United States Patent [19]
`Vance et al.
`
`lIllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
`5,541,158
`Jul. 30, 1996
`
`US005541158A
`[11] Patent Number:
`[45] Date of Patent:
`
`[54] METHOD FOR INCREASING THE
`HEMATOCRIT OF A NORMAL MAMMAL
`
`[75] Inventors: John F. A. Vance, Stowe, Vt.; Robert
`I. Abels, West?eld, N.J.; Freedolph D.
`Anderson, Neshanic Station, N.J.;
`William L. Harris, Port Murray, N .J .;
`Dorothy Thompson, Hulmeville, Pa.
`
`[73} Assignee: Ortho Pharmaceutical Corporation,
`Raritan, NJ.
`
`[21) Appl. No.: 409,223
`[22] Filed:
`Sep. 19, 1989
`
`Related U.S. Application Data
`
`[63] Continuation-impart of Ser. No. 36,646, Apr. 10, 1987,
`abandoned.
`
`[51] Int. Cl.6 ........................ .. A61K 38/16; A61K 35/14;
`A01N 37/18; C07K 1/00
`[52] U.S. Cl. ................................... .. 514/8; 514/2; 514/21;
`530/380; 530/397
`[58] Field of Search ................... .. 514/8, 2, 21; 530/397,
`530/380
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,013,718
`
`5/1991 Adamson et a1. ........................ .. 514/8
`
`FOREIGN PATENT DOCUMENTS
`2171303 8/1986 United Kingdom.
`2171304 8/1986 United Kingdom.
`
`OTHER PUBLICATIONS
`
`Hauer ct al. (1984) Vox Sang. 46:8-12.
`Udupa et a1. (1984) J. Lab. Clin. Med. 1031574-580.
`Egrie ct a1. (1985) Prog. Clin. Biol. Res. 191:339-350.
`Rodgers et al. (1975) Proc. Soc. Exp. Biol. Med.
`148:380-382.
`Schustack et a1. (1985) Clin. Nephrology 23(6):303-306.
`Hedstrand et al. (1977) Scand. J. Haematol. 19:417-423.
`Cottino et al. (1985) Acta Gerontol. 35(3/4):2l7—222
`(abstract only).
`
`Maeda et al. (1989, Jul. 29) The Lancet 2(8657):284.
`Levine et al. (1988) Surgery 104:365-369.
`Masunaga et al., Acta Haematol JPN, 49(4):807-815 (1986).
`Essers et al., Proc. Eur. Dial. Transplant Assoc., 11:398-402
`(1974).
`Winearls et al., Lancet, pp. 1175-1178 (Nov. 22, 1986).
`Eschbach et al., N. Eng. J. Med, 316:73-78 (1987).
`Eschbach et al, J. Clin. Invest, 74:434-441 (1984).
`Egrie et al., ImmunobioL, 1722213-224 (1986).
`Spivak et al., Johns Hopkins Med. J., 1461311-320 (1980).
`Schustack et al., Clin. Nephrol., 23(6):303—306 (1985).
`Estrella et al., Clinica Chirnica Acta, 164:1-6 (1987).
`Hedstrand et al., Scand. J. HaematoL, 19(5):4l7—423
`(1977).
`Udupa et al., J. Lab. Clin. Med., 103(4):574-580 (1984).
`Feleppa, Scand. J. Haemat., 10:186-188 (1973).
`Feleppa, Pharm. Res. Comm, 4(4):?163-367 (1972).
`Beru et al., J. Biol. Chem, 260(16):925l-9257 (1985).
`Rotruck et al., J. Agric. Food Chem, 27(l):27-33 (1979).
`Kickler et al., JAMA, 260(1):65—67 (1988).
`Levine et al., J. Trauma, 29(8):1134-1139 (1989).
`Maeda et al., Lancet, p. 284 (Jul. 29, 1989).
`Schwenk et al., DICP, 23(7-8):528-536 (1989).
`Levine et al., Surgery, 104(2):?165-369 (1988).
`Goodman & Gilman’s, The Pharmacological Basis of Thera
`peutics, 7th Ed., pp. 1131-1134 (1985).
`
`Primary Examiner-Robert A. Wax
`Assistant Examiner-Lisa J. Hobbs
`Attorney, Agent, or Firm—John W. Walleu, 111
`
`[57]
`
`ABSTRACT
`
`A method for increasing the hematocn't of a normal mammal
`using erythropoietin (EPO) is provided. The method com
`prises the steps of administering to the mammal a hcmatocrit
`increasing elfective amount of EPO, in a pharmaceutically
`acceptable form. Additionally administered is an effective
`amount of iron, in a pharmaceutically acceptable form,
`suf?cient to increase the serum iron content of the mammal
`to an erythropoiesis supportable level. The method is useful
`for increasing the amount of blood that can be donated for
`transfusion purposes, in particular autologous transfusion.
`
`22 Claims, 4 Drawing Sheets
`
`Pharmacosmos, Exh. 1041, p. 1
`
`

`
`US. Patent
`
`Jul. 30, 1996
`
`Sheet 1 0f 4
`
`5,541,158
`
`1 A HEMATOLOGIC PARAMETERS IN
`ERYTHROPOIETIN TREATED SUBJECTS
`PRIOR TO IRON LOADING
`
`I
`
`1s -
`
`17
`
`HGB(G/DL) 16
`
`15'
`
`14
`
`Pharmacosmos, Exh. 1041, p. 2
`
`

`
`US. Patent
`
`Jul. 30, 1996
`
`Sheet 2 of 4
`
`5,541,158
`
`FIG 1 B HEMATOLOGIC PARAMETERS IN
`I
`ERYTHROPOIETIN TREATED SUBJECTS
`PRIOR TO IRON LOADING
`
`70
`
`60
`
`50
`
`40
`
`TOT. RETIC
`(x 10, coo/cu MM)
`30
`
`20 104M
`
`2 a 4 5 ($1 I is '91'01'11k1'31'41'51'61'7
`DAYS
`
`Pharmacosmos, Exh. 1041, p. 3
`
`

`
`US. Patent
`
`Jul. 30, 1996
`
`Sheet 3 0f 4
`
`5,541,158
`
`2A
`
`-
`
`HEMATOLOGIC PARAMETERS nv
`ERYTHROPOIETIN TREATED SUBJECTS
`AFTER IRON LOADING
`
`1a —
`
`17
`
`HGB(G/DL) 16—
`C
`
`15
`
`14~
`
`Pharmacosmos, Exh. 1041, p. 4
`
`

`
`US. Patent
`
`Jul. 30, 1996
`
`Sheet 4 0f 4
`
`5,541,158
`
`FIG 2 B HEMATOLOGIC PARAMETERS IN
`I
`ERYTHROPOIE TIN TREATED SUBJECTS
`AFTER IRON LOADING
`
`70
`
`60 -
`
`50
`
`40
`ror. RETIC
`(x 10, ova/cu MM)
`30
`
`20
`
`2 3 4 5 é 7"
`
`is l91'01|11|21'31I41|51I61|7
`DAYS
`
`Pharmacosmos, Exh. 1041, p. 5
`
`

`
`5,541,158
`
`1
`METHOD FOR INCREASING THE
`HEMATOCRIT OF A NORMAL MAMMAL
`
`BACKGROUND OF THE INVENTION
`
`2
`Speci?cally, it has now been discovered that the hemat~
`ocrit and rate of erythropoiesis of normal mammals, such as
`humans, may be increased by administering to the mammal
`a hematocrit increasing eifective amount of erythropoietin,
`in a pharmaceutically acceptable form. The erythropoietin is
`administered in conjunction with the step of administering to
`the mammal, iron, in a pharmaceutically acceptable form
`and in an amount sui?cient to increase the serum iron
`content to an erythropoiesis supportable level. Thus, for
`example, the hematocrit may be maintained at an acceptable
`level while withdrawing blood for autotransfusion tech
`niques.
`Prior to the discovery associated with the present inven
`tion, it was suggested that the administration of erythropoi—
`etin (EPO) is bene?cial in EPO de?cient patients. Early
`studies of the effect of EPO-rich plasma in patients with
`end-stage renal disease have been reported with variable
`results (Esser, U. et al. Proc. Eur. Dial. Transp. Ass., 1974,
`11:398-402). These prior methods of using EPO have been
`con?ned to the treatment of diseased or unhealthy individu
`als, especially those exhibiting anemia and having low
`hematocrits. It has heretofore been thought that EPO therapy
`would not be useful for normal individuals, i.e. those indi
`viduals who are non-anemic and have a normal hematocrit
`(i.e., greater than about 34%). In fact, the potential bene?t of
`EPO therapy to correct anemia was doubted because of the
`concern that erythropoietin inhibitors might block the effect
`of the EPO. Such doubt about EPO treatment in normal
`mammals was apparently con?rmed in our early experi
`ments, wherein EPO treated mammals failed to respond.
`This doubt was unexpectedly extinguished, however, with
`our discovery that by employing, in conjunction with EPO,
`iron, in an amount sufficient to support erythropoiesis, a
`dramatic increase in the hematocrit of EPO treated normal
`subjects resulted.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIGS. 1A and B depict the hematologic parameters of
`erythropoietin treated subjects prior to employing the teach
`ings of this invention; and
`FIGS. 2A and B depict the hematologic parameters of
`erythropoietin treated subjects after treatment in accordance
`with this invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The present invention provides a method for increasing
`the hematocrit, and the rate of erythropoiesis of a normal
`mammal by treating said mammal with effective amounts of
`iron and EPO.
`The hematocrit is a measure of the red blood cell mass of
`a mammal’s blood and is generally determined by simply
`loading a sample of whole blood into a capillary tube and
`centrifuging the tube to pack red blood cells in the bottom
`of the tube. The volume of packed red blood cells is then
`compared to the total volume of whole blood. In general, in
`normal (i.e., non-anemic) and otherwise healthy, grown,
`human females, hematocrit levels are greater than 37% and
`for normal human males the hematocrit levels are greater
`than 42%. For the purposes herein, anemia shall be de?ned
`as a hematocrit which is less than about 34%. A more direct
`measurement of the rate of erythropoiesis is the total reticu
`locyte count in the blood expressed as the total cells per unit
`volume. Normal levels for grown humans are approximately
`5O—l50><l03 cellslmm3. As described herein, the present
`
`This application is a continuation-in-part of US. Ser. No.
`036,646, ?led Apr. 10, 1987, now abandoned, the contents of
`which are hereby incorporated by reference into this appli
`cation.
`The present invention relates to a method for increasing
`the hematocrit and the rate of erythropoiesis of a normal
`mammal and speci?cally relates to a method for doing so by
`administering effective amounts of erythropoietin and iron.
`It has long been the practice in both pre and post-operative
`therapy to attempt to expand the patient’s red blood cell
`population. Typically, such red blood cell expansion has
`been achieved by the transfusion of homologous blood
`received from various donors. Recently, however, there has
`been heightened concern about the safety of homologous
`transfusion, aroused by the discovery that acquired immu
`node?ciency syndrome (AIDS) as well as other infectious
`diseases such as hepatitis, can be transferred by blood
`transfusions. Further, the present method of screening blood
`donors does not entirely eliminate the possibility of trans
`fusion transmitted infections in donated homologous blood.
`For instance, tests for non-A, non-B Hepatitis have been
`found to be relatively unreliable and tests for antibody to the
`AIDS virus have failed to detect blood donors with viremia.
`Accordingly, interest in recent years has increased in red
`blood cell expanding techniques other than homologous
`transfusion, and speci?cally in so~called autologous trans
`fusion.
`Autologous transfusion has been accomplished in the past
`by essentially two ways: 1) through the intraoperative sal
`vage and retransfusion of the patient’s blood during surgery;
`and 2) through donation by the patient of the required
`amount of blood before surgery i.e. pre-deposit or pre
`donation. The clinical and laboratory procedures used in
`collecting, preserving and transfusing autologous blood are
`the same as those used routinely in providing homologous
`blood to patients. Such procedures require administration
`arrangements for gaining access to the medical services and
`for insuring that the autologous blood, once collected, gets
`to the correct patient who pre-deposited it. Prior to the
`present invention, a major problem with autologous trans
`fusion has been the inability to obtain the required amount
`of blood due to induction of anemia (hematocrit less than
`34%) by phlebotomy.
`Accordingly, there is a need for a less inconvenient but
`satisfactory alternative to homologous blood transfusion.
`Use of the present invention prevents or attenuates the
`induction of anemia which occurs when blood is withdrawn
`for transfusion and hence allows the collection of the
`required number of units of blood, particularly in patients
`with a low baseline hematocrit.
`
`10
`
`20
`
`25
`
`35
`
`40
`
`45
`
`55
`
`SUMMARY OF THE INVENTION
`
`In accordance with the present invention, a method is
`provided as an alternative to homologous blood transfusion
`which method avoids the risk of the patient’s receiving
`contaminated homologous transfusions. Use of the present
`method prevents or attenuates the drop in hematocrit due to
`phlebotomy and permits the patient to pre~donate greater
`amounts of blood for transfusion purposes, in particular
`autologous transfusion.
`
`65
`
`Pharmacosmos, Exh. 1041, p. 6
`
`

`
`5,541,158
`
`10
`
`20
`
`30
`
`3
`invention increases the hematocrit and reticulocyte count
`above the normal levels and hence is useful as an alternative
`to homologous transfusions or the current autologous trans
`fusion techniques. By employing the present methods, the
`reticulocyte content of a patient’ s blood may be increased by
`as much as ten-fold over normal levels.
`Various forms of erythropoietin exist and any of them
`may be used in the present methods. The erythropoietin
`employed in the present invention is a 30,400 dalton gly
`coprotein hormone which is produced in the adult kidney
`and which is responsible for maintaining the body’s red
`blood cell (erythrocyte) mass at an optimal level. EPO has
`an activity which stimulates erythropoiesis (i.e. the forma
`tion of red blood cells) resulting in the differentiation of
`blood stem cells into red blood corpuscles. Because of this
`activity, EPO has been widely examined as a therapeutic in
`the clinical treatment for those disorders characterized by
`low or defective red blood cell production, such as anemia,
`and in particular renal anemia. However, such investigations
`were limited prior to the advent of recombinantly produced
`EPO because of the unavailability of large amounts of pure,
`human EPO.
`An example of the earlier method of obtaining EPO by
`puri?cation of urine is described in US. Pat. No. 4,397,840.
`25
`Methods for producing human EPO by recombinant DNA
`techniques are described in International Patent Application
`Publication Nos. WO 85/02610 and WO 86/03520. Recom
`binant techniques for producing EPO provide the advantage
`of a source of relatively pure EPO which can be produced in
`large quantities inexpensively. Recombinant monkey EPO
`has been produced also and shown to elevate the hematocrit
`of Balb C mice (see Egrie, J. C. et al., Prog. Clin. Bio. Res.,
`1985, 191 :339-50). The EPO of choice is the recombinantly
`derived human EPO described in the above publication.
`It is contemplated that biologically active fragments,
`analogs, or chemically synthesized derivatives of EPO may
`be used in the present methods rather than the naturally
`occurring molecule produced by recombinant DNA tech
`niques, provided that such fragments or derivatives retain
`the biological activity of naturally occurring EPO. Certain
`EPO analogs are described in US. Pat. No. 4,703,008.
`Therefore, use of such biologically active EPO analogs,
`fragments or derivatives is considered to be within the scope
`of the present invention, provided that such analogs, frag
`ments or derivatives possess the biological activity of caus
`ing bone marrow cells to increase production of reticulo
`cytes and red blood cells, and to increase hemoglobin
`synthesis or iron uptake. As used herein, “erythropoietin”
`shall include such fragments, analogs or derivatives.
`In practicing the present invention, the EPO is preferably
`administered to the subject parenterally. Suitable routes of
`parenteral administration are intravenous, intramuscular and
`subcutaneous injection. The EPO may be administered in
`any pharmaceutically acceptable form, such as a physiologi
`cally buffered solution. A suitable physiological buifered
`solution is an isotonic saline solution having a pH of about
`7.6. at 1:10 dilution.
`Although at the present time, EPO is only available in
`parenteral dosage forms, it is envisioned that oral dosage
`forms of EPO may become available in the future. Thus, oral
`administration of EPO is considered to be within the scope
`of the present invention. Such oral dosage forms would be
`designed to provide effective blood levels of EPO similar to
`the present parenteral dosage forms. Such oral dosage forms
`may be in the form of a coated tablet, such as enteric coated
`tablets, to prevent degradation of the EPO by gastric acid
`
`35
`
`45
`
`55
`
`60
`
`65
`
`4
`and enzymes. Additionally, new forms of EPO may be made
`to produce a more lipophilic chemical derivative of EPO. A
`more lipophilic derivative is more rapidly absorbed so that
`the residency time in the degradative gastric medium is
`minimized.
`The amount of EPO administered to the subject is an
`amount su?icient to effectively increase the hematocrit level
`of that subject. Thus, for a normal adult male it is that
`amount which increases the hematocrit above the level
`existing in that individual before EPO therapy was begun.
`For example, use of the present method may increase the
`subject’s hematocrit by about 10% over a period of two
`weeks. In general, a hematocrit increasing effective amount
`of EPO is in the range of about 15-1500 units per kilogram
`of body weight (U/kg) as a single dose given several days
`each week. Preferably, the dose is in the range 100-700 U/kg
`as a single dose administered several times weekly.
`It has been discovered that the EPO must be administered
`to the normal subject in conjunction with iron, if the
`subject’s iron stores are low prior to the start of therapy, in
`order to achieve an increase in the hematocrit. The iron may
`be administered in any pharmaceutically acceptable form.
`Oral preparations of ferrous sulfate are preferred, in the form
`of a tablet, elixir, syrup or oral solution. Any of the various
`ferrous salts, as hydrated or dry salts, may be used as
`alternatives to the sulfate salt. Such salts include fumarate,
`gluconate, succinate, glutamate, lactate, citrate, tartrate,
`pyrophosphate, cholinisocitrate, and carbonate. Reduced
`iron (metallic iron, elemental iron) in the form of carbonyl
`iron powder may also be used. Reducing agents (e.g.,
`ascorbic acid) and some chelating agents (e. g., succinic acid
`or sulfur containing amino acids) may be added to the iron
`formulation to increase absorption of the ferrous iron.
`Parenteral preparations of iron such as solutions of iron
`dextran, iron sorbitex, green ferric ammonium citrate, fer
`rous gluconate, iron adenylate and iron polyisomaltose may
`also be used.
`The preferred route of administration for the iron is oral
`due to various disadvantages inherent in the parenteral
`preparations. However, circumstances may exist where the
`parenteral route is preferred. In particular, the iron may be
`administered parenterally when in mixture with the EPO.
`The amount of iron to be administered must be su?icient
`to increase the available iron to a level su?'icient to support
`erythropoiesis. Iron available for erythropoiesis is in the
`form of serum iron loosely bound to transferrin, a glyco
`protein B-globulin which transports iron throughout the
`blood stream and, in particular, transports iron to the bone
`marrow for erythropoiesis. The primary source of transferrin
`bound iron is dietary iron which is generally absorbed in the
`small intestine (most easily in the ferrous form) and then
`passed into and through the mucosal cells of the small
`intestine directly into the blood stream where it immediately
`is bound to the transferrin. A secondary source of iron is
`available in the form of ferritin, a stored form of iron
`consisting of ferric hydroxide-ferric phosphate and attached
`to a protein called apoferritin.
`In normal humans, the quantity and total capacity of
`transferrin to bind iron generally greatly exceeds the amount
`of iron actually bound. For example, transferrin concentra
`tions in normal sera are in the range of 0.2 to 0.4 g/lOO ml.
`Normally, the absolute transferrin concentration is not deter
`mined but instead is expressed in terms of the capacity of the
`transferrin to bind iron, i.e. the quantity of iron that could be
`bound if all the transferrin were saturated with iron. The
`term employed for this saturation capacity is the total iron
`
`Pharmacosmos, Exh. 1041, p. 7
`
`

`
`5
`binding capacity (TIBC). Accordingly, the percent satura
`tion of transferrin is the serum iron expressed as a percent
`age of the TBC. Normal ranges for such percent saturation
`in humans are from about 20—55%.
`Without being bound by such theory, based on our work
`it is now believed that once an effective amount of EPO is
`present in a mammal’s blood, the controlling parameter
`affecting the rate of erythropoiesis is the available iron
`bound to transfern'n which in turn may be expressed as the
`percent saturation of transferrin. It follows then that for EPO
`therapy to be effective in the normal individual, the percent
`saturation level of transferrin may have to be increased
`above such individual’s normal levels. For example, the
`percent saturation level may be increased by a factor of
`about 1.2-3.0 in order to achieve a level high enough to
`support erythropoiesis. Preferably, such levels would be
`increased by a factor in the range 1.54.5.
`In most normal humans, iron administered at the rate of
`100-1500 mg per day will produce the requisite increase in
`saturation levels. Preferably, the iron should be administered
`at the rate of 300-900 mg per day. The iron therapy should
`begin about 1—21 days prior to EPO therapy. Preferably, iron
`therapy is begun 14 days before EPO therapy and continued
`throughout the entire treatment period. It will be understood
`that the choice of therapy is dependent on such factors as the
`iron status of the individual as well as any other precondi
`non.
`Various treatment regimens may be used in the practice of
`the present invention. One treatment regimen which may be
`used is as follows. A suitable amount of iron suflicient to
`increase or maintain the serum iron content of the patient to
`an erythropoiesis supportable level is administered to the
`patient 14 days prior to EPO therapy at one or more doses
`per day, for example, three times a day. EPO is also
`administered on day 14 at one or more doses per day,
`preferably one dose per day. It is also possible to administer
`iron only several times a week, e.g. two or three doses per
`week. This treatment regimen is then continued each day
`until the hematocrit of the patient reaches a desired level.
`Variations of this regimen, such as alternating EPO and/or
`iron treatment on every second or third day, would be
`determined by the skilled practitioner. A preferred total
`treatment period is about 21 days beginning with the ?rst
`day of EPO therapy. If the patient has adequate iron stores
`prior to the start of therapy then it is not necessary to start
`him on iron therapy before EPO is administered. Pre~therapy
`with iron will be necessary in patients having low iron
`stores.
`The invention will be further clari?ed by consideration of
`the following examples, which are intended to be purely
`exemplary of the use of the invention.
`EXAMPLE 1
`Recombinantly produced human-EPO (Amgen Corpora
`tion, Thousand Oaks, Calif.) was used. The speci?c activity I
`of the recombinant human~EPO was 129,000 units per
`milligram of hormone. One unit of EPO is that quantity
`which provides a response similar to 5 micromoles of Cobalt
`as is described in The Johns Hopkins Medical Journal
`(1980), Vol. 146 at pp. 311-320. The recombinant human
`EPO was greater than 99% pure and formulated in a buffered
`saline solution containing 2.5 mg/ml human serum albumin.
`Placebo doses were formulated identically except for the
`lack of EPO.
`Four healthy adult volunteers (age 18-45) were selected
`for the study. One additional patient was selected as a
`
`20
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`35
`
`50
`
`55
`
`5,541,158
`
`6
`control and treated with placebo. The following is a partial
`list of the criteria used to select the patients. Each patient did
`not have clinically signi?cant abnormal values for the fol
`lowing hematology tests: hemoglobin, hematocrit (did not
`exceed the upper limit of the laboratory normal range); total
`erythrocyte count (did not exceed the upper limit of the
`laboratory normal range); total lcucocyte count, including
`differential; platelet count; and reticulocyte count. Each
`patient did not have clinically significant abnormal values
`for the standard serum chemistry tests, urinalysis, serum iron
`and TIBC. At baseline, ferritin levels were low. Additionally,
`each patient had a normal erythropoietin plasma level.
`The normal human subjects were ?rst treated with EPO
`alone (i.e., without iron supplementation) over a 17 day
`period. The dosing regimen was 200 U/kg of body weight by
`I.V. bolus injection on days 1 and 4-17 for three of the
`subjects. The fourth subject received 300 U/kg. The control
`subjects were treated identically to those subjects who
`received EPO. As can be seen from FIG. 1 and Table 1, there
`was no signi?cant difference in hematocrit, hemoglobin or
`reticulocyte levels between Day 1 and Day 17.
`In the second part of the study, each subject (including the
`control subject) was given ferrous sulfate tablets at approxi
`mately 300 milligrams 3 times each day (i.e., 900 mg per
`day) for two weeks prior to EPO administration and con
`tinued daily throughout the treatment period. After iron
`loading, the subjects were treated with the same EPO dose
`regimen described above. As can be seen from FIG. 2 and
`Table 2, the subjects had a brisk reticulocytosis and increase
`in hematocrit and hemoglobin after iron loading.
`A comparison of the results listed in Tables 14 reveals
`the importance of adequate iron availability in normal
`human patients in order to enable a clinically meaningful
`erythropoietic response to EPO. The data in Tables 1 and 2
`are the summaries of the data of the four subjects tested,
`except that the serum iron values in Table 1 for Days 1 and
`10 are only for three of the patients. The Day 10 and Day 17
`results are expressed as the unit change (increase or
`decrease) from the Baseline values. Tables 3 and 4 represent
`the data for placebo treated patients before and after iron
`loading, respectively. The Table 3 values are the mean
`values for three patients (except for Day 10 ferritin and
`serum iron, where only two patients were measured). The
`Table 4 values are for one patient. The Baseline values for
`Tables 1 and 3 are pre-study for ferritin and Day 1 (Pre-dose)
`for the other variables. The Baseline values for Tables 2 and
`4 are Day 1 (Pre-dose) for all variables.
`
`TABLE 1
`
`SUMMARY OF RETICULOCYTE COUNT, HEMATOCRIT,
`FERRITIN AND SERUM IRON RESULTS FOR EPO
`SUBJECTS BEFORE IRON LOADING
`
`Baseline
`
`Day 10
`Change
`
`Day 17
`Change
`
`60
`
`Retieulocyte
`Count
`(x l03/mm3)
`Hematocrit (%)
`
`Ferriu'n
`(ng/ml)
`Serum Iron
`(meg/d1)
`
`65
`
`Mean
`Std. Dev.
`
`Mean
`Std. Dev.
`Mean
`Std. Dev.
`Mean
`Std. Dev.
`
`96.70
`50.34
`
`46.62
`4.48
`15.98
`7.67
`62.67
`25.11
`
`23.08
`41.02
`
`1.32
`2.69
`—9.72
`5.81
`—37.33
`38.79
`
`67.45
`70.75
`
`2.08
`3.84
`—11.45
`6.92
`—43.50
`22.17
`
`Pharmacosmos, Exh. 1041, p. 8
`
`

`
`5,541,158
`
`7
`
`TABLE 2
`
`SUMMARY OF RETICULOCYTE COUNT, HEMATOCRIT,
`FERRITIN AND SERUM IRON RESULTS FOR
`EPO SUBJECT S AFl‘ER IRON LOADING
`
`Baseline
`
`Day 10
`Change
`
`Day 17
`Change
`
`Reticulocyte
`Count
`(x l03/mrn3)
`Hematocrit (%)
`
`Fen'itjn
`g/ml)
`Serum Iron
`(mcg/dl)
`
`Mean
`Std. Dev.
`
`Mean
`Std. Dev.
`Mean
`Std. Dev.
`Mean
`Std. Dev.
`
`91.78
`42.38
`
`45.70
`2.97
`47.60
`12.03
`105.00
`54.26
`
`386.18
`120.57
`
`4.52
`1.61
`—l9.50
`4.67
`—75.25
`52.09
`
`577.14
`75.09
`
`7.40
`2.16
`—30.65
`8.64
`—76.50
`55.38
`
`TABLE 3
`
`PLACEBO TREATED PATIENTS
`BEFORE IRON LOADING
`
`Baseline
`
`Day 10
`Change
`
`Day 17
`Change
`
`122.6
`
`—4l.6
`
`—36.7
`
`48.4
`6.1
`
`52.3
`
`—O.9
`2.7
`
`4.7
`
`—-0.9
`0.2
`
`—28.3
`
`Reticulocyte
`Count
`(X l03/rnrn3)
`I-Iernatocrit (%)
`Ferritiu
`(mg/ml)
`Serum Iron
`(mcg/dl)
`
`TABLE 4
`
`PLACEBO TREATED PATIENT
`AFI‘ER IRON LOADING
`
`Baseline
`
`Day 10
`Change
`
`Day 17
`Change
`
`10
`
`20
`
`25
`
`30
`
`35
`
`8
`patients. Prior to iron loading, EPO had a minimal effect on
`erythropoiesis: there was no signi?cant di?erence in reticu~
`locyte count and hematocrit when compared to the control.
`After iron loading there were signi?cant increases in mean
`reticulocyte count and mean hematocrit in the EPO subjects.
`The combined effect of EPO and iron was so marked that the
`study medication was discontinued early for all subjects.
`These experiments conclusively demonstrate that in nor
`mal subjects with adequate iron stores, a combined treat
`ment of EPO and iron is effective in stimulating erythro
`poresrs.
`
`EXAMPLE 2
`
`This example concerns a double-blind, placebo-con
`trolled study to determine whether recombinant human EPO
`can facilitate presurgical autologous blood donation. The
`study population consisted of 54 patients between the ages
`of 18 to 25 years who were scheduled for orthopedic surgery,
`which was to occur within 25 to 35 days after receiving the
`?rst dose of study medication. The dosing regimen consisted
`of 6 doses of recombinant human EPO (600 U/kg) or
`placebo (diluent) by IV bolus, each dose administered every
`3 to 4 days over a 21 day period.
`At each study visit a complete blood count was obtained
`from each patient prior to dosing. If the patient’s hematocrit
`level was greater than or equal to 34%, a unit of blood was
`collected and stored for autologous transfusion. A unit of
`blood is approximately equal to one pint. If the patient’s
`hematocrit was less than 34%, no blood donation was made.
`The hematocrit cutoff level of 34% was chosen because it is
`the American Association of Blood Banks standard for
`autologous donation (levels below this number render a
`patient too anemic to donate blood). However, each patient
`received study medication at each of the 6 visits whether or
`not a unit of blood was donated. All patients received oral
`iron supplementation three times daily throughout the study.
`Prior to the present invention, the amount of blood a
`patient could pre-donate before elective surgery was limited
`by the time frame in which his blood could be kept “fresh”
`and also the capacity of his body to produce new blood
`during this time frame. A patient could pre-donate blood any
`time before surgery but usually, autologous blood was not
`pre-donated more than about 35 days prior to elective
`surgery. The reason for this is that blood stored in liquid
`form is generally not viable after 35 days. If surgery is
`scheduled more than 35-42 days from donation, the blood
`must be frozen, but most surgeons prefer to work with fresh
`blood rather than frozen blood. In order to prevent the
`patient from becoming anemic, the typical patient was only
`able to donate about 3-4 units of blood (less than half a
`person’s total blood supply) over the 35 day period that his
`blood was kept in liquid form.
`The present invention not only provides a means for
`increasing the yield of blood that can be pre-donated but it
`also attenuates the problem of presurgical anemia that
`occurs with prior methods of autologous transfusion. By
`using the present method, about 5-6 units of blood can be
`obtained from a patient within a 35 day or shorter time
`period and that patient’s hematocrit will be maintained at or
`above normal levels thereby preventing a state of anemia
`from occurring. Use of the present method will attenuate the
`drop in hematocrit due to phlebotomy. Such a treatment
`regimen will allow the patient to go to surgery with a higher
`hematocrit and more stored blood than is possible if the
`present method is not used.
`A preferred regimen for autologous transfusion may
`begin, for example, by starting a patient on iron therapy
`about one week before the ?rst blood donation. The iron may
`be administered from one to three times per day. It should be
`
`Reticulocyte
`Count
`(x lO3/rnm3)
`Hernatocrit (%)
`Fen-itin
`(mg/ml)
`Serum Iron
`(mcg/dl)
`
`48.2
`
`45.3
`19.8
`
`39.0
`
`48.4
`
`l2.2
`
`40
`
`0.4
`3.0
`
`3.0
`
`1.5
`—2.3
`
`38.0
`
`When considering the control data presented herein, it
`should be noted that it is well known that iron supplemen
`tation alone in pharrnacologic doses will not increase the
`hematocrit of a normal patient having adequate iron stores.
`This phenomenon was recognized in the study of a disease
`called primary hemochromatosis in which patients absorb
`excess iron through the gastrointestinal tract. These patients
`may, in fact, die of the effects of excessive tissue iron stores.
`However, despite massive iron overload, they do not
`develop polycythernia (an increased number of red blood
`cells in the blood). This is demonstrated in the classic article
`by Finch et al. on ferrokinetics (Finch, C. A. et al., “Ferro
`kinetics in Man”, in Medicine, Vol. 49, No. 11, pp. 17-48
`(1970)). Speci?cally, page 43 of Finch et al. indicates that
`there is a normal hematocrit in 10 patients with primary
`hemochromatosis despite their state of iron overload. This
`“experiment of nature” indicates that increasing iron stores
`beyond physiological values does not increase erythropoie
`sis beyond normal levels.
`'
`Tables 1 and 2 show that in the face of depleted iron
`stores, EPO has no hematologic effect in normal human
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Pharmacosmos, Exh. 1041, p. 9
`
`

`
`5,541,158
`
`9
`noted that iron supplementation, although preferred, may not
`be necessary in all cases, especially when the patient already
`has adequate iron stores or very high iron stores, such as in
`hemochromatosis. It is believed that EPO alone will have an
`adequate effect in such individu