|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the University of Illinois at Chicago, Chicago,
IL; VA Chicago West Side Division, Chicago, IL; Pharmachemie BV,
Haarlem, The Netherlands.
Augmentation of the fetal hemoglobin (HbF) levels is of
therapeutic benefit in patients with sickle cell anemia. Hydroxyurea (HU), by increasing HbF, lowers rates of pain crisis, episodes of acute
chest syndrome, and requirements for blood transfusions. For patients
with no HbF elevation after HU treatment, augmentation of HbF levels by
5-aza-2'-deoxycytidine (5-aza-CdR, decitabine) could serve as an
alternate mode of treatment. Eight adult patients participated in a
dose-escalating phase I/II study with 5-aza-CdR at doses ranging from
0.15 to 0.30 mg/kg given 5 days a week for 2 weeks. HbF, F cell, F/F
cell, Sickle cell anemia occurs as a result of a mutation
in the The abnormal hemoglobin ( The clinical severity is altered by Laboratory and clinical studies using hydroxyurea (HU) in primates and
in patients with sickle cell anemia demonstrated a modest increase in
HbF.9,10 Patients randomized to HU in a clinical had lower
rates of painful crises, fewer episodes of the acute chest syndrome,
and fewer requirements for blood transfusions.11-14 Some
patients had an insignificant change in HbF and reported clinical
well-being. However, the reports of secondary malignancies in patients
with polycythemia rubra vera treated with HU have raised concerns about
its long-term safety.15-18
Other agents that enhance HbF production include erythropoietin, as a
single agent or in combination with HU. Butyrate and other short-chain
fatty acids also stimulate fetal globin expression with increases in
DeSimone and coworkers demonstrated, in primates, the augmentation of
HbF to levels of 70% of total hemoglobin using 5-azacytidine (5-aza-C), and a similar response in HbF with the analog
5-aza-2'-deoxycytidine (5-aza-CdR).23,24 These primate
studies were followed by phase I clinical trials with 5-aza-C in
patients with sickle cell disease and The insufficient documentation of adverse events led us to conduct a
clinical trial (phase I/II) with 5-aza-CdR in patients who were
nonresponders to HU, because HU serves as the standard of comparison
for drugs capable of inducing HbF.13,32 The purpose of the
study was to determine the dose of 5-aza-CdR required to increase the
HbF level using the end points of Patient selection, inclusion and exclusion criteria
Patients were excluded from participation if they were pregnant or of
child-bearing age and were unwilling to use contraception. Other
reasons for exclusion were liver or renal disease (alanine transaminase
> 300 IU or albumin < 2.0 g/dL; creatinine > 2.5 mg/dL); history
of malignancy; positive for human immunodeficiency virus or acquired
immunodeficiency syndrome; cerebrovascular accident within the last
year; proven response to HU with increase in HbF or clinical
improvement. Patients on chronic blood transfusion therapy were also
excluded. Patients were allowed to withdraw from the study at any time
for reasons of their own.
Drug administration
Treatment Eligible patients were enrolled in cohorts of 3. Patients were allowed to re-enter successive cohorts after laboratory tests showed a return to baseline -chain synthesis. The starting dose of 5-aza-CdR
was 0.15 mg/kg per day, based on preclinical studies in our
laboratory.24 Patients in cohorts 1 and 2 received this dose intravenously for 5 days a week for 2 consecutive weeks, followed
by 2 weeks off therapy. Treatment was repeated for 2 consecutive weeks,
followed by 6 weeks of observation. The dose was escalated in
subsequent cohorts by 0.05 mg/kg per day. Patients in cohorts 3 and 4 were treated for 2 weeks followed by 6 weeks of observation. Baseline
HbF, F cells, F reticulocytes, and /( S+) synthesis
ratios were determined on all patients. HbF and F cells were repeated
weekly, and the -chain synthesis ratios were repeated on days 15 and
22 of each cycle.
Laboratory testing and monitoring Clinical evaluation and laboratory tests for toxicity were repeated weekly and as necessary. Toxicity was evaluated using the National Cancer Institute Common Toxicity Criteria. Complete blood cell and reticulocyte counts were obtained by standard procedures.The F cell number was determined by the acid elution method.33 HbF was measured by alkali denaturation in 2 independent laboratories.34 In some samples containing more than 5% HbF, it was also quantified by high-performance liquid chromatography (HPLC). Globin chain synthesis was measured by incorporation of [3H]-leucine into globin chains of reticulocytes followed by separation of globin chains by HPLC using a C4 column.35
Three men and 5 women ranging in age from 18 to 46 years completed the trial. The starting dose was 0.15 mg/kg per day; the dose was increased in subsequent cohorts by 0.05 mg/kg per day, and the highest dose tested was 0.30 mg/kg per day. Six patients received both single- and 2-cycle treatment regimens and 2 patients were treated only once at the 0.15-mg dose level. Patients receiving 0.15 and 0.20 mg/kg per day were treated for 2 cycles. Patients receiving higher doses were treated for only a single cycle. The 2-cycle protocol was replaced by single-cycle therapy after completion of the 0.2-mg/kg dose to achieve a more rapid dose escalation. Toxicity None of the patients experienced any untoward effects during administration of the drug or of nausea or vomiting at any time during the treatment period. There was no report of use of antiemetics. All patients showed a decrease in white blood count and 2 developed absolute neutropenia (absolute neutrophil count < 500), with resolution within 3 days and none developed fever or infection. Hemoglobin levels increased by at least 1 g/dL in 6 patients (Table 1). Platelet counts were stable during treatment and subsequently increased an average increment of 220% by the end of the cycle.
Effect of 5-aza-CdR on HbF Table 1 shows the maximum HbF and other hematologic parameters achieved at each dose level. A typical temporal HbF response to 5-aza-CdR (patient 3, treated at 0.20 mg/kg per day for 2 cycles) is presented in Figure 1. Also included in the figure are absolute neutrophil count, hemoglobin, and absolute reticulocyte count. The HbF response begins after only 1 week of treatment and is biphasic, with the first peak occurring 3 to 4 weeks after beginning the cycle. The maximum HbF is attained after the second cycle and is approximately 35% greater than the initial peak.
The average Table 2 shows the maximum HbF for
each of the patients who were nonresponsive to HU. These 5 patients had
little or no elevations in HbF despite compliance with HU. The HbF
(percentage of total hemoglobin ± SD) measured before and during
compliant HU therapy remained unchanged. However, after 5-aza-CdR
administration, the average maximum HbF was 12.7% ± 1.8%, a
5.6-fold increase over baseline and a 35-fold greater increase over
that seen during HU therapy. The peak HbF was observed 5 weeks after
the start of 5-aza-CdR therapy as compared to the lack of response
after more than 24 weeks with HU.
The 2 HU responders who were unable to sustain the levels (patients 6 and 7, Table 1) were treated with 5-aza-CdR and attained HbF of 15.9% (0.15 mg/kg) and 20.2% (0.30 mg/kg), respectively. This represents an average 52% increase over that seen with HU. Only patient 8 in the study had not been previously treated with HU. This patient achieved a maximum HbF of 18.1% with the lowest dose of 5-aza-CdR (0.15 mg/kg). Six patients were treated at 2 dose levels of 5-aza-CdR (Figure
2). Because there were large individual
variations in baseline HbF and response to therapy, the effect of the
higher dose level was always compared with each patient's response at
the lower dose. Although a dose-response relationship was not clearly
demonstrated, increasing the dose of 5-aza-CdR consistently resulted in
an increased maximum HbF within a given patient.
Effect of 5-aza-CdR on platelets, neutrophils, and reticulocytes Generally, 5-aza-CdR is considered to be a cytotoxic drug and has been reported to have a marked effect on myelopoiesis at the chemotherapeutic concentrations used for the treatment of hematologic malignancy (> 10 4 mol/L).36 To define the
toxicity of low-dose 5-aza-CdR on blood cell production we analyzed the
changes in platelet, neutrophil, and absolute reticulocyte counts after
5-aza-CdR treatment. Figure 3 presents the temporal changes in these
blood cell elements during the course of 5-aza-CdR treatment. Each
point on each curve is the average value for that element obtained at
weekly intervals for all patients and all treatment doses. Unlike other
cytotoxic agents, low-dose 5-aza-CdR treatment resulted in a transient
increase in platelet count that peaked at 220% above baseline 5 weeks
(week 6) after initiation of treatment (P < .0001), and
returned toward baseline by the seventh week. This increase in
platelets was mirrored by a transient decline in neutrophils with a
nadir of 32% of baseline at 6 weeks (P < .0001). Table 1
shows the individual neutrophil counts at the nadir. In patients whose
neutrophil counts decreased below 1000, the nadir lasted only 1 to 3 days, and baseline values were re-established by the seventh week even
when they were treated with a 2-cycle regimen (Figure 2). The changes
in platelet and neutrophil counts first appeared at about the third
week of a cycle. There was a reduction in absolute reticulocyte count,
with a nadir of 59% of baseline at 6 weeks. The decrease in absolute reticulocyte count, although not as steep as that seen in neutrophil count, was observed after only 1 week of treatment. Total hemoglobin levels increased (a mean of 1 g/dL approximately 12%) by the 6th week
(Table 1, P < .0001 compared to baseline). None of the
patients developed any sickle cell disease-related events during the
period of study.
We have demonstrated that low-dose 5-aza-CdR is effective in augmenting HbF in the 8 patients studied. The only observed drug-related adverse change was a transient neutropenia, which was not accompanied by any clinical sequelae. The surprising finding is that 5-aza-CdR can augment HbF in patients who do not respond to HU. The maximum HbF attained in these patients was 6-fold higher than baseline. The mechanism of action of 5-aza-CdR is likely to be different from
mere cytotoxicity. 5-aza-CdR has been shown to cause DNA hypomethylation through inhibition of DNA methyltransferase
(DMT).37 Hypomethylation is correlated with changes in
gene expression, and increased Administration of low doses of 5-aza-CdR, as used in our patients, may be essential for optimal stimulation of HbF production and optimal therapeutic effect. Higher doses may result in greater neutropenia and may also blunt the HbF response. Jutterman and colleagues have recently demonstrated that after incorporation into DNA, 5-aza-CdR binds covalently to DMT blocking DNA polymerase progression, and at high level, adduct formation results in growth arrest and cell death.43 The limited covalent adduct formation occurring during low-dose 5-aza-CdR therapy can be efficiently repaired allowing DNA polymerase progression, and survival of hypomethylated cells. Although 5-aza-CdR was effective in augmenting HbF in our patients, we do not advocate its use in patients who are responding well to HU. We do see a potential role for 5-aza-CdR in patients who are not responding to HU and in those with pretreatment HbF below 2.0%, for the latter patients appear to be the majority of the nonresponders to HU.12,14 Patients who are candidates for elective surgery and have multiple red blood cell antibodies may benefit from preoperative treatment with 5-aza-CdR, because of the rapid escalation in both hemoglobin and HbF. Although only a small number of patients were studied, our results are encouraging. We have started a second phase of the study to determine whether HbF levels can be maintained optimally for 36 weeks by increasing or decreasing doses and cycle length according to duration of effect and toxicity in each patient. This phase I/II dose escalation trial demonstrated the ability of 5-aza-CdR to stimulate HbF production with minimal toxicity and in patients who had not responded to HU. Studies to demonstrate a sustained effect of 5-aza-CdR by repeated intermittent doses are in progress. An oral formulation of 5-aza-C in combination with tetrahydrouridine to ensure bioavailability would be a welcome alternate drug for patients with sickle cell anemia.24 Obviously, long-term safety of the drug should continue to be a consideration before any drug can be offered for routine treatment of patients.
Submitted December 7, 1999; accepted June 6, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mabel Koshy, University of Illinois at Chicago, 840 South Wood St (M/C 787), Chicago, IL 60612; e-mail: mkoshy{at}uic.edu.
1.
Platt O, Brambilla DJ, Rosse WF, et al.
Mortality in sickle cell disease: life expectancy and risk factors for early death.
N Engl J Med.
1994;330:1639-1644 2. Koshy M, Leikin J, Dorn L, Lebby T, Talischy N, Telfer MC. Evaluation and management of sickle cell disease in the emergency department (an 18-year experience): 1974-1992. Am J Ther. 1994;1:309-320[Medline] [Order article via Infotrieve]. 3. Koshy M, Thomas C, Goodwin J. Vascular lesions in the central nervous system in sickle cell disease (neuropathology). J Assoc Acad Minor Phys. 1990;1:71-78[Medline] [Order article via Infotrieve]. 4. Koshy M, Burd L, Wallace D, Moawad A, Baron J. Prophylactic red-cell transfusions in pregnant patients with sickle cell disease A randomized cooperative study. N Engl J Med. 1988;319:1447-1452[Abstract]. 5. Koshy M. Sickle cell disease and pregnancy. Blood Rev. 1995;9:157-164[Medline] [Order article via Infotrieve]. 6. Watson J, Staldman AW, Bilello FP. The significance of the paucity of sickle cells in newborn Negro infants. Am J Med Sci. 1948;215:419-423[Medline] [Order article via Infotrieve].
7.
Conley CL, Weatherall DJ, Richardson SN, Shepard MK, Charache S.
Hereditary persistence of fetal hemoglobin: a study of 79 affected persons in 15 Negro families in Baltimore.
Blood.
1963;21:261-281 8. Perrine SP, Pembry ME, John P, Perrine S, Shoup F. Natural history of sickle cell anemia in Saudi Arabs. Ann Intern Med. 1978;88:1-6. 9. Letvin NL, Linch DC, Beardsley GP, McIntyre KW, Nathan DG. Augmentation of fetal hemoglobin in anemic monkeys by hydroxyurea. N Engl J Med. 1984;310:869-873[Abstract]. 10. Platt OS, Orkin SH, Dover G, Beardsley GP, Miller B, Nathan DG. Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia. J Clin Invest. 1984;74:652-656.
11.
Charache S, Dover GJ, Moore RD, et al.
Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia.
Blood.
1992;79:2555-2565
12.
Charache S, Terrin ML, Moore RD, et al.
Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia.
N Engl J Med.
1995;332:1317-1322
13.
Steinberg MH, Lu Z, Barton FB, Terrin MI, Charache S, Dover GJ.
Fetal hemoglobin in sickle cell anemia: determinants of response to hydroxyurea. Multi-center study of hydroxyurea.
Blood.
1997;89:1078-1088 14. Charache S. Mechanism of action of hydroxyurea in the management of sickle cell anemia in adults. Semin Hematol. 1997;34:15-21[Medline] [Order article via Infotrieve].
15.
Ho PTC, Murgo AJ, Silver RT.
Hydroxyurea and sickle cell crisis.
N Engl J Med.
1995;333:1008 16. Murphy S, Peterson P, Iland H, Laszlo J. Experience of the Polycythemia Vera Study Group with essential thrombocythemia: a final report on diagnostic criteria, survival and leukemia transition by treatment. Semin Hematol. 1997;34:29-39[Medline] [Order article via Infotrieve]. 17. Lofvenberg E, Wahlin A. Management of polycythaemia vera, essential thrombocythemia and myelofibrosis with hydroxyurea. Eur J Haematol. 1988;41:375-381[Medline] [Order article via Infotrieve]. 18. Furgerson JL, Vukelja SJ, Baker WJ, O'Rourke TJ. Acute myeloid leukemia evolving from essential thrombocytopenia in two patients treated with hydroxyurea. Am J Hematol. 1996;51:137-140[Medline] [Order article via Infotrieve].
19.
Perrine SP, Miller BA, Faller DV, et al.
Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia.
Blood.
1989;74:454-459
20.
Constantoulakis P, Knitter G, Stamatoyannopoulos G.
On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-azaC and AraC.
Blood.
1989;74:1963-1971
21.
Dover GJ, Brusilow S, Charache S.
Induction of fetal hemoglobin production in subjects with sickle cell anemia by oral sodium phenylbutyrate.
Blood.
1994;84:339-343
22.
Atweh GF, Sutton M, Nassif I, et al.
Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease.
Blood.
1999;93:1790-1797
23.
DeSimone J, Heller P, Hall L, Zwiers D.
5-Azacytidine stimulates fetal hemoglobin (HbF) synthesis in anemic baboons.
Proc Natl Acad Sci U S A.
1982;79:4428-4431 24. DeSimone J, Heller P, Molokie RE, Hall L, Zwiers D. Tetrahydrouridine, cytidine analogues, and hemoglobin F. Am J Hematol. 1985;18:283-288[Medline] [Order article via Infotrieve].
25.
Ley TJ, DeSimone J, Noguchi CT, et al.
5-Azacytidine increases
26.
Charache S, Dover G, Smith K, Talbor CC, Moyer M, Boyer S.
Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the
27.
Ley TJ, DeSimone J, Anagou NP, et al.
5-Azacytidine selectively increases
28.
Lowrey CH, Nienhuis AW.
Brief report: treatment with azacytidine of patients with end-stage
29.
Carr BI, Reilly J, Smith S, Winberg C, Riggs A.
The tumorigenicity of 5-azacytidine in the male Fischer rat.
Carcinogenesis.
1984;5:1583-1590 30. Carr BI, Rahbar S, Asmeron Y, Riggs A, Winberg CD. Carcinogenicity and haemoglobin synthesis induction by cytidine analogues. Br J Cancer. 1988;57:395-402[Medline] [Order article via Infotrieve]. 31. Laird PW, Jackson-Grusby L, Fazeli A, et al. Suppression of intestinal neoplasia by DNA hypomethylation. Cell. 1995;81:197-205[Medline] [Order article via Infotrieve].
32.
Bunn HF.
Induction of fetal hemoglobin in sickle cell disease.
Blood.
1999;93:1787-1789 33. Kleihauer E, Braun E, Betke K. Demonstration von fetalem haemoglobin in den erythrocyten eines blutausstrichs. Klin Wochenschr. 1957;35:637-638[Medline] [Order article via Infotrieve].
34.
Singer K, Chernoff AL, Singer L.
Studies on abnormal hemoglobins, I: their demonstration in sickle cell anemia and other hematologic disorders by means of alkali dentauration.
Blood.
1951;6:413-428
35.
DeSimone J, Schroeder WA, Shelton JB, et al.
Speciation in the baboon and its relation to gamma-chain heterogeneity and to the response to induction of HbF by 5-azacytidine [abstract].
Blood.
1984;63:1088-1095 36. Covey JM, Zaharko DS. Comparison of the in vitro cytotoxicity (L1210) of 5-aza-2'-deoxycytidine with its therapeutic and toxic effects in mice. Eur J Clin Oncol. 1985;21:109-117.
37.
Creusot F, Acs G, Christman JK.
Inhibition of DNA methyltransferase and induction of friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2'-deoxycytidine.
J Biol Chem.
1982;257:2041-2048
38.
Lavelle D, DeSimone J, Heller P, Zwiers D, Hall L.
On the mechanism of HbF elevations in the baboon by erythropoietic stress and pharmacological manipulation.
Blood.
1986;67:1083-1089
39.
Fauser AA, Messner HA.
Identification of megakaryocytes, macrophages and eosinophils in colonies of human bone marrow containing neutrophilic granulocytes and erythroblasts.
Blood.
1979;53:1023-1027
40.
Nicola NA.
The production of committed hemopoietic colony-forming cells from multi-potential precursor cells in vitro.
Blood.
1982;60:1019-1029
41.
Visvader J, Adams JM.
Megakaryocytic differentiation induced in 416B myeloid cells by GATA-2 and GATA-3 transgenes or 5-azacytidine is tightly coupled to GATA-1 expression.
Blood.
1993;82:1493-1501 42. Dover GJ, Boyer SH, Charache S, Heintzelman H. Individual variation in the production and survival of F-cells in sickle cell disease. N Engl J Med. 1978;299:1428-1435[Abstract].
43.
Juttermann R, Li E, Jaenisch R.
Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA methylation.
Proc Natl Acad Sci U S A.
1994;91:11797-11801
© 2000 by The American Society of Hematology.
| |||||||||||
 |
|
 |
|
  D. Lavelle, Y. Saunthararajah, and J. DeSimone DNA methylation and mechanism of action of 5-azacytidine Blood, February 15, 2008; 111(4): 2485 - 2485. [Full Text] [PDF]
|
|
|
|
|
|
H. Fathallah, R. S. Weinberg, Y. Galperin, M. Sutton, and G. F. Atweh Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin Blood, November 1, 2007; 110(9): 3391 - 3397. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hsu, R. Mabaera, C. H. Lowrey, D. I. K. Martin, and S. Fiering CpG Hypomethylation in a Large Domain Encompassing the Embryonic {beta}-Like Globin Genes in Primitive Erythrocytes Mol. Cell. Biol., July 1, 2007; 27(13): 5047 - 5054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. P. Kransdorf, S. Z. Wang, S. Z. Zhu, T. B. Langston, J. W. Rupon, and G. D. Ginder MBD2 is a critical component of a methyl cytosine-binding protein complex isolated from primary erythroid cells Blood, October 15, 2006; 108(8): 2836 - 2845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Gollob, C. J. Sciambi, B. L. Peterson, T. Richmond, M. Thoreson, K. Moran, H. K. Dressman, J. Jelinek, and J.-P. J. Issa Phase I Trial of Sequential Low-Dose 5-Aza-2'-Deoxycytidine Plus High-Dose Intravenous Bolus Interleukin-2 in Patients with Melanoma or Renal Cell Carcinoma Clin. Cancer Res., August 1, 2006; 12(15): 4619 - 4627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Rupon, S. Z. Wang, K. Gaensler, J. Lloyd, and G. D. Ginder Methyl binding domain protein 2 mediates {gamma}-globin gene silencing in adult human betaYAC transgenic mice PNAS, April 25, 2006; 103(17): 6617 - 6622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Fathallah and G. F. Atweh Induction of Fetal Hemoglobin in the Treatment of Sickle Cell Disease Hematology, January 1, 2006; 2006(1): 58 - 62. [Abstract] [Full Text] |
Top
Abstract
Introduction
-globin synthesis prior to therapy was 3.19% ± 1.43% and
increased to 13.66% ± 4.35% after treatment. HbF increased from
3.55% ± 2.47% to 13.45% ± 3.69%. F cells increased from
21% ± 14.8% to 55% ± 13.5% and HbF/F cell increased from 17%
to 24%. In the HU nonresponders HbF levels increased from 2.28% ± 1.61% to 2.6% ± 2.15% on HU, whereas on 5-aza-CdR HbF increased to 12.70% ± 1.81%. Total hemoglobin increased by 1 g/dL in 6 of 8 patients with only minor reversible toxicities, and all
patients tolerated the drug. Maximum HbF was attained within 4 weeks of
treatment and persisted for 2 weeks before falling below 90% of the
maximum. Therefore 5-aza-CdR could be effective in increasing HbF in
patients with sickle cell anemia who failed to increase HbF with HU.
Demonstration of sustained F levels with additional treatment cycles
without toxicity is currently being performed.
(Blood. 2000;96:2379-2384)
,
neutrophils/1000; 
, Hb g/dL; *, treatment with 0.2 mg/mL; 
, HbF
%; 
, absolute reticulocytes/10-5.
, before
treatment; 
, hydroxurea;
, 0.15 mg/kg; 
, 0.20 mg/kg; 
, 0.25 mg/kg; 
, 0.30 mg/kg.
-