WO2004060392A1 - Lactoferrin protein with iron for treating iron deficiency and anemia - Google Patents

Abstract

The invention is directed to therapeutic and/or nutritional compositions comprising lactoferrin proteins complexed with one or more iron molecules. The lactoferrin protein can be recombinantly produced in host cells transformed with a neucleic acid encoding the lactoferrin protein. The invention includes methods of making the composition comprising recombinantly produced lactoferrin, and methods of treating a patient in need of iron supplementation.

Description

Lactoferrin Protein With Iron for Treating Iron Deficiency and Anemia

Field of the Invention

The present invention relates to lactoferrin protein for the treatment for iron deficient anemia or iron deficiency The lactoferrin protein can be recombinantly produced.

References The following references are cited herein, and to the extent they may be pertinent to the practice of the invention, are incorporated herein by reference. General reference is made to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1993) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. throughout for basic laboratory procedures. References also specifically intended for inclusion are references cited in USSN 10/077,381 that is herein incorporated by reference in its entirety. All additional references cited herein are incorporated by reference in their entirety.

Background Of The Invention Human Lactoferrin is a single chain polypeptide of 692 amino acids organized into two globular lobes, representing its N-terminal and C-terminal halves (the N-lobe and C-lobe) [Baker, E. N., B. F. Anderson, et al. (1991). Structure, function and flexibility of human lactoferrin. Int J Biol Macromol 13 (3): 122-9]. Each lobe is itself folded into two domains (N- lobe: N1 and N2; C-lobe: C1 and C2) that enclose the iron binding sites. This two-lobe, four- domain structure (provides the key to understanding the dynamic properties of lactoferrin. Lactoferrin undergoes a conformational change as iron is bound (closed form) or released (open form).

Complete amino acid sequences have been delineated for lactoferrin from eight species (human, bovine, buffalo, camel, goat, horse, mouse and pig) [Shimazaki, K.-i. (2000). Lactoferrin : structure, function, and applications : proceedings of the 4th International

Conference on Lactoferrin: Structure. Function, and Applications: held in Sapporo, Japan, 18-22 May 1999. Amsterdam ; New York, Elsevier Science B.V.]. The homology between human and bovine is 70%. Human lactoferrin (HLf) and bovine lactoferrin (BLf) each have 3 to 5 potential glycosylation sites. However, only two sites (137 and 478) in HLf and four sites (233, 368, 476 and 545) in BLf are normally found glycosylated. Crystallographic studies with HLf and recombinant HLf expressed in Aspergillus indicates that carbohydrate moieties do not play a role in polypeptide conformation [Sun, X. L., H. M. Baker, et al. (1999). Structure of recombinant human lactoferrin expressed in Aspergillus awamori. Acta Crvstalloqr D Biol Crvstalloqr 55: 403- 7]. With a human kidney-derived cell line that constitutively expresses recombinant HLf, it was shown that both glycosylated and nonglycosylated rHLf are completely saturated with iron [van Berkel, P. H., M. E. Geerts, et al. (1995). Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis. Biochem J 312: 107-14].

HLf is present in many biological fluids and mucous secretions (milk, tears, saliva, synovial fluid, genital and nasal secretions) as well as in neutrophils. However, the concentration of HLf is highest in milk (1-3 mg/ml) [Lonnerdal, B. (1985). Biochemistry and physiological function of human milk proteins. Am J Clin Nutr 42: 1299-317].

There have been studies done in vitro, in experimental animals and in humans in an attempt to determine the function of lactoferrin since its first discovery by Sorensen et al more than 60 years ago [Sorensen, M. and M. Sorensen (1939). The proteins in whey. Compt Rend Trav Lab Carlsberq 23: 55-59]. It is becoming increasingly clear that lactoferrin has multiple functions depending upon on its cellular or extracellular environment. Lactoferrin has been indicated to exhibit antibacterial, antiviral, antifungal and antiparasitic activities. Although most of the studies were conducted in vitro (for review, see [Shimazaki, K.-i. (2000). Lactoferrin: structure, function, and applications : proceedings of the 4th International Conference on Lactoferrin: Structure. Function, and Applications: held in Sapporo, Japan, 18-22 May 1999, Amsterdam; New York, Elsevier Science B.V; Naidu, A. S. (2000). Lactoferrin: natural, multifunctional, antimicrobial. Boca Raton, FL, CRC Press], there are some in vivo indications as well [Kirstila, V., M. Lenander-Lumikari, et al. (1996). Effects of oral hygiene products containing lactoperoxidase, lysozyme, and lactoferrin on the composition of whole saliva and on subjective oral symptoms in patients with xerostomia. Acta Odontol Scand 54: 391-7; Iwasa, M., M. Kaito, et al. (2002). Lactoferrin inhibits hepatitis C virus viremia in chronic hepatitis C patients with high viral loads and HCV genotype 1b. Am J Gastroenterol 97: 766-7; Tanaka, K., M. Ikeda, et al. (1999). Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: a pilot study. Jpn J Cancer Res 90: 367-71 ; Yamauchi, K. et al. (2000). Oral administration of bovine lactoferrin for treatment of tinea pedis. A placebo-controlled, double-blind study. Mycoses 43:197-202; Masci, J. R. (2000). Complete response of severe, refractory oral candidiasis to mouthwash containing lactoferrin and lysozyme. Aids 14: 2403-4]. Lactoferrin has also been implicated as an anti-inflammatory/antioxidant agent. This function is thought to exert through the sequestration of free iron in the inflammatory sites [Morris, C. J., J. R. Earl, et al. (1995). Reactive oxygen species and iron-a dangerous partnership in inflammation. Int J Biochem Cell Biol 27: 109-22; Gutteridge, J. M.(1989). Iron and oxygen: a biologically damaging mixture. Acta Pediatr Scand Suppl 361: 78-85] or direct binding to lipopolysaccharides (LPS) [Baveye, S., E. Elass, et al. (1999). Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process. Clin Chem Lab Med 37: 281-6]. Lactoferrin has been also shown to decrease cutaneous inflammatory reaction by inhibiting the migration of epidermal Langerhans cells in mice and human volunteers [Kimber, I., M. Cumberbatch, et al. (2002). Lactoferrin: influences on langerhans cells, epidermal cytokines, and cutaneous inflammation. Biochem Cell Biol 80: 103-7; Cumberbatch, M., R.J. Dearman, et al. (2000). Regulation of epidermal Langerhans cell migration by lactoferrin. Immunology 100: 21-8; Griffiths, C. E., M. Cumberbatch, etal. (2001). Exogenous topical lactoferrin inhibits allergen- induced Langerhans cell migration and cutaneous inflammation in humans. Br J Dermatol 144: 715-25].

More or less, most of the reported functional characteristics are related to the iron binding capacity of the protein. Lactoferrin's iron affinity is about 300 times that of transferrin, an iron binding protein found in blood. Lactoferrin receptors have also been found in several types of human cells (for review, see [Testa, U. (2002). Proteins of iron metabolism. Boca Raton, Fla., CRC Press]), including monocytes-macrophages, small intestinal brush border membrane, parenchymal liver and breast epithelial cells. The hypothesis of supplying iron through Lac-Fe complex during the infancy has been discussed [Lonnerdal, B. and S. Iyer (1995). Lactoferrin: molecular structure and biological function. Annu Rev Nutr 15: 93-110].

Iron deficiency (ID) is the most common nutritional deficiency in the World and is estimated to affect nearly 25% of the World population. The severity of ID can be demonstrated in that it affects 3.5 billion people, while Vitamin A and Iodine deficiency affects 0.3 billion people and 0.8 billion people respectively. Iron deficiency is the highest among young children and women of childbearing age (particularly pregnant women).

The development of iron deficiency can be divided into three stages. The first stage is defined as reduction in iron stores (serum ferritin) but results in no clinical consequences. The second stage represents the exhaustion of iron stores. While the hemoglobin (Hb) level is still within the reference range, it may well be below "normal" for that individual. It is usually called Biochemical iron deficiency (ID). If ID is left untreated, it will eventually develop into IDA. At this stage, there is no iron left in the marrow and Hb level is below the reference range. IDA is the most common form of anemia. In the United States, 20% of all women of childbearing age have IDA (compared to 2% of adult men). Current treatment for Iron Deficiency Anemia (IDA) is taking iron pills (e.g. as ferrous iron) at the range from 300 mg - 1 g, which corresponding to elemental iron of 60 mg-200 mg. Due to the very poor bio-availability and possible uptake of inorganic iron (1-7%), a significant quantity of iron pills have to been consumed by IDA patients. Heavy iron load in the gut causes many problems, including gastro-intestinal discomfort, nausea, vomiting, diarrhea, constipation, other toxic effects and possibly promotes bacteria infections. In addition, a large number of people use proton-pump inhibitor to control the reflex, which in turn will alter the pH in the gut to become more neutral. Such change in pH will inevitably reduce the absorption of iron in the gut. Another alternative to overcome side-effect problems is to deliver iron through intravenous (iv) injection. However, it is a costly alternative and is not necessarily favored by patients.

Therefore, due to the poor compliance with the current treatment, a lot of physicians feel that there is a need to find an alternative in treating IDA.

Summary Of The Invention In one aspect, the invention includes a composition formulated for administration to a human in need of iron supplementation comprising a lactoferrin protein, wherein the lactoferrin protein comprises at least one iron molecule in complex with said protein. The lactoferrin protein can be recombinantly produced in a host cell transformed with a nucleic acid sequence encoding said lactoferrin protein and isolated from the host cell. The lactoferrin protein can be saturated with iron. The recombinant production can comprise a host cell selected from the group consisting of a plant cell, a bacterial cell, an animal cell, and a yeast cell. Where the host cell is a plant cell, the plant cell can be a monocot. The lactoferrin protein can be extracted from a product of a mature monocot seed and the lactoferrin protein can comprise at least one plant- derived glycosyl group. The formulation can be selected from the group consisting of parenteral, enteric, edible, suppository, inhalant, intranasal and topical formulations. The formulation can comprise a nutritionally or pharmaceutically acceptable carrier.

A method of making a composition for administering to a patient in need of iron supplementation comprising the steps of a. expressing a nucleic acid encoding a human lactoferrin protein or portion thereof in a host cell; b. isolating the lactoferrin protein from the host cell; and c. contacting the recombinantly produced lactoferrin protein with one or more iron molecules to form a complex of the protein with iron. The host cell can be selected from the group consisting of a plant cell, a bacterial cell, an animal cell, and a yeast cell.

The host cell can be a plant cell and the plant can be a monocot.

The method can further comprise the step of: d. formulating the recombinantly produced lactoferrin for parenteral or oral delivery to the patient comprising contacting the protein and iron with a pharmaceutically or nutritionally acceptable carrier.

The invention further contemplates a method of treating iron deficiency in a patient comprising administering to the patient a composition comprising a lactoferrin protein and an iron salt, wherein the composition is formulated in a pharmaceutically or nutritionally acceptable carrier.

The invention also contemplates a method of treating iron deficiency in a patient comprising administering to the patient a composition comprising a lactoferrin protein recombinantly produced in a host cell transformed with a nucleic acid sequence encoding said lactoferrin protein and an iron salt, wherein the composition is formulated in a pharmaceutically or nutritionally acceptable carrier. The lactoferrin protein can be saturated with iron.

The invention is also a method of treating iron deficiency in a patient comprising administering to the patient a composition comprising a lactoferrin protein produced in a monocot seed transformed with a nucleic acid sequence encoding said lactoferrin protein and extracted from a product of a mature monocot seed. The method can further comprise administering an iron salt before, after or during administration of the lactoferrin protein.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

Brief Description of the Figures

Figure 1 is a restriction map of the pAPI164 plasmid that expresses the human lactoferrin codon-optimized coding sequence under the control of a rice glutelin (Gt1 ) promoter, a Gt1 signal peptide, and a nopaline synthase (NOS) terminator/polyadenylation site. Figure 2 is a Southern blot analysis of transgenic rice event 164-12-1 expressing recombinant human lactoferrin. All plant DNAs were digested with Hinά\\\ and EcoRI and DNAs from six generations of selfed material corresponding to the 164-12-1 transgenic plant event. DNA probes utilized in this figure were the lactoferrin gene, the vector backbone and the hygromycin selectable marker gene respectively. Figure 3 is a Southern blot analysis where the same DNAs (spanning 6 generations) described in Figure 2 were digested exclusively with EcoRI. Only the lactoferrin gene was utilized as a probe for this blot.

Figure 4 shows the results of a SDS-PAGE analysis for the expression of recombinant human lactoferrin. Total proteins from rice seed extracts were suspended in Laemli sample buffer, run on a gradient gel and stained with Coomassie blue. Lane 1 is the molecular weight marker; lanes 3 - 6 are purified human derived lactoferrin (Sigma Chemical, USA); lanes 8 - 13 are single seed extracts from homozygous independent transgenic rice lines and lane 14 is a seed extract from non-transformed rice varietyTP-309. Figure 5 shows the results of a Western blot analysis of various tissues of the transgenic rice plants, demonstrating the tissue specificity of rLF expression in mature rice seeds. Lane 1 is the molecular weight marker; lane 2 is human lactoferrin (Sigma, USA); lane 3 is an extract from leaf; lane 4 is an extract from sheath; lane 5 is an extract from root; lane 6 is an extract from seed and lane 7 is an extract from 5-day germinated seeds. Figure 6 is a graph illustrating pH-dependent iron release by native human lactoferrin

("nHLF") and purified recombinant human lactoferrin produced in transgenic rice seeds ("rHLF"). Purified nHLf and rHLf proteins were iron saturated and the pH of the solution lowered to pH 2. Figure 7 indicates binding of rHLf to Caco-2 cells. The Kd for native HLf and rHLf were 0.16 +/- 0.03 and 0.27 +/- 0.04 uM, respectively. Number of binding sites for nHLf and rHLf were 0.91 +/- 0.05 and 1.92 +/- 0.11 pmol/ million of cells respectively.

Figure 8 indicates digesion stabilities of rHLf. Figure 8A: SDS-PAGE and Western blot analysis before and after in vitro digestion. 'B': before the digestion; 'Pep': post pepsin treatment (pH 3.8 for 30min); 'Pan': after pepsin treatment, pH adjusted to 7.0 and pancreatin added for 60 min, then heat inactivated. Figure 8B: Survival rates of HLf were determined by ²⁵l-Lf, ⁵⁹Fe-Lf binding and ELISA, respectively. Results are shown as a percentage against starting amounts. Figure 8C: Anti microbial activity after low pH treatment. 5 μg/ml of eithet nHLf or rHLf was incubated with EPEC in synthetic broth for 24 hours and relative growth determined.

Figure 9 indicates the binding and uptake of rHLf to Caco-2 cells after in vitro digestion. Figure 9A shows the determination of Dissociation constant for both nHLf and rHLf. Figure 9B shows the number of binding sites for HLf on Caco-2 cells. Figure 9C shows the total uptake of HLf and Fe to Caco-2 cells within 24 h. Figure 9D shows degradation of HLf after uptake into Caco cells determined by the amount of free ¹²⁵l remaining in the cell fractions.

Figure 10 indicates the thermal stability of rHLf. Figure 10A shows the iron-holding capacity after the thermal treatments as measured by A₂₈o.'A₄₆5 ratio. Figure 10B: the survival rates after thermal trearment as determined by ELISA. Figure 10C: SDS-PAGE and Western blot analysis after the thermal treatment. 'N' indicates nHLf and 'R' indicates rHLf. Figure 10D: anti-microbial activity after the thermal treatments; 5 μg/ml of either nHLf or rHLf was incubated with EPEC in synthetic broth fro 24 hours. Relative growth of EPEC to control is indicated.

Figure 11 demonstrates the pH stability of rHLf. Figure 11 A: the iron holding capacity after low pH treatments. Figure 11 B: SDS_PAGE and Western blot after low pH treatments. Figure 11C: anti-microbial activity after low ph treatments; 5 μg/ml of either nHLf or rHLf was incubated with EPEC in synthetic broth fro 24 hours. Relative growth of EPEC to control is indicated.

Detailed Description of the Invention

I. Definitions

Unless otherwise indicated, all terms are generally consistent with same meaning that the terms have to those skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1993) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

All publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies that might be used in connection with the invention. Specifically, USSN 10/077,381 is incorporated herein by reference in its entirety, and specifically for its definitions section, for its descriptions of recombinant production of human lactoferrin, and for its other supportive information and descriptions with regard to this invention.

II. Compositions Containing Plant-derived human lactoferrin complexed with iron

The present invention provides plant-produced recombinant lactoferrin, and plant- produced recombinant lactoferrin complexed with iron for administration to persons in need of iron supplementation. The invention also includes methods of making such compositions, and methods of treating iron deficiencies by administering recombinant lactoferrin, either alone or complexed with iron. The recombinant production can be plant-produced.

Lactoferrin can be provided from any source. It can be native or synthetic lactoferrin. It can be recombinantly produced in a host cell. It can be otherwise synthetically produced. It can be provided from an animal. Recombinantly produced lactoferrin can be made in host cells including, bacterial, yeast, animal and plant cells. For production of lactoferrin in bacterial, yeast and animal cells, see USPN 5,571,691 and USPN 6,228,614, both herein incorporated by reference in their entirety. Production in animal cells can be accomplished generally as described in Ausubel F.M. et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993. Lactoferrin protein produced in plants can be produced in the seeds or grain of transgenic plants expressing the nucleic acid coding sequence for the lactoferrin protein. Following expression, lactoferrin protein is isolated from these host transgenic grains. The recombinant lactoferrin can be administered to the patient requiring iron supplementation in conjunction with administration to the patient of reduced amounts of ferrous sulfate or other iron containing compositions. The amount of ferrous sulfate or other iron-containing compositions is reduced in comparison to the amount of iron that would otherwise be administered if the iron deficient patient did not receive lactoferrin protein. Alternatively, the lactoferrin protein can be complexed with iron before administration to the iron deficient patient, and the protein-iron complex can be then administered to the patient. Production of plant-produced recombinant lactoferrin can be based on the expression of human lactoferrin (HLF) under the control of a seed specific promoter in plants, preferably monocot plants such as rice, corn or barley. Most preferred is the use of rice (Oryza species). The lactoferrin produced in plant may be glycosylated, containing one or more plant glycosylation groups. The invention relies on the use of heterologous nucleic acid constructs including the coding sequence for lactoferrin or lactoferrin polypeptide of nutritional and/or therapeutic value, exemplified herein.

The compositions of the invention include recombinant lactoferrin complexed with iron. The composition may be unsaturated or saturated with iron. The molecular ratio of lactoferrin protein to iron in the composition is 1 : at least 0.5, preferably 1 : at least 0.8, and most preferably at 1 : at least 1. It is possible to administer the lactoferrin protein and in a separate or simultaneous administration also administer the necessary iron. Although the invention is not limited to theories of how the invention works, it is loosely presumed that the lactoferrin complexes with iron in vivo if the protein is not already saturated before delivery. In one preferred approach, the composition is administered to a person in need of iron supplementation, for example a person exhibiting symptoms of iron deficiency, either mild or severe; the composition may also be administered to persons at risk for iron deficiency, for example, persons who have low metabolic iron or who are undergoing a therapy which typically reduces one's biological iron. Typically the composition, e.g., recombinant lactoferrin or recombinant lactoferrin complexed with iron is administered in an amount that corresponds to the amount necessary for restoration of healthy iron metabolism in the patient. The composition can be administered enterically or parenterally, including by inhalation, intranasal, intravenous, intramuscular, orally, or topically. The HLf can be administered prior to saturation with iron, then followed or accompanied with an administration of an iron salt (e.g. ferrous sulfate). Alternatively, the HLf can be administered in equilibrium or saturation or semi-saturation with an appropriate amount of an iron salt. The Fe binds tightly to LF and it will be released at pH 2. In order to avoid the pre-mature release of Fe during the delivery, entero-coating technology can be employed in the manufacturing of oral formulations such as tablets or capsules. The HLf can be administered orally as a pill or capsule, in a food or drink formulation, a powder formulation, or a formulation for parenteral administration (e,g, including intravenous injection and other non- oral administration routes including inhalation or intranasal administration).The iron, if not complexed with lactoferrin before delivery, may also be delivered enterically or parenterally. The lactoferrin and iron, if delivered separately can be delivered by the same or different delivery means (e.g. generally any one of the many means for delivery that fit into the categories of enteric or parenteral delivery).

The dosing can be established through routine experimentation, based on the severity of the condition and the health, age, weight and sex of the patient. Preferred dosages are from about 20-120 mg/kg, more preferably about 50-100 mg/kg, based on the lactoferrin.

In one protocol, human subjects can be orally administrated with 4.8 g of purified recombinant human lactoferrin daily for a six weeks, approximately the equivalent to 68 mg rHLf/kg/day body weight if an average weight of 70 Kg is used. The tested dosage used in rhesus monkey experiments that showed no adverse effects was 1 g/kg body weight. A 13- week oral repeated administration toxicity study [Yamauchi, K., T. Toida, et al. (2000). 13-Week oral repeated administration toxicity study of bovine lactoferrin in rats. Food Chem Toxicol 38: 503-12] of BLf in rats concluded that at a 2 g/kg/day regimen, no adverse effect was observed. Recombinant LF (rLF) has been produced as a fusion protein in Aspergillus oryzae [Ward P.P., et al, (1992). Production of biologically active recombinant human lactoferrin in Aspergillus oryzae. Bio/technology 10:784-89) and in a baculovirus expression system [Salmon V., et al, (1997). Characterization of human lactoferrin produced in the baculovirus expression system. Protein Expr Purif 9:203-10]. Lactoferrin has also been expressed in tobacco (Nicotiana tabacum L. cv Bright Yellow) cell culture [Mitra A. and Z. Zhang, (1994). Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial proteins. Plant Physiol. 106: 977-981], tobacco plants [Salmon V., etal., 1998. Production of human lactoferrin in transgenic tobacco plants. Protein Expr Purif 13:127-351 and potato (Solanum tuberosum) plants [Chong D.K. and W.H. Langridge, (2000). Expression of full-length bioactive antimicrobial human lactoferrin in potato plants. Transgenic Res 9: 71-8].

In contrast to most other proteins, lactoferrin has also been shown to be resistant to proteolytic degradation in vitro, with trypsin and chymotrypsin remarkably ineffective in digesting lactoferrin, particularly in its iron-saturated form. Some large fragments of lactoferrin were formed, but proteolysis was clearly limited.

The amino acid sequence of recombinant lactoferrin is identical to that of native lactoferrin and it binds iron in a manner similar to that of native lactoferrin. The carbohydrate side chains of the molecule are similar to those of rice proteins, but slightly different from those of native human lactoferrin. However, these carbohydrate structures are not needed for iron binding to the lactoferrin receptor and rice recombinant human lactoferrin binds to the intestinal receptor in a manner similar to that of native lactoferrin.

Expression vectors for generation of transgenic plants expressing human lactoferrin protein are described in commonly owned USSN 10/077,381 that is herein incorporated by reference in its entirety. Exemplary methods for constructing chimeric genes and transformation vectors carrying the chimeric genes are given in USSN 10/077,381 including promoters, signal and transport sequences, protein coding sequences, variant human lactoferrin protein-encoding nucleic acid sequences, codon optimization, transcription factor coding sequences, additional expression vector components, the generation of transgenic plants, the plant hosts, and detecting expression of recombinant human lactoferrin protein.

The invention provides, in one embodiment, a composition containing flour, extract, or malt obtained from mature monocot seeds and a seed-produced human lactoferrin protein in substantially unpurified form, saturated with iron. Where the lactoferrin protein is expressed at a level of between about 0.1 to 1 percent of the seed weight, the composition will contain the same or preferably a higher percentage of lactoferrin protein, e.g., 0.1 to 20 % of the composition depending on the composition added. In particular, a grain composition will yield an amount of lactoferrin protein that is comparable to that in the mature seed; the extract composition, by contrast, in which most of the starch has been removed, will typically show a severalfold increase in percentage of lactoferrin protein, e.g., 10-40% of the total weight of the extract. The malt composition will contain an intermediate level, typically greater than grain, but less than extract.

Any of the recombinantly produced lactoferrin proteins, including either the seed- produced lactoferrin or the extract of lactoferrin from the mature seed, can be contacted with sufficient ferrous sulfate or other iron salt to form an iron saturated recombinantly produced human lactoferrin, useful for administration to patients having an iron deficiency condition. In determining the amount of grain, extract, or malt composition to be used in the composition, it is useful to determine the amount of any lactoferrin protein present and add an amount of composition bringing the final level of lactoferrin protein to a desired level per dosage amount in the composition. USSN 10/077,381 describes different compositions possible from plant-produced recombinant human lactoferrin, e.g. including flour composition, extract composition, malt composition, and including also various other processes of extraction of recombinant protein from plant seeds.

The following examples illustrate but are not intended in any way to limit the invention.

Example 1

An Expression Vector for Generation of Transgenic Plants

In general, expression vectors were constructed using standard molecular biological techniques as described in Ausubel FM et al. (1993). Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. The vectors contain a heterologous protein coding sequence for lactoferrin under the control of a rice tissue-specific promoter, as further described below. A. An Expression Vector For Human Lactoferrin Expression In Transgenic Rice

The hLF gene [Rey, M.W., et al, (1990). Complete nucleotide sequence of human mammary gland lactoferrin. Nucleic Acid Res 18: 5288] was codon optimized and synthesized by Operon Technologies (CA, USA). Human milk lactoferrin gene (Genebank accession number: HSU07642) was re-synthesized with codons most frequently used in translation of rice seed proteins in order to obtain optimal level of expression [Nandi S, Y.A. Suzuki, et al. (2002). Expression of human lactoferrin in transgenic rice grains for the application in infant formula. Plant Sci 163: 713-22]. Although numbers of codons changed accounted for 22.46% of the entire sequence, amino acid composition remains identical (Table 1) to non-recombinant human lactoferrin.

Table 1: Amino acid composition of normal human milk lactoferrin (HLf), recombinant HLf and bovine Lf (BLf).

The plasmid containing the codon-optimized gene was called Lac-ger. Lac-gerwas digested with Sma\IXho\ and the fragment containing the lactoferrin gene was cloned into pAPI141 that was partially digested with Λ/ael and completely digested with Xho\. For expression of hLF in rice seeds, the codon-optimized gene was operably linked to the rice endosperm-specific glutelin (Gt1) promoter and NOS terminator. The resulting plasmid was designated pAPI164 (Figure 1). Production system

A detailed procedure for delivering the foreign gene into rice genome was found in [Wu L., et al, (2002). Expression and inheritance of nine transgenes in rice. Transgenic Res 11 : 533- 41 ; Nandi S., et al, (2002) Plant Sci 163: 713-22]. Briefly, rice variety Taipei 309 (Oryza sativa, Japonica) was selected as the production system for recombinant human lactoferrin (rHLf) and transgenic rice events were eventually generated by the particle bombardment of embryogenic rice calli with the plasmid pAPI164 and a companion marker plasmid containing the hygromycin phosphotransferase gene as a selectable marker. Fully developed, fertile rice plants were obtained by this prodecure. DNA Characterization

DNA from plants propagated for seven generations of the best performing rHLf expressing line (164-12-1) was subjected to Southern blot analysis to determine the stability of transgenes over six subsequent generations. A restriction map of pAPI164 is shown in Fig. 1 with relelvant restriction sites indicated. As demonstrated in Figure 2, when the lactoferrin gene is utilized as a probe, many bands above and below the H/nc/lll/EcoRI fragment which includes the Gt1 promoter, Gt1 signal peptide, synthetic HLf gene and NOS terminator can be visualized. This may be due to the loss of one or both original restriction sites(s) surrounding the chimeric gene for lactoferrin expression or rearrangement during the integration of the fragment into the rice genome. Probing the blot with the vector backbone of pAP1164 or the hygromycin gene as probes indicated that the transgene locus of 164-12-1 was inherited from R₀ to R₆ generation as a single linkage unit (single locus). This was supported by a Southern blot illustrated in Figure 3 where the plasmid was digested with either the single restriction enzymes EcoRI or Hind\\\. All inherited banding patterns were identical over the six generations. Agronomic Characterization

Agronomic characteristics of both transgenic and non-transgenic plants, such as plant height, percentage of fertility, number of effective tillers, filled grains/panicle, non-filled grains/plant, time to maturity and 1000 grain weight were determined and summarized in Table 2.

Table 2: Agronomic characteristics comparison of non-transformed and transgenic rice plants (164-12) harboring rHLf gene.

High Level Protein Expression of rHLf and Characterization of Tissue Specificity of rHLf Expression

Expression of rHLf was under the control of the seed maturation-specific promoter Gt1. The high level expression of rHLf was evident in Figure 4 as independent transgenic rice events were screened. Total soluble proteins from mature rice seed extracts were run on Laemli gels and stained with Comassie blue to visualize the proteins. An ~82kD recombinant lactoferrin protein was obtained in all transgenic lines as indicated by the stained gel. Expression levels of rHLf corresponded to ~ 0.05% / weight of seed. To confirm the tissue specificity of the Gt1 promoter, proteins from roots, shoots or leaves were extracted and analyzed by Western blot analysis. Figure 5 shows clearly that there was no detectable level of rHLf expression in any other tissues except the mature grain and grain that has been germinated for five days. Time course studies

The timing of expression and accumulation of rHLf during grain development was analyzed by a time course study. rHLf from immature and mature seeds were analyzed by ELISA . It appears that rHLf expression begins around 7 days after flowering (dpa) and plateaus around 24 DAF. rHLf levels were able to maintained through maturation and drying process (data not shown). Stability over generations

The stable expression of rHLf was monitored for seven generations. The expression level was maintained at around 5 g/kg of brown rice (data not shown).

Example 2 Chemical. Physical and Analytical Data of Recombinant Lactoferrin in Comparison with Non- recombinant Human Lactoferrin and Bovine Lactoferrin.

Recombinant HLf from rice grain was purified to near homogeneity. Briefly, seed extracts were prepared by grinding the polished rice seeds and suspending the resulting rice flour in phosphate buffered saline (PBS pH 7.4) and 0.5M NaCI (75g/l) at room temperature for 2.5 hrs. After filtration through cheesecloth to remove large particulate matter, the resulting homogenate was centrifuged at 10,000xg and filtered through a 0.45 um nitrocellulose membrane. The filtrate was loaded onto a SP-Sepharose column eguilibrated with 0.4M NaCI in 0.05M NaPO4 (pH 8.0). Lactoferrin protein was eluted by a linear gradient (0.4M - 1.0M NaCI) and the lactoferrin containing fractions dialyzed against PBS. The purified lactoferrin was analyzed by SDS-PAGE and judged to be graeter then 90% homogeneous (data not shown). The overall properties were then compared with its purified non-recombinant counterpart isolated from human milk (Sigma Chemical, catalogue #L48940). The data are summarized in Table 3. Table 3: Physical, chemical and analytical data.

Table 4: Overall characteristics of lactoferrin glycan from Human Lactoferrin (HLf) and recombinant HLf. The overall glycosylation pattern based on the characterization of HLf isolated from human milk has been detailed [Hutchens T.W-, S.V. Rumball, etal, (1994). Lactoferrin: Structure and Function. New York, Plenum Press]

Iron Saturation And pH Dependent Iron Release

Purified lactoferrin was incubated with 2M excess ferric iron (FeCI3: NTA = 1 :4) and sodium bicarbonate (Fe: HCO3- = 1 :1 ) for 2h at room temperature. Excess free iron was removed by using a PD-10 desalting column (Pharmacia, USA) and the iron saturation level was determined by the A280/A456 ratio. Both native HLF and rHLF were completely saturated by iron. Holo HLF was incubated in buffers with a pH between 2 and 7.4, at room temperature for 24 h. Free iron released from HLF was removed and the iron saturation level was determined by A280/A456 ratio.

The results showed that iron release was similar for both native HLF and rHLF. Iron release began around pH 4 and was completed around pH 2 (Fig. 6). The iron binding was reversible since iron-desaturated rHLF was re-saturated by raising the pH to 7 (data not shown). The similarity in pH dependent iron release of rHLF to that of the native HLF standard demonstrated that rHLF is able to adapt the appropriate tertiary structure for proper iron binding and release (Salmon V., ef al, (1997) Protein Expr Purif 9: 203-10). Binding and uptake assay by Caco-2 (a human intestinal cell line) cells. 50,000 Caco-2 cells/well were seeded and grown in Minimum Essential Medium

(GIBCO, Rockville, MD) containing 10% fetal bovine serum in 48 well tissue culture plates for 3 weeks. For binding studies, Caco-2 cells were incubated with varying concentrations (0-2 μM) of 1251-HLf in the presence or absence of 100-fold excess of unlabeled nHLf for 2 hours at 4 oC and cells were washed 5 times with ice-cold PBS. Cells were solubilized with 0.5 ml of 0.1% SDS and radioactivity was quantified in a gamma counter. For uptake studies, 0.4 μM of 1251- HLf was incubated with Caco-2 cells for 0 to 24 hours at 37 °C and the cells were washed, dissociated in the same way as in the binding study. 0.5 ml of 24% TCA solution was added to the dissociated cells and free iodine was removed by the centrifugation. Free and protein-bound ¹²⁵l were quantified separately to evaluate how much of HLf was degraded in the cells. Receptor binding of rHLf to the Caco-2 cell line was saturable and specific, indicating that rHLf bound to the Lf receptor (Figure 7). The binding constant was similar for rHLf and nHLf, but the number of binding sites was slightly higher for rHLf, which may be due to the slight difference in plant-based glycosylation of rHLf. Uptake of HLf by Caco-2 cells was identical with respect to rHLf and nHLf. In vitro Digestion Stability of rHLf: effect on antimicrobial activity and binding/uptake to Caco-2 cells

SDS-PAGE and ELISA revealed that nHLf and rHLf resist digestion by pepsin (at pH 3.8) and pancreatin, whereas human serum albumin is completely digested after in vitro digestion (Figure 8A). Western blots revealed that immunoreactivity was also maintained after digestion. Although some smaller molecules were generated during digestion of HLf, most of the immunologically detectable HLf retained its intact size. More than 50 % of rHLf and nHLf was immunologically detectable by ELISA, but ¹²⁵l-HLf was around 40 % and ^5SFe-HLf was only 20 % detectable, indicating that ELISA detects small peptide fragments of HLf, which are removed by the PD-10 column and that about 50-60 % of Fe was released from detectable HLf after in vitro digestion. The iron-holding capacity was not significantly different (Figure 8B). Lactoferrin is known to inhibit the growth of a variety of bacterial species based on its iron chelation and direct bactericidal properties. The antimicrobial effect of rHLF extracted from rice seeds was tested following treatment using an in vitro digestion model with an enzymatic system containing pepsin (an enzyme active in stomach) and pancreatin (an enzyme active in deodenum).

LF proteins were dissolved in PBS at 1mg/ml, and either left untreated, pepsin treated (0.08mg/ml at 37°C for 30 min), or pepsin/pancreatin treated (0.016 mg/ml at 37°C for 30 min). LF proteins were sterilized by passing through a membrane filter with a pore size of 0.2 μm [Rudloff, 1992]. The filter sterilized LF (0.5μg/ml) was incubated with 104 colony forming unit (CFU) enteropathogenic E. coli (EPEC)/μl in 100 μl sterile synthetic broth (1.7%: AOAC) containing 0.1% dextrose and 0.4 ppm ferrous sulfate at 37°C for 12h and colony forming units (CFU) determined.

Starting with an enteropathogenic E. coli (EPEC) concentration of 10⁴ CFU (colony forming units), the untreated samples of rHLF reached up to 10⁶⁵ CFU after 12 h of incubation at 37°C in comparison to nHLF, which produced up to 10⁶ CFU. An in vitro digestion model using an enzymatic system containing pepsin (enzyme active in stomach) and pancreatin (enzyme active in deodenum) with moderate shaking to imitate the transit of protein through infant gut [Rudloff S., (1992). Solubility and digestibility of milk proteins in infant formulas exposed to different heat treatments. J Pediatr Gastroenterol Nutr 15: 25-33] was used. rHLf and nHLf were treated with active pepsin and pancreatic enzymes and exposed to 10⁴ CFU EPEC cells for 12 h at 37°C (Fig. 6) Both the native human lactoferrin standard (nHLf) and the recombinant rice-derived lactoferrin (rHLf) remained active in inhibiting growth of enteropathogenic E. coli, indicating that both nHLf and rHLf are resistant to protease digestion (Figure 8C). The dissociation constant (Kd) and the number of binding sites for HLf to its receptor were determined from the Caco-2 human intestinal cell line binding and uptake study (Figure 9). Both Kd and the number of bindings sites were not significantly different between nHLf and rHLf after in vitro digestion (Figure (9A, 9B). Digestion did not appear to affect on the Kd but made the number of binding sites much lower. Total Lf uptake was not significantly different between nHLf and rHLf after in vitro digestion (Figure 9C), though uptake was about one third when compared with undigested nHLf. Total iron uptake from nHLf was twice as high as that from rHLf. Percent degradation of HLf was similar regardless of digestion or not, and the native or recombinant form (Figure 9D). Thermal Stability of rHLf: effect on antimicrobial activity and binding/uptake to Caco-2 cells. 1.0 mg/ml of holo-HLf in PBS was treated by the following conditions: (a) 62 °C for 15 minutes, (b) 72 °C for 20 seconds, (c) 85 °C for 3 minutes, or (d) 100 oC for 8 seconds. Survival ratio of HLf determined by ELISA were more than 90% following treatment at 62 °C for 15 minutes, at 72 °C for 20 seconds, or at 85 °C for 3 minutes, but it was considerably lower after 100 °C for 8 seconds (Figure 10A). This high temperature precipitated both types of HLf and only 10% of HLf was detectable by ELISA. More than 80% of iron was still bound to both rHLf and nHLf after all thermal treatments with the exception of 100 °C for 8 sec. In 10% of survived HLf after 100 °C for 8 sec, the iron saturation level of nHLf was above 80% whereas that of rHLf was only about 40% (Figure 10B). SDS-PAGE and Western blots revealed no difference in immunoreactivity between nHLf and rHLf at 62 °C for 15 minutes, at 72 °C for 20 seconds, and at 85 °C for 3 minutes, but at 100 °C for 8 seconds, rHLf almost completely lost its immunological activity, whereas nHLf still maintained detectable immunoreactivity (Figure 10C).

There was no significant difference in anti-microbial activity between nHLf and rHLf after heat-treatment (Figure 10D). Anti-microbial activity of HLf was not affected by treatment at either 62 °C for 15 min, 72 °C for 20 sec or 85 °C for 3 minutes.

In the Caco-2 human intestinal binding cell assay regarding HLf thermal stability (data not shown), the Kd and the number of binding sites for nHLf and rHLf were not significantly different at 62 °C and 72 °C though there is a trend that nHLf exhibits somewhat lower Kd and binding sites than rHLf. As the temperature was increased (such as 85 °C and 100 °C), more rHLf bound to Caco-2 cells, most likely by non-specific binding due to more rHLf being denatured than nHLf. Uptake properties were similar for nHLf and rHLf even in the group treated at 100 °C where uptake of both types of HLf was highest among all the thermal treatments. Free iodine levels in the cells were also evaluated since it reflects degradation of HLf. About 20% of HLf was degraded in the untreated sample. There was no significant difference between nHLf and rHLf. Interestingly, samples treated at 100 °C were degraded twice as much as untreated samples of nHLf and rHLf, which may indicate that denaturation of HLf caused by heat treatment will make the protein more susceptible to proteases in the cells. pH Stability of rHLf: effect on antimicrobial activity and binding/uptake to Caco-2 cells 1.0 mg/ml of holo-HLf in PBS was adjusted to pH 2, 4, 6, or 7.4 by the addition of 1 M

HCI and incubated for 1 h at room temperature. The pH was then adjusted to 7.0 with 1 M NaHC03. Free iron released from HLf, was removed by a desalting column.

After low pH treatment, 100% of both nHLf and rHLf survived. The iron-holding capacity was maintained in all samples and the iron saturation level was above 95% (Figure 1 A). SDS- PAGE and Western blots revealed that there was no difference between nHLf and rHLf for any of the treatments (Figure 11 B). A slightly smaller immunoreactive molecule (-70 kD) was detected after exposure of nHLf to pH 2 and 4 and of rHLf to pH 2.

Antimicrobial activities of nHLf and rHLf were stable after exposure to low pH in the range of pH 2.0 to 7.4. As the pH was lowered, the activity of rHLf appeared to be higher and constant, whereas nHLf did not show any pH dependency (Figure 11 C).

In the Caco-2 human intestinal binding cell assay regarding HLf pH stability (data not shown), the Kd and the number of binding sites for nHLf were not significantly different from those for rHLf but a trend was always lower for nHLf within the range of pH 2.0 to 7.4, which is similar to control and thermal treatment samples. The Kd and the number of binding sites for nHLf and rHLf were not affected by pH treatment (dropping to pH 2.0 for 1 hour). Uptake properties were similar for nHLf and rHLf in the pH range of 2.0 to 7.4. Degradation of HLf in Caco-2 cells was also evaluated and there was no significant difference between nHLf and rHLf.

Apart from the differences in glycosylation, all the above information and data indicate that there are no significant compositional or functional differences between nHLf and rHLf.

Example 3

Non-clinical Studies

Pharmacokinetics of HLf was studied in rats, mice and rabbits. Results showed that HLf was rapidly cleared out of the plasma and taken up by the liver in all studies. Toxicity was assessed by a thirteen-week oral toxicity study with bLf and no adverse effects were reported in all the rats.

Pharmacokinetics and Product Metabolism (Biological Disposition) in Animals

Pharmacokinetics of iodine containing and native lactoferrin from human milk was studied in intact mice. Both native and l-lactoferrin, were similarly distributed in intact mice body. Exogenous lactoferrin penetrated mainly into liver tissue, then simultaneously with bile into the small intestine and further into the large intestine [Kazachkina N.I., E.R. Nemtsova, et al. (1991). Pharmacokinetics of human milk lactoferrin in intact mice and tumor-carrying animals. Vopr Med Khim 37: 35-7]. Pharmacokinetic behavior of HLf was also studied after it was intravenously administrated to freely moving rats [Beljaars L., H.I. Bakker, etal. (2002). The antiviral protein human lactoferrin is distributed in the body to cytomegalovirus (CMV) infection-prone cells and tissues. Pharm Res 19: 54-62]. It was found that human lactoferrin (hLF) was rapidly cleared from the plasma in a dose-dependent manner. Immunohistochemical analysis revealed that HLf was predominantly sequestered to the liver, whereas minor distribution to lungs, kidneys and spleens were also observed in these rats. In the same report, the pharmacokinetics of hLF was also studies after intraperitoneal administration. The highest uptake of hLf was found to be in liver. Plasma peak concentration was measured at 2-4 h after injection, whereas at 24 h after the first injection, the plasma hLf levels were undetectable [Beljaars et al, (2002) Pharm Res 19: 54-62]. In a previous study, the rapid clearance of intravenously injected HLf from plasma and re-disposed in liver in rat was also suggested [Peen E., A. Johansson, et al. (1998). Hepatic and extrahepatic clearance of circulating human lactoferrin: an experimental study in rat. EurJour Haematol 61:151-91.

Lactoferrin turnover was studied in the rabbit with ¹²⁵l- and ³¹l- labelled.human lactoferrin [Karle H., N. E. Hansen, et al. (1979). Turnover of human lactoferrin in the rabbit. Scand Jour Haematol 23: 303-12]. Plasma lactoferrin activity showed a rapid decrease during the first 24 h, followed by a 'final slope' with a T1/2 of about 25 h after intravenously injection. The rapid turnover was confirmed in whole body studies. Concomitantly with the initial disappearance from the plasma, there was a marked accumulation of protein-bound activity only in the liver [Karle et al, (1979) Scand Jour Haematol 23: 303-12].

Example 4

Toxicological Effects

Bovine lactoferrin (LF), which is an iron-binding glycoprotein in cow milk, was administered orally to groups of 12 males and 12 female rats at dose levels of 200, 600 and 2000 mg/kg/day once daily for 13 weeks and its toxicity on repeated administration was examined. Throughout the administration period, there were no deaths caused by administration of the test compound, nor were any adverse effects noted in the general condition of the animals. The study findings concerning body weight and food consumption, ophthalmology, urinalysis including water consumption, hematology, blood chemistry, necropsy, organ weights and histopathology revealed that there were no apparent changes due to administration of LF

Example 5

Artificially Induced Anemia in Rats The concept of using Lf-Fe complex to treat anemia was supported by a study conducted with artificially induced anemic rats. Anemic rats were fed FeSO4 or Lf-Fe daily for 70 days. The data indicated that Fe in the form of Lf-Fe was able to restore the Hb and hematocrit and was at least 5 times more bio-available then FeSO4 [Kawakami H., M. Hiratsuka, et al. (1988). Effects of iron-saturated lactoferrin on iron absorption. Agric Biol Chem 52: 903-8). Example 6

The turnover of ¹²⁵l-labelled human milk-derived lactoferrin was measured in ten adults and simultaneous organ radioactivity counting was performed. Of the administered ¹²⁵l label 99% was recovered in the urine, as free iodine, within the first 24 hours, suggesting rapid catabolism. Organ radioactivity counting indicated that lactoferrin was rapidly incoporated by both liver and spleen [Bennett R.M. and T. Kokocinski (1979). Lactoferrin turnover in man. Clin Sci (Lond) 57: 453-60}.

Example 7

Safety and Efficacy

Three clinical trials had been conducted with HLf (2 trials) or BLf (1 trial) to evaluate the therapeutic effects for Helicobacter pylori infection, see Opekun, A. R., H. M. T. El-Zaimaity, ef al. (1999). Novel therapies for Helicobacter pylori nfection. Aliment. Pharmacol. Ther. 13: 35- 41 , ileostomies; see Troost, F. J., W. H. Saris, et al. (2002). Orally Ingested Human Lactoferrin Is Digested and Secreted in the Upper Gastrointestinal Tract In Vivo in Women with Ileostomies. J Nutr 132: 2597-2600; and HCV, see Tanaka, K., M. Ikeda, et al. (1999). Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: a pilot study. Jpn J Cancer Res 90: 367-71. Two open-label studies (low and high dose) were performed to assess the safety and efficacy of orally administrated rHLf to cure or suppress H. pylori infection in adult subjects. The rHLf was given by mouth five times throughout a 24-h period. Six subjects received 250 mg per dose (1.25 g/day) and six subjects 1 g per dose (5 g/day). No significant adverse effects were observed [see Opekun et al. (1999) Aliment. Pharmacol. Ther. 13: 35-41]. In order to determine the survival rate of orally administered recombinant human lactoferrin (rhLF) in the upper gastrointestinal (Gl) tract and in the small intestine in vivo in humans. Female ileostomy patients [n = 8; median age 44 (43-57) years] ingested a test beverage containing 5 g rhLF and collected full ileostomy output for 24 h. The results showed that dietary rhLF is digested in the upper Gl tract and does not reach the colon. Intact LF appearing in ileostomy effluent is likely to originate from an endogenous source [Troost, F. J., W. H. Saris, et al. (2002). Orally Ingested Human Lactoferrin Is Digested and Secreted in the Upper Gastrointestinal Tract In Vivo in Women with Ileostomies. J Nutr 132: 2597-2600]. No adverse effects were reported in the study.

Example 8 Objectives

To demonstrate the efficacy of two different dosages of Ventria LF-Fe in the treatment of ID or IDA as compared to ferrous sulfate.

Test and Control Articles

Test Articles: 600 mg Ventria LF-FE

Control Article: 300 mg Ferrous Sulfate

All test and control articles will be supplied by the sponsor in masked containers and labeled with the patient number. Dosage:

Group 1 - 600 mg Ventria LF-FE three times a day for 6 weeks

Group 2 - 1200 mg Ventria LF-FE twice a day for 6 weeks

Group 3 - 300 mg Ferrous sulfate three times a day for 6 weeks Subjects

Number and Source

Approximately 60 patients with ID or IDA will be analyzed as defined by hemoglobin and serum ferrritin levels. Subjects must not be currently taking any iron supplements. Inclusion Criteria

Willing to give consent.

At least 18 years of age

Any sex or race

Able to make the required study visits

Able to follow instructions

Hemoblobin concentration is <80 g/L and serum ferritin <12 ug/L

Normal C-reactive protein level

Exclusion Criteria

• Under the age of 18 • Presence of moderately severe disease, such as renal disease, evidence of ulcer, cancer, infection or inflammatory disease, Sickle cell anemia.

• Currently taking medications or other herbal preparations containing iron

• Currently taking aspirin, NSAIDs, gastric acid suppressants.

• Women who are nursing, pregnant, or trying to become pregnant; • Regular blood donors.

Additionally the principle investigator may declare any patient ineligible for a sound medical reason.

Study Procedure

This study is designed as a randomized double-masked, parallel comparison of 1800 mg Ventria LF-FE, 2400 mg Ventria LF-FE and 900 mg Ferrous sulfate in patients with ID or IDA. The study will last 6 weeks with patients taking the medication as prescribed. Blood will be drawn on Day 0, 7, 14, 21 , 28, 35 and 42 for assay of clinical markers. The medication will be supplied in identical, masked containers with the patient number. No other iron medications will be allowed during the study.

Screening Examination Design

• Prospective, randomized, double/triple-masked clinical trial.

• All subjects will be asked to maintain constant diet and exercise for the duration of this study. • The duration of the trial will be 6 weeks on the test product (2 arms with two different dosages) and leading iron supplement. Blood will be drawn every week and analyzed immediately for group difference (without breaking the mask).

• Randomization will be equal at 1 :1 :1.

• Subjects will be recruited from general population. • A trial coordinator will oversee all the subjects at each visit.

• The trial will be monitored by representatives from Ventria Bioscieήce.

• The subjects are not to take any acidic drink, like orange juice while taking LF-Fe. They are required to drink a cup of water before taking the pills.

Any side effects will be reported to principal investigator (PI) and to IRB (when serious and adverse) using appropriate reporting format.

Statistical Analysis Sample Size: • Clinical endpoints: A statistically significant improvement in iron deficiency as shown through objective and subjective measurement;

• Power = 0.8, alpha=0.05;

• A total of 60 subjects (20 subjects/arm) will be needed for the study; • Using a drop-out/loss-to-follow-up rate of 10%, we would need to begin the study with approximately 66 subjects;

Treatment Information

Test material and ferrous sulfate:

Test material and ferrous sulfate will be supplied in identical bottles. Dosing:

One gram of fully iron-saturated lactoferrin (HoloLac) will bind 1.4 mg of Fe. The current plan is to prepare LF-Fe capsule containing 600 mg lactoferrin saturated with Fe (840 ug Fe). In one tested arm, subjects will be asked to take 3 capsules (1.8 gram of LF and 2.520 mg Fe) a day. In another tested arm, subjects will be asked to take 4 capsules (total iron intake is 3.36 mg Fe). Subjects in the third arm will be treated with commercially available iron pills.

Example 9

Objectives:

Demonstrate the efficacy of two different dosages of Ventria LF-Fe in the treatment of ID or IDA.

Test and Control Articles

Test Articles: 600 mg Ventria LF-FE

All test articles will be supplied by the sponsor in containers labeled with the patient number.

Dosage: Two (2) capsules with 0.6 gm Ventria LF-FE four times a day for 6 weeks

Subjects

Number and Source

Approximately 5 patients with ID or IDA will be analyzed as defined by hemoglobin and serum ferrritin levels. Subjects must not be currently taking any iron supplements.

Inclusion Criteria Willing to give consent and make study visits

Females at least 18 years of age

Able to make the required study visits

Able to follow instructions

Hemoglobin concentration is <80 g/L and serum ferritin <12 ug/L

Normal C-reactive protein level

Exclusion Criteria • Under the age of 18

• Presence of moderately severe disease, such as renal disease, evidence of ulcer, cancer, infection or inflammatory disease, Sickle cell anemia.

• Currently taking medications or other herbal preparations containing iron

• Currently taking aspirin, NSAIDs, gastric acid suppressants. • Women who are nursing, pregnant, or trying to become pregnant;

• Regular blood donors.

Additionally the principle investigator may declare any patient ineligible for a sound medical reason.

Study Procedure

This study is designed as open label trial to determine the efficacy of 4.8 gm of Ventria LF-FE in raising Hb and/or serum ferritin levels in women with ID or IDA.

The study will last 6 weeks with patients taking the medication as prescribed. Blood will be drawn on Day 1 , 8, 15, 22, 29, 36 and 43 for assay of clinical markers.

No other iron medications will be allowed during the study.

Screening Examination Patients who have been tested for iron markers and are deficient or anemic can be recruited for the study.

Potential subjects should be given information about the study and asked to return on the study start date. Dav 1

All subjects have pregnancy test All subjects have blood drawn for following tests: CBC SMAC-12 Hemoglobin Hematocrit Serum ferritin TIBC cRP - C reactive protein

All subjects sign informed consent

Subjects are given the study schedule

Subjects are given study dietary supplement and instructions on dosing

Day 8. 15. 22. 29. 36

Subjects return to doctor's office

Complete questionnaire on compliance, sioe effects and physical status

Receive additional test material for dosing as required

All subjects have blood drawn for following tests: Hemoglobin

Hematocrit

Serum ferritin

TIBC

Day 43, Study completion

Subjects return to doctor's office.

Complete questionnaire on compliance, side effects and physical status

All subjects turn in any unused test material

All subjects have pregnancy test All subjects have blood drawn for the following tests:

All subjects have blood drawn for following tests:

CBC

SMAC-12 Hemoglobin Hematocrit Serum ferritin TIBC cRP - C reactive protein

Plus extra serum for antibody levels.

Example 10

Human lactoferrin is a major part of the diet of breast-fed infants; the addition of recombinant human lactoferrin to the diet will be an extension of their normal lactoferrin intake. The study is to evaluate the effect of recombinant human lactoferrin in a rice meal, given twice daily from 6 to 12 months of age, on iron status of infants.

The field intervention will be a double-blind study that will provide a weaning meal based on rice to 300 infants (150 per group) during 6 months of intervention. Subjects will enter at 6 months age; a signed consent form from parents or guardians will be required. At entry, they will be assigned to two groups, receiving either: a) iron-fortified rice, or b) iron-fortified rice with lactoferrin.

Using the formula for unpaired differences between means with an α-level = 0.05 and a power (1-β) = 0.9, each group will have 25 children that will allow detection of differences between groups with 95% of confidence interval and 90% power; 20% has been added to allow for program dropouts.

Infants will be given two portions of rice gruel per day, each containing 35 g of polished rice and 1.05 mg of ferrous sulfate. The two groups (n=150) will be given either regular rice or rice with recombinant human lactoferrin at a level of 5 mg/gram, i.e. the lactoferrin intake will be 700 g/day. Breast-fed infants consume -500-1000 mg/day. The lactoferrin rice will provide additional iron, but as it is not saturated with iron, it can also enhance the bioavailablility of "native" iron in rice. Lactoferrin has a very high affinity for iron and thus will quickly acquire iron in "free" form (ferrous sulfate) in the presence of bicarbonate ions (pancreatic fluid). A commercial rice cereal will be used for feeding with the addition of 1.05 mg ferrous sulfate, 1 teaspoon of either control rice protein extract or lactoferrin rice protein extract. Field workers will visit the mothers in their homes every morning and supervise the feeding of the first meal, and leave the second one to the mothers to feed the infants in the afternoon.

Mothers in their homes will be visited twice a week and a field worker will record information on infants' morbidity and feeding patterns. Children will be examined every month by the pediatrician and at this time anthropometry will be measured at a Community Hospital. Free medical care is available for all participants at the clinic. At 9 and 12 months, blood samples will be drawn for assessment of iron, zinc and copper status. Hemoglobin will be measured directly on a Hemocue instrument, and serum will be separated from red blood cells (RBC) at the Clinic and frozen for later transport to UCDavis. In the Pi's lab, serum ferritin, zinc, copper and CRP (elevated levels are indicative of infection) will be analyzed, as well as Cu-Zn- superoxide dismutase in RBCs. Statistical analysis will be done using the SPSS/PC statistical Program V. 7.5 for

Windows. Data analysis includes descriptive statistics, paired t-test and chi-square tests, ANOVA, multivariate analysis of variance (MANOVA). Initial Hb will be considered as covariate.

300 infants (150 per group) will be recruited at 5 months of age and start to receive a weaning meal based on iron-fortified rice twice daily from 6 to 12 months of age. Children should have been born at term in a health center, with adequate weight for gestational age, and breast-fed during the first 6 months of age. Infants should not have any chronic disease, or congenital malformations. In this community, 95% of deliveries are attended in health centers; from these records we will visit potential subjects' homes to invite the family to participate in the study. Recruitment will also be done through health centers and community organizations. Parents who want their infant to participate in the study will receive a complete description of the study, including a detailed consent form to read and discuss with family member and/or friends. Any questions will be addressed by the investigators. If a potential subject qualifies for the study and the parents or guardians have decided to participate, they will be asked to sign the informed consent form.. At entry, infants will be examined by the pediatrician in the project, and also evaluated by a nutritionist; a venous blood sample will be drawn to assess iron, zinc and copper status. Any infant with a hemoglobin <85 g/L will be excluded and given medicinal iron drops (such a low Hb is very unusual at this age).

All procedures in this study will be overseen by highly trained individuals; minimizing potential risks to the subjects. Potential risks to the infants in the study include those associated with blood drawing. The participants may receive a bruise and, rarely, an infection at the site of the venipuncture.

Benefits to the infants include medical attention during the period from 6 months to 12 months of age, a very important stage for the health and development of the child. He or she will also receive medical and nutritional consultations, as well as lab analyses

Results from the study will provide data on the capacity of lactoferrin to provide iron to growing infants, and on the activity of lactoferrin-fortified rice in preventing infection in infants. Rice with high levels of human lactoferrin is likely to have anti-infective activity, and to improve iron status in infants, and the results from this study may thus be of great benefit to this vulnerable population worldwide. Brief Description of the Seouences

Description SEQ ID NO

Codon optimized lactoferrin coding 1 sequence:GGGCGGCGGGGGCGCTCGGTGCAGTGGTGCGCCGTGTCCCAGC

CCGAGGCGACCAAGTGCTTCCAGTGGCAGCGCAACATGGGGAAGGTGCGC

GGCCCGCCGGTCAGCTGCATCAAGCGGGACTCCCCCATCCAATGCATCCAG

GCCATCGCGGAGAACCGCGCCGACGCGGTCACCCTGGACGGCGGGTTCAT

CTACGAGGCGGGGCTCGCCCCGTACAAGCTCCGCCCGGTGGCGGCGGAG

GTGTACGGCACCGAGCGCCAGCCGCGCACGCAGTACTACGCGGTGGCCGT

CGTCAAGAAGGGCGGGTCCTTCCAGCTCAACGAGCTGCAGGGCCTGAAGT

CGTGCCACACGGGCCTCCGGCGGACGGGGGGCTGGAACGTGCCCATCGG

CACCCTGCGCCCCTTCCTGAACTGGACCGGCCCGCCGGAGCCGATCGAGG

CCGCCGTGGCCCGCTTCTTCAGCGCCTCCTGCGTCCCCGGCGCCGACAAG

GGCCAGTTCCCGAACCTCTGCCGGCTCTGCGCCGGGACGGGCGAGAACAA

GTGCGCCTTCTCCTCGCAGGAGCCGTACTTCTCCTACTCGGGCGCGTTCAA

GTGCCTCCGCGACGGGGCCGGCGACGTGGCGTTCATCCGCGAGTGCACCG

TGTTCGAGGACCTCTCCGACGAGGCGGAGCGGGACGAGTACGAGCTGCTG

TGCCCCGACAACACCCGCAAGCCGGTGGACAAGTTCAAGGACTGCCACCTG

GCGCGGGTGCCCTCGCACGCGGTCGTCGCCCGCAGCGTCAACGGCAAGGA

GGACGCGATCTGGAACCTCCTCCGCCAGGCCCAGGAGAAGTTCGGGAAGG

ACAAGTCCCCCAAGTTCCAGCTCTTCGGGAGCCCCAGCGGCCAGAAGGACC

TCCTCTTCAAGGACTCCGCGATCGGCTTCTCCCGCGTCCCCCCGCGCATCG

ACTCCGGCCTGTACCTCGGCTCCGGGTACTTCACCGCGATCCAGAACCTGC

GGAAGAGCGAGGAGGAGGTGGCGGCGCGGCGGGCCCGCGTCGTGTGGTG

CGCCGTGGGCGAGCAGGAGCTGCGGAAGTGCAACCAGTGGAGCGGCGTGA

GCGAGGGGTCGGTGACCTGCTCGTCCGCCAGCACCACCGAGGACTGCATC

GCGCTCGTCCTCAAGGGGGAGGCCGACGCGATGAGGCTCGACGGGGGGTA

CGTCTACACCGCCGGCAAGTGCGGCCTGGTCCCGGTCCTGGCGGAGAACT

ACAAGTCGCAGCAGTCCAGCGACCCCGACCCGAACTGCGTGGACCGCCGC

GTCGAGGGCTACCTCGCCGTGGCCGTCGTGCGCCGGTGCGACACCTCCCT

GACGTGGAACAGCGTCAAGGGCAAGAAGAGCTGCCACACCGCCGTGGACC

GCACCGCCGGCTGGAACATCCCGATGGGCCTCCTCTTCAACCAGACCGGCT

CCTGCAAGTTCGACGAGTACTTCTCCCAGTCCTGCGCCCCCGGCTCGGACC

CCCGCTCCAACCTGTGCGCCCTCTGCATCGGGGACGAGCAGGGCGAGAAC

AAGTGCGTGCCCAACAGCAACGAGCGGTACTACGGCTACACGGGGGCCTT

CCGCTGCCTGGCGGAGAACGCCGGGGACGTCGCGTTCGTGAAGGACGTGA

CCGTGCTGCAAAACACGGACGGGAACAACAACGAGGCGTGGGCGAAGGAC

CTCAAGCTCGCGGACTTCGCCCTGCTGTGCCTCGACGGCAAGCGCAAGCCC

GTCACCGAGGCGCGGTCCTGCCACCTGGCGATGGCCCCCAACCACGCCGT

CGTCTCCCGCATGGACAAGGTCGAGCGCCTCAAGCAGGTGCTCCTGCACCA

GCAGGCCAAGTTCGGCCGGAACGGCAGCGACTGCCGGGACAAGTTCTGCC

TGTTCCAGTCGGAGACCAAGAACCTCCTCTTCAACGACAACACCGAGTGCCT

GGCGCGCCTCCACGGCAAGACCACCTACGAGAAGTACCTCGGCCCGCAGT

AGGTCGCCGGCATCACCAACCTCAAGAAGTGCTCCACCTCCCCCCTCCTGG

AGGGGTGCGAGTTCCTCCGCAAGTGA

Amino acid sequence based on codon optimized lactoferrin coding sequence:

GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQAIAEN

RADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSFQL

NELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAAVARFFSASCVPG

ADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFKCLRDGAGDVAFIREST

VFEDLSDEAERDEYELLCPDNTRKPVDKFKDCHLARVPSHAVVARSVNGKEDAI

WNLLRQAQEKFGKDKSPKFQLFGSPSGQKDLLFKDSAIGFSRVPPRIDSGLYLG SGYFTAIQNLRKSEEEVAARRARVVWCAVGEQELRKGNQWSGLSEGSVTCSS

ASTTEDCIALVLKGEADAMSLDGGYVYTAGKCGLVPVLAENYKSQQSSDPDPN

CVDRPVEGYLAVAVVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQ

TGSCKFDEYFSQSCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAF

RCLAENAGDVAFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTE

ARSCHLAMAPNHAWSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSE

TKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK

Rice Gt1 promoter and Gt1 leader coding sequence

CATGAGTAATGTGTGAGCATTATGGGACCACGAAATAAAAAGAACATTTTGAT

GAGTCGTGTATCCTCGATGAGCCTCAAAAGTTCTCTCACCCCGGATAAGAAA

CCCTTAAGCAATGTGCAAAGTTTGCATTCTCCACTGACATAATGCAAAATAAG

ATATCATCGATGACATAGCAACTCATGCATCATATCATGCCTCTCTCAACCTA

TTCATTCCTACTCATCTACATAAGTATCTTCAGCTAAATGTTAGAACATAAAGC

CATAAGTCACGTTTGATGAGTATTAGGCGTGACACATGACAAATCAGAGACT

CAAGCAAGATAAAGCAAAATGATGTGTACATAAAACTCCAGAGCTATATGTCA

TATTGCAAAAAGAGGAGAGCTTATAAGACAAGGCATGACTCACAAAAATTCA

CTTGCCTTTCGTGTCAAAAAGAGGAGGGCTTTACATTATCCATGTCATATTGC

AAAAGAAAGAGAGAAAGAACAACACAATGCTGCGTCAATTATACATATCTGTA

TGTCCATCATTATTCATCCACGTTTCGTGTACCACACTTCATATATCATAAGA

GTCACTTCACGTCTGGACATTAACAAACTCTATCTTAACATTTAGATGCAAGA

GCCTTTATCTCACTATAAATGCACGATGATTTCTCATTGTTTCTCACAAAAAG

CGGCCGCTTCATTAGTCCTACAACAACATGGCATCCATAAATCGCCCCATAG

TTTTCTTCACAGTTTGCTTGTTCCTCTTGTGCGATGGCTCCCTAGCG

Claims

WHAT IS CLAIMED IS:

1. A composition formulated for administration to a human in need of iron supplementation comprising a lactoferrin protein, wherein the lactoferrin protein comprises at least one iron molecule in complex with said protein.

2. A composition as in claim 1 , wherein the lactoferrin protein is recombϊnantiy produced in a host cell transformed with a nucleic acid sequence encoding said lactoferrin protein and isolated from the host cell.

3. A composition as in claim 1 or 2, comprising a plurality of lactoferrin protein.

4. A composition as in claim 1 or 2, wherein tne lactoternn protein is saturated with iron.

5. A composition as in claim 3, wherein the plurality of lactoferrin protein are saturated with iron.

6. A composition as in claim 2, wherein the recombinant production comprises a host cell selected from the group consisting of a plant cell, a bacterial cell, an animal cell, and a yeast cell.

7. A composition as in claim 6, wherein the host cell is a plant cell and the plant cell is a monocot.

8. A composition as in claim 7, wherein the lactoferrin protein is extracted from a product of a mature monocot seed,

9. A composition as in claim 7, wherein the lactoferrin protein comprises at least one plant-derived glycosyl group.

10. A composition as in claim 1 or 2, wherein the formulation is selected from the group consisting of parenteral, enteric, edible, suppository, inhalant, intranasal and topical formulations.

11. A composition as in claim 1 or 2, wherein the formulation comprises a nutritionally or pharmaceutically acceptable carrier.

12. A method of making a composition for administering to a patient in need of iron supplementation comprising the steps of a. expressing a nucleic acid encoding a human lactoferrin protein or portion thereof in a host cell; b. isolating the lactoferrin protein from the host cell; and c. contacting the recombinantly produced lactoferrin protein with one or more iron molecules to form a complex of the protein with iron.

13. A method as in claim 12, wherein the host cell is selected from the group consisting of a plant cell, a bacterial cell, an animal cell, and a yeast cell.

14. A method as on claim 13, wherein the host cell is a plant cell and the plant is a monocot.

15. A method as in claim 12, further comprising the step of: d. formulating the recombinantly produced lactoferrin for parenteral or oral delivery to the patient comprising contacting the protein and iron with a pharmaceutically or nutritionally acceptable carrier.

16. A method of treating iron deficiency in a patient comprising administering to the patient a composition comprising a lactoferrin protein and an iron salt, wherein the composition is formulated in a pharmaceutically or nutritionally acceptable carrier.

17. A method of treating iron deficiency in a patient comprising administering to the patient a composition comprising a lactoferrin protein recombinantly produced in a host cell transformed with a nucleic acid sequence encoding said lactoferrin protein and an iron salt, wherein the composition is formulated in a pharmaceutically or nutritionally acceptable carrier.

18. A method as in claim 16 or 17, wherein said lactoferrin protein is saturated with iron.

19. A method of treating iron deficiency in a patient comprising administering to the patient a composition comprising a lactoferrin protein produced in a monocot seed transformed with a nucleic acid sequence encoding said lactoferrin protein and extracted from a product of a mature monocot seed.

20. A method as in claim 19, further comprising administering an iron salt before, after or during administration of the lactoferrin protein.