mg kg 1, 33.9% at 2 mg kg 1, 30.2% at ] at 0.5 mg kg 1 ). Renal clearance was low, 5 mg kg 1, and 25.7% at 10 mg kg 1 (Table 2). <0.5 ml min 1, for all s.c. and i.v. doses on days 1 and 5. Total clearance of lenograstim decreased with dose following i.v. administration on both days 1 and 5 (day 1CL=75.0 [s.d., 16.9] ml min 1 at 0.5 mg kg 1 to 19.5 Neutrophil response [s.d. 2.7] ml min 1 at 10 mg kg 1 ; day 5 CL=79.1 [s.d. Mean ANC nadirs were observed on day 1 within 15.1] ml min 1 at 0.5 mg kg 1 to 26.0 [s.d min of i.v. administration of lenograstim 5.4] ml min 1 at 10 mg kg 1 ). The mean apparent t 1/2 ( l 1 after 0.5 mg kg 1 ; l 1 after 2, 5 of lenograstim on day 1 and day 5 was h when and 10 mg kg 1 ), and within min of s.c. adminis- given s.c. and h when given i.v., with the longest tration (2.5, 2.2, 1.8 and l 1, respectively). mean apparent t 1/2 of 3.3 h (s.d. 1.3) after 2 mg kg 1 s.c. ANCs then began to rise in a dose-dependent manner on day 5 and the shortest of 0.9 h (s.d. 0.1) after (Figure 1). Greater ANC increases were observed following 0.5 mg kg 1 i.v. on day 1. The median terminal t 1/2 of each s.c. dose compared with its corresponding i.v. each lenograstim s.c. dose (34.8 h [range ] at dose, and higher ANC peaks were attained. While ANCs Table 1 Pharmacokinetic variables of lenograstim following subcutaneous and intravenous dosing. Subcutaneous administration Intravenous administration Dose 1 (mg kg 1 ) Day 1 Day 5 Day 1 Day 5 C max (ng ml 1 ) Mean s.d. Mean s.d. Mean s.d. Mean s.d t max (h) Median Range Median Range Median Range Median Range AUC(0, 24h) (ng ml 1 h) Mean s.d. Mean s.d. Mean s.d. Mean s.d mg kg 1 doses, n=3; all other doses, n=6. s.d.=standard deviation; C max =maximum serum concentration; t max =time to C max ; AUC(0, 24h)=area under the serum concentration time curve Blackwell Science Ltd Br J Clin Pharmacol, 47,

4 Mean ANC ( x 10 9 l 1 ) A. C. Houston et al. 50 were greater following s.c. than i.v. administration; rises above the normal ranges (AP= i.u., LDH= i.u., UA= i.u.) were seen following all s.c. doses except the 0.5 mg kg 1 s.c. dose, and after the 10 mg kg 1 i.v. dose. All laboratory values returned to normal within 7 10 days of the final dose. No significant changes in blood pressure, pulse rate or 20 temperature were recorded during the study, and electrocardiograms were normal. 10 Lenograstim was generally well tolerated when given s.c. and i.v. at doses of up to 5 mg kg 1, and there was 0 no apparent difference in the incidence of adverse events 1* 2* 3* 4* 5* 6 (Table 3). The total number of adverse events increased Days (lenograstim* treatment) with dose, and the most common were headache, Figure 1 Mean absolute neutrophil count (ANC) following back/spine pain, generalized pain and pharyngitis. Seven subcutaneous (s.c. ---) and intravenous (i.v. ) administration of volunteers (one with 5 mg kg 1 s.c.; three with lenograstim (0.5 (&), 2 (2), 5 (+) mg kg 1, n=6; mg kg 1 s.c.; and three with 10 mg kg 1 i.v.) required ($) mg kg 1, n=3) to healthy volunteers for 5 days. ibuprofen (400 mg) for pain relief. Twenty-eight adverse events were considered by the investigators to be highly remained relatively constant from day 2 following the probably related to lenograstim treatment (22 bone pain, 0.5, 2 and 5 mg kg 1 i.v. doses, rises were observed until 2 generalized aches, 1 joint pain, 1 neck pain, 1 left day 6 after the 5 and 10 mg kg 1 s.c. doses. A mean subcostal pain, 1 pharyngitis); all occurred at the ANC peak of l 1 on day 6 followed the 10 mg kg 1 dose level (11 s.c.; 17 i.v.). 5 mg kg 1 s.c. doses; the mean ANC peak for 5 mg kg 1 i.v. on day 2 was l 1. Discussion Safety Increases in alkaline phosphatase (AP), lactate dehydrogenase (LDH), and uric acid (UA) concentrations were observed. Changes were maximal on days 5 and 6 and The results of this study in healthy volunteers demonstrate that the pharmacokinetics of both s.c. and i.v. lenograstim are dose- and time-dependent. However, the maximum serum concentration of lenograstim is higher and attained sooner following i.v. administration, yet t 1/2 is longer and Lenograstim dose (mgkg 1 ) s.c. i.v. s.c. i.v. s.c. i.v. s.c. i.v. Table 3 Adverse events following subcutaneous (s.c.) and intravenous (i.v.) dosing of lenograstim. Number of subjects Number of subjects with adverse events Number of adverse events Headache Back/spine pain Pharyngitis Abdominal pain Diarrhoea Rash Bone pain Unclassified pain Other Includes data for volunteer who withdrew after one dose, but not for volunteer who withdrew before any treatment. 2 Includes data for volunteer who withdrew before any treatment. 3 Volunteers received either 10 mg kg 1 s.c. or i.v. dosing during the first treatment phase. 4 Does not include data for volunteer who withdrew before any treatment. 5 Adverse events where total incidence is < Blackwell Science Ltd Br J Clin Pharmacol, 47,

5 Pharmacokinetics of lenograstim ANC rises are more pronounced when the s.c. route Previous studies agree that lenograstim is generally well is used. tolerated [4]. Bone pain in the present study in healthy Previously published data on the pharmacokinetics of volunteers appeared more severe than that seen previously lenograstim are limited. The present results are in with filgrastim in studies of patients with bone marrow agreement with an earlier study in healthy Japanese adults suppression [10]. A possible explanation for the difference that also showed the pharmacokinetics of lenograstim is that the bone pain is related to increased bone marrow (1 40 mg day 1 i.v.) to be dose- and time-dependent activity: a greater intensification of activity, and hence [5]. The time-dependent component may be associated pain, may occur in healthy adults compared with patients with the accompanying rise in ANCs through which with bone marrow suppression. there is an increase in the capacity of neutrophil receptormediated In summary, the good pharmacokinetic profile, neutro- endocytosis and degradation [6]. Increasing phil response, and tolerability of both s.c. and i.v. clearance of lenograstim with rising ANCs, to a maximum administered lenograstim seen in this study support the of 2 ml min 1 kg 1 at cells l 1, has been use of either route to treat or prevent neutropenia. observed [2]. Similarly, when nonglycosylated rhug- However, the s.c. route offers benefits of a greater and CSF (filgrastim, 5 15 mg kg 1 s.c.) was administered to more prolonged neutrophil response. children with severe chronic neutropenia, increased clearance, from 0.40 to 0.74 ml min 1 kg 1, Lenograstim was supplied by Chugai Pharmaceutical Co. Ltd, Japan, and the study was supported by a grant from Chugai accompanied rises in ANCs [7]. The present study Rhône-Poulenc, France. showed the renal clearance of lenograstim to be low, but there is evidence that degradation by hepatic enzymes and proteolytic enzymes in mature neutrophils are further References important mechanisms [6]. Clearance of G-CSF may be 1 Steward WP. Granulocyte and granulocyte-macrophage a homeostatic mechanism to control neutrophil levels. colony-stimulating factors. Lancet 1993; 342: The longer apparent t 1/2 following s.c. compared with 2 Frampton JE, Yarker YE, Goa KL. Lenograstim: a review of i.v. administration is probably due to the prolonged release its pharmacological properties and therapeutic efficacy in of the former from its site of administration to the systemic neutropenia and related clinical settings. Drugs 1995; 45: circulation. The apparent t /2 of the lower lenograstim i.v. 3 Frampton JE, Lee CR, Faulds D. Filgrastim: a review of its doses (0.5, 2, 5 mg kg 1 ) in the present study of healthy pharmacological properties and therapeutic efficacy in volunteers (#1 h) is similar to that found for similar doses neutropenia. Drugs 1994; 48: of filgrastim (1 3 mg kg 1 ) administered i.v. to cancer 4 Morstyn G, Souza LM, Keech J, et al. Effect of granulocyte patients (#1.5 h) [8]. Longer apparent t 1/2 shave,however, colony stimulating factor on neutropenia induced by been observed following higher i.v. doses of filgrastim cytotoxic chemotherapy. Lancet 1988; i: (10 60 mg kg 1 )incancerpatients(#3.5 4 h) [6]. It is 5 Sekino H, Moriya K, Sugano T, Wakabayashi K, Okasaki A. suggested that the apparent t Recombinant human G-CSF (rg-csf). Shinryo to Shinyaku 1/2 sofrhug-csfsareaffected 1989; 26: by some disease states, and that clearance mechanisms are 6 Layton JE, Hockman H, Sheridan WP, Morstyn G. saturated at higher doses [2, 9]. Evidence for a novel in vivo control mechanism of The dose-dependent increase in C max following leno- granulopoiesis: mature cell-related control of a regulatory grastim administration in the present study is in agreement growth factor. Blood 1989; 74: with data for filgrastim [3]. The observed t max following 7 Stute N, Sanatan VM, Rodman JH, Schell MJ, Ihle JN, s.c. administration (4 9 h) fits well with the study by Evans WE. Pharmacokinetics of subcutaneous recombinant Stute et al. [7] which reports the t max of filgrastim to granulocyte colony-stimulating factor in children. Blood be 4 12 h. 1992; 79: Kearns CM, Wang WC, Stute N, Ihle JN, Evans WE. The initial nadir and subsequent rapid increase in ANC Disposition of recombinant granulocyte colony-stimulating observed following lenograstim administration in this factor in children with severe neutropenia. J Paediatr 1993; study is similar to previous time-dependent neutrophil 123: responses reported from studies in both healthy subjects 9 Morstyn G. The impact of colony-stimulating factors as and cancer patients [4, 5, 10 12]. Lenograstim has been cancer chemotherapy. Br J Haematol 1990; 75: shown to be more potent than filgrastim in increasing 10 Hollingshead LM, Goa KL. Recombinant granulocyte ANCs both in vitro [13] and in vivo [14]. The more colony-stimulating factor: a review of its pharmacological properties and prospective role in neutropenic conditions. pronounced elevation in ANCs seen in the present study Drugs 1991; 42: in response to s.c. compared with i.v. administration of 11 Sato N, Sawada K, Takahashi TA, et al. A time course study lenograstim indicates that the s.c. route may be more for optimal harvest of peripheral blood cells by granulocyte appropriate when aiming to achieve a large and sustained colony-stimulating factor in healthy volunteers. Exp Haematol neutrophil recovery. 1994; 22: Blackwell Science Ltd Br J Clin Pharmacol, 47,

6 A. C. Houston et al. 12 Lane TA, Law P, Maruyama M, et al. Harvesting and of the biological potency of glycosylated versus nonenrichment of haemopoietic progenitor cells mobilized into glycosylated rg-csf. Drug Invest 1994; 7: the peripheral blood of normal donors by granulocyte- 14 Höglund M, Smedmyr B, Bengtsson M, et al. Mobilization macrophage colony-stimulating factor (GM-CSF) or G-CSF. of CD34+ cells by glycosylated and nonglycosylated G-CSF potential role in allogeneic marrow transplantation. Blood in healthy volunteers a comparative study. Eur J Haematol 1995; 85: ; 59: Nissen C, Dalle Carbonare V, Moser Y. In vitro comparison Blackwell Science Ltd Br J Clin Pharmacol, 47,