WO2020031141A1

WO2020031141A1 - Method for treating iron deficiency

Info

Publication number: WO2020031141A1
Application number: PCT/IB2019/056782
Authority: WO
Inventors: Jeffrey Bruce Wicking; Zoraida Defreitas
Original assignee: Cura Global Health (Bvi) Limited
Priority date: 2018-08-10
Filing date: 2019-08-09
Publication date: 2020-02-13

Abstract

The present invention provides a method for treating iron deficiency and iron deficiency anemia in a subject, the method comprising providing to the subject an iron supplement or product comprising fungal biomass harvested from cultured filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass; wherein normal transferrin saturation capacity is maintained in the subject during treatment.

Description

METHOD FOR TREATING IRON DEFICIENCY

Related Application

[001] This application claims priority from Australian Provisional Patent Application No

2018902920 entitled 'Method for treating iron deficiency' filed on 10 August 2018 The entire disclosure of AU 2018902920 is hereby incorporated by reference herein.

Technical Field

[002] The technology relates to use of an iron supplement or product comprising fungal biomass containing iron to treat iron deficiency and iron deficiency anemia in a subject.

Background

[003] Iron plays a crucial role in many physiological functions, such as oxygen

transportation, as an enzyme cofactor and in adenosine triphosphate (ATP) synthesis. Iron deficiency is the most common nutritional deficiency in the world, especially among women and children in developing countries. Negative consequences of iron deficiency anemia (IDA) include increased mortality, reduced cognitive and physical development of children and decreased performance and work productivity in adults.

[004] The most widely used approach to combat iron deficiency anemia is to increase the consumption of foods with high amounts of iron. However, in many cases these foods are neither available for economic reasons nor suitable as is the case of vegetarian people. Ferrous sulfate (FeS0₄) is effectively absorbed by humans but causes many side effects due to quick increases of iron in the blood and in the intestine when taken as a supplement. High levels of un-absorbed iron in the intestine are associated with diarrhea, inflammation and constipation. Furthermore, high surges of iron into serum are known to overcome the capacity of transferrin, a protein that carries iron from the serum to various tissues and organs.

[005] Under normal iron status, transferrin is available to carry the iron present in circulation, so the total iron binding capacity of the serum is high and the percent of iron (transferrin) saturation is low. However, in acute and chronic iron overload conditions, the capacity of transferrin to bind iron decreases, causing high transferrin (iron) saturation (TS) and production of non-transferrin bound iron (NTBI). Normal TS levels in women and men are < 45% and < 50%, respectively, but in acute and chronic iron overload conditions, they can be exceeded. [006] It is important to keep low iron saturation since high TS has recently been

associated with reduced telomere shortening. It has been observed that individuals with high (>45%) and normal to high (35-45%) TS had shorter leucocyte telomere lengths compared with those with low to normal (<30%) TS. Furthermore, it has been

recommended to control TS below 35% to delay biological aging (cellular aging) and reduced the risk of aging-associated diseases induced by oxidative stress.

[007] Besides the known negative side effects, such as diarrhea, constipation and nausea, ferrous sulfate decreases the total iron binding capacity of transferrin creating prooxidative conditions, due to the formation of NTBI. It is now known that circulating NTBI is likely to appear despite the presence of available binding sites on transferrin if the rate of iron influx into plasma exceeds the rate of iron acquisition by transferrin. The amount of NTBI represents labile iron which is free to react with DNA, protein, lipids and organic compounds causing chronic oxidative stress, cellular aging, increase susceptibility to infections and consequently developing disease.

[008] The present applicant has developed a new nutritional supplement containing iron made by culturing filamentous fungi in the presence of iron to accumulate iron in the fungal biomass. The technology is described in WO 2014/040122 which is incorporated herein by reference.

[009] Ultimine™ iron supplement is a product produced according to WO 2014/040122 or WO 2017/205890 by Cura Global Health, Inc and is a source of iron naturally encapsulated by Aspergillus oryzae biomass, also known as koji culture, or Aspergillus niger biomass. (ULTIMINE™ is a trade mark owned by Cura Global Health, Inc). More than 90% of the iron can be stored within the cells of fungal culture in the mycelia as organic iron. The present inventors have found that the organic iron from the Ultimine™ product is absorbed and causes less iron surges into the blood. A potential benefit of this slow release behaviour is the reduction of reactive non-transferrin bound iron (NTBI) and its side effects.

[010] The present inventors have developed an improved method for treating iron deficiency and iron deficiency anemia using an iron supplement produced from filamentous fungi.

Summary

[01 1] In a general aspect, there is provided a method for treating iron deficiency and iron deficiency anemia in a subject, the method comprising providing to the subject an iron supplement or product comprising fungal biomass harvested from cultured filamentous fungi having organic iron accumulated in the fungal biomass; wherein normal transferrin saturation capacity is maintained in the subject during treatment.

[012] In a first aspect, there is provided a method for treating iron deficiency and iron deficiency anemia in a subject, the method comprising providing to the subject an iron supplement or product comprising fungal biomass harvested from cultured filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass; wherein normal transferrin saturation capacity is maintained in the subject during treatment.

[013] In a second aspect, there is provided a method of providing iron supplementation to a subject, the method comprising providing to the subject an iron supplement comprising fungal biomass harvested from filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass; wherein normal transferrin saturation capacity is maintained in the subject during supplementation.

[014] In a third aspect there is provided a method of providing iron supplementation to a subject, the method comprising providing to the subject an iron supplement comprising fungal biomass harvested from filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass; wherein iron levels in the subject are increased without formation of non-transferrin bound iron during supplementation.

[015] In a fourth aspect there is provided use of an iron supplement comprising fungal biomass harvested from filamentous fungi having organic iron accumulated in the fungal biomass to provide iron supplementation in a subject.

[016] In a fifth aspect there is provided use of an iron supplement comprising fungal biomass harvested from filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass to provide iron supplementation to a subject wherein normal transferrin saturation capacity is maintained in the subject during treatment, wherein iron levels in the subject are increased without formation of non-transferrin bound iron during treatment.

[017] In a sixth aspect there is provided use of an iron supplement comprising fungal biomass harvested from filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass to treat iron deficiency and iron deficiency anemia to a subject, wherein normal transferrin saturation capacity is maintained in the subject during treatment, and wherein iron levels in the subject are increased without formation of non-transferrin bound iron during treatment.

[018] In an embodiment, the filamentous fungus is Aspergillus oryzae. [019] In an embodiment, the nutritional product is a supplement in the form of a tablet, capsule, gummy, caplet, powder, granule, ampoule, vial, ready-to-use solution or suspension, drink, medicinal food, a food in the form of a drink, powder, soup, cereal, bread, cookies, meat analogue, ready-to-eat meals or lyophilized material.

[020] In an embodiment, the iron supplement or product is a medicinal food containing fungal biomass with accumulated organic iron

[021] In an embodiment, the nutritional product is a medicinal food in the form of a drink, ready-to-use solution or suspension, powder or meal.

[022] In an embodiment, the iron supplement or product is a capsule formulated for daily consumption.

[023] In an embodiment, the iron supplement or product contains sufficient iron to provide the desired supplementation. It will be appreciated that the nutritional product can be provided periodically to the subject in any suitable dosage regime.

[024] In an embodiment, from about 1 to 195 mg iron is provided to the subject on a daily basis.

[025] In an embodiment, from 1 , 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, to 195 mg iron is provided to the subject on a daily.

[026] In an embodiment, about 65 mg iron is provided to the subject on a daily basis.

[027] In an embodiment, the iron supplement or product is taken orally once per day.

[028] In an embodiment, the iron supplement or product is taken orally several time a day to provide the iron supplementation required.

[029] In an embodiment, the treatment is maintained for at least 3 weeks.

[030] In an embodiment, the treatment is maintained for at least a month.

[031] In an embodiment, the treatment is maintained for at least 3 months.

[032] In an embodiment, the treatment is maintained for at least 6 months.

[033] In an embodiment, the treatment is maintained for at least 1 year.

[034] In an embodiment, the iron levels in the subject are increased without formation of non-transferrin bound iron during treatment.

[035] An advantage of the present invention is that normal transferrin saturation capacity is maintained in the subject during treatment. [036] An advantage of the present invention is that potential iron overload (toxicity) can be reduced or eliminated but still provide the iron supplementation required in a subject.

[037] An advantage of the present invention is that it can maintain normal iron status in a human subject without the production of harmful reactive free iron and with fewer gastrointestinal side effects.

[038] Another possible advantage of the present invention is a potential to also increase glomerular filtration rate (eGFR) in subjects with lower eGFR status and/or impaired kidney function.

[039] An advantage of the present invention is that it can maintain normal iron status in a human subject without the production of non-transferrin bound iron.

[040] An advantage of the present invention is that it can maintain normal iron status in a human subject while reducing the potential for oxidative stress, chronic disease and premature aging.

[041] An advantage of the present invention is that normal transferrin saturation capacity is maintained without production of harmful free iron in the subject during treatment.

Definitions

[042] Throughout this specification, unless the context clearly requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[043] Throughout this specification, the term 'consisting of means consisting only of.

[044] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present technology. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present technology as it existed before the priority date of each claim of this

specification.

[045] Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the technology recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

[046] In the context of the present specification the terms 'a' and 'an' are used to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, reference to 'an element' means one element, or more than one element. [047] In the context of the present specification the term 'about' means that reference to a figure or value is not to be taken as an absolute figure or value, but includes margins of variation above or below the figure or value in line with what a skilled person would understand according to the art, including within typical margins of error or instrument limitation. In other words, use of the term 'about' is understood to refer to a range or approximation that a person or skilled in the art would consider to be equivalent to a recited value in the context of achieving the same function or result.

[048] Those skilled in the art will appreciate that the technology described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the technology includes all such variations and modifications. For the avoidance of doubt, the technology also includes all of the steps, features, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features and compounds.

[049] In order that the present technology may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief Description of the Drawings

[050] Figure 1 shows Recruiting Process.

[051] Figure 2 shows Clinical Trial Design.

[052] Figure 3 changes in serum iron (a) throughout 8 hours after acute supplementation with 65 mg Iron from Ultimine™ product were significantly lower at all time points compared to FeS0₄. n=16. ^** P< 0.01 , ^*P<0.0001. The relative bioavailability of both iron sources has been reported to be the same (b).

[053] Figure 4 shows changes in serum iron throughout 8 hours and 4 hours (a, b, respectively) after acute supplementation with various doses (10, 20 and 65 mg iron) from Ultimine™ product. Differences between treatments at each time point was analyzed with two-way repeated measures of ANOVA. ^*P<0.01 (Figure 3 a).

[054] Figure 5 shows percent (a) and change (b) of TS after supplementation with 65 mg Iron from either Ultimine™ product or FeS04. n=16. ^*P <0.01.

[055] Figure 6 shows changes in NTBI (Mean ± SEM) throughout 8 hours after acute supplementation with 65 mg of iron from Ultimine™ product or FeS0₄ with a semi-purified meal. Differences between treatments at each time point was analyzed with two-way repeated measures of ANOVA. ^*P<0.01 , ^**P<0.0001.

[056] Figure 7 shows correlation between SI and NTBI (A) and TS and NTBI (B). Values represent 194 pairs combining ULT and FeS0₄. [057] Figure 8 shows mean (± SEM) frequency of reported side effects over 3 weeks of supplementation (n=16).

[058] Figure 9 shows mean (± SEM) frequency of combined nausea, constipation and diarrhea reported over 3 weeks of supplementation (n=16). Different letters indicate statistical significance, alpha=0.05. Linear-mixed models mean frequency effects amongst groups were compared with one-way ANOVA and Tukey multiple comparison test.

[059] Figure 10 shows a process for obtaining an iron supplement containing organic iron made by culturing filamentous fungi in the presence of iron to accumulate iron in the fungal biomass.

Description of Embodiments

FUNGAL IRON-CONTAINING PRODUCT

Process

[060] General steps of a process to form a fungal biomass containing iron are set out in Figure 10. Further production information can be found in WO 2014/040122 or

WO 2017/205890, incorporated herein by reference.

Fungal strains

[061 ] Aspergillus oryzae (A.o.) or Aspergillus niger ( A.n .) and Rhizopus orligosporus (R.o) or Rhizopus oryzae (R.oz) have the ability to uptake iron from natural sources such as soil or in controlled solid or liquid fermentations containing high concentrations of iron.

[062] Although the filamentous fungi Aspergillus oryzae or Aspergillus niger have been found to particularly useful, it will be appreciated that other filamentous fungi suitable for human consumption can be used to produce an iron supplement or product containing organic iron for use in the present invention.

[063] All culture strains that are suited for the production of food can be used, including but not limited to strains of Aspergillus oryzae that are approved and employed

commercially for soy sauce and miso manufacture, including Aspergillus oryzae 2355 and 40151 from Chinese Center of Industrial Culture Collection (CICC); Aspergillus oryzae 22787 from American Type Culture Collection (ATCC) can be used. Aspergillus niger var. 2206 and 10557 for citric acid production from CICC and Aspergillus niger 66876 for phytase production from ATCC can also be used. Fungal culture

[064] Stains of Aspergillus oryzae, Aspergillus niger, Rhizopus orligosporus (R.o) or Rhizopus oryzae were cultured and maintained in media composed of chemicals including but not limited to; carbon source such as dextrose, nitrogen source such as urea, phosphorous source such as potassium phosphate and micro nutrients. The media can be composed of organic materials such as ground whole corn, wheat bran, soybean hulls, molasses of beet, cane and fruits juice process by-product, and any other food process by product consist of starch, sugar and protein. Such organic raw materials can be pretreated by enzymes, including amylases, gluco-amylases, phytase and protease. The insoluble iron compounds that can be added during the preparation of the growth media may include ferrous sulphate, ferric sulphate and other soluble iron compounds. And/or insoluble iron compounds such as elemental iron powder, ferric pyrophosphate, ferric orthophosphate or iron oxides may be included. The quantity of the iron compound ranges from about 1 gram per liter of media up to 3 grams per liter. The forms of iron that are used in the process should be suitable for human consumption.

[065] Fungal spores were prepared by inoculating a solid media, such as cooked rice, soybean, and sorghum and the combination of them with moisture of 40-70%. In 2-3 weeks, the spores germinated and were ready to be collected. The fungal spores were collected into sterilized distilled water. The pre-cultures fermenters were prepared with 1 - 10% volume of the final production fermenters. The media for the pre-cultures can be the same as the production media as described above. Incubation for 18-28 hours of pre culture fermentation time is suitable to generate healthy pre-cultures after the spores were introduced into the pre-culture media. The pre-culture is added to the production fermenter and fungus is allowed to grow to produce the desired fungal mass containing iron.

Apparatus

[066] Large scale fermentation can be carried out in any suitable fermentation vessel or apparatus. For the iron enriched biomass production, the fermentation is preferably carried out under aerobic conditions for 48-72 hours. Sterilized or filtered air can be pumped into the fermenter at 0.5 to 1 .0 vvm during the fermentation period to improve growth and yield. The culture is preferably agitated or stirred during fermentation. The combination of air, agitation and design of the fermentation vessel is well understood for commercial microbial culture. Fungal fermentation

[067] Fermentation can be carried out for 24-72 hours or until cell autolysis begins at a temperature of 28-35°C. A temperature of 28-30°C has been found to be suitable. It will be appreciated that incubation times and temperature may vary depending on the fungus type and strain used.

[068] Depending on the nutritional profile of the raw materials, other nutrients may be needed to supplement the growth media for an aerated fungal fermentation. These nutrients may include organic and inorganic nitrogen sources, phosphors source and micro minerals.

Production of iron enriched fungal products as an iron supplement

[069] Fungi, including filamentous fungi, have the ability to further uptake relatively bio- unavailable and strongly cytotoxic iron. It should be noted that, direct supplementation of soluble inorganic iron salt in human diets can result in a cytotoxic reaction. Therefore, using fungi to uptake insoluble iron and transform it to an organic form may reduce the side effects of the direct consumption of iron salts.

[070] The insoluble iron can be added during the fermentation. The common choice of the insoluble iron includes elemental iron powder, ferric pyrophosphate, ferric orthophosphate or iron oxides. To increase the level of iron in the fungal product, insoluble iron can be incrementally fed during the fermentation. The dosing of iron depends on the type of iron used but the dosing level needs to not compromise the growth of fungi. After harvest, the fungal mycelium can be thoroughly washed to remove excess iron. A mild acid, pH 2-3, wash can be effective in this regard.

Fungal biomass harvesting

[071 ] After fermentation, fungal biomass containing iron can be harvested by a dewatering machine such as a centrifuge, belt press etc. Washing with water and/or mild acid such as hydrochloric acid 0.01 M can be used to remove iron residues. The iron enriched fungal product can then be dried at 60-80°C using forced air, fluid bed dryer, etc. The final moisture of the product is preferably less than about 10%.

[072] If there is any residue of iron powder that had not been utilized by fungi; a magnetic mechanism recovery system can be used to remove the iron if desired. Ferric

pyrophosphate and orthophosphate residues may also be removed by filtration if desired. Formulation

[073] For example, the nutritional product may be formulated for oral delivery. Non limiting examples of particular formulation types include tablets, capsules, caplets, powders, granules, gummies, ampoules, vials, medicinal foods, ready-to-use solutions or

suspensions, drinks, meat analogue, ready-to-eat meals and lyophilized materials. The solid formulations such as the tablets or capsules may contain any number of suitable acceptable excipients or carriers. Food application may include powder, flake or extruded forms, or/and blended with other minerals, vitamins and food ingredients.

METHODOLOGY

Subjects

[074] Females, 18-40 years of age were recruited via Iowa State University (ISU)-wide email. Consented subjects completed an online health screen questionnaire including demographics (age, gender, education and ethnicity), medication use, approximate height and weight, recent blood donations and dietary supplement consumption. Eligible subjects were recruited following an in-person interview for inclusion criteria of a body mass index (BMI) of 18.5-30 kg/m²; no medication use (except non-iron combination oral

contraceptives); no blood donation within 2 months; nonsmoking; nonpregnant or lactating; no history of chronic diseases; no gastrointestinal-associated conditions or dietary intolerances; no intake of vitamin, mineral, or herbal supplements one week prior and during the study period. Subjects were excluded based on following criteria: hemoglobin (Hb) <12 g/dL, serum ferritin (SF) ³40 pg/L, abnormal kidney, liver and basic metabolic panel indicators. Informed written consent was obtained from each participant and the study was approved by the Institutional Review Board at Iowa State University (ISU, Ames, IA).

[075] A total of 91 consented subjects were screened, of which only 17 were eligible to participate based on the set inclusion criteria and were randomized to each treatment (Figure 1 ). One subject dropped out following FeS0₄ and placebo treatments, due to reported side effects of gastrointestinal discomfort. Therefore, 16 subjects were included in the final statistical analyses. A sample size of 15 subjects per group were needed to detect intra-subject differences of 30% in NTBI response with alpha=0.05 and a power of 80 % (b=0.20). Study Design

[076] A 9-week intervention study with the aim to measure influx of iron into serum and production of NTBI over 8 hours after acute iron intake on days 1 and 42; followed by change of iron status, safety, inflammation, oxidative stress and gastrointestinal distress over three-weeks of iron supplementation. Seventeen female subjects were enrolled in a double-blinded cross-over study, whereby they were randomized to receive daily capsules containing 65 mg of iron from either FeS0₄ or Ultimine™ product for 3-week periods, with a 3-week placebo/washout period when they received a placebo pill prior to treatment crossover (Figure 2).

[077] A gastrointestinal side effects questionnaire (GISQ) was randomly distributed electronically, to participants over two weekdays and one weekend day. Subjects acted as their own controls and side effects from iron supplementation were determined based on complete responses. General compliance was recorded by retrieved containers with any remaining capsules.

[078] The following parameters were measured:

Primary endpoint: Acute serum iron and NTBI changes over 8 hours.

Iron status endpoints: Hemoglobin (Hb), serum iron, total iron binding capacity (TIBC), and % TS.

Gastroinstestinal side effects: Nausea, heartburn, abdominal discomfort, headaches, fatigue, diarrhea and constipation; compliance.

Supplemental safety endpoints: Kidney function (blood urea nitrogen [BUN];

glomerular filtration rate [eGFR]; and creatinine ); liver function (alanine

aminotransferase [ALT] and aspartate transaminase [AST]); inflammatory indicators (C-reactive protein [CRP], and hepcidin), and oxidative stress indicators

(thiobarbituric acid reactive substances; TBARS; and protein carbonyls [PCOs]).

Iron supplements and placebo

[079] Each iron supplement contained either 65 mg iron as FeS0₄ (Nature Made^®,

Mission Hills, CA) or 65 mg iron as Ultimine™ commercial product of iron-enriched

Aspergillus oryzae containing 8.7% organic iron). Placebo capsules were made with dextrose monohydrate (sugar). All pills were prepared in opaque colored, bovine pharmaceutical-grade gelatin capsules (Capsuline, Pompano Beach, FL). Pill containers with 21 capsules were given to subjects on day one of each treatment period for 3-week consumption. Subjects were explicitly instructed to only take one capsule daily, even if they forgot to take it on prior days, and asked to bring the remaining of the capsules in the bottles.

Iron status, inflammatory, safety and oxidative stress biochemical indicators

[080] Biochemical indicators were assessed at both baseline (week one) and final (week three) of treatment period one; and baseline (week six) and final (week nine) of treatment period two. Washout period (placebo) was evaluated as week three and week six (Figure 2). Whole blood and serum were collected and sent to a certified diagnostic laboratory (Quest Diagnostics, Lenexa, KS) for Hb, serum iron, TIBC, %TS, CRP, ALT, AST, BUN, eGFR and creatinine analyses. Serum aliquots were collected at all four visits and stored at -80°C until oxidative indicators were measured within three months of collection. Circulating hepcidin, CRP and soluble transferrin receptor (sTFR) concentrations were measured using commercial enzyme linked immunosorbent assays (Hepcidin-25 ELISA, Peninsula

Laboratories International, Inc.; CRP ELISA, American Laboratory Products Company, Salem, NH; sTFR ELISA, Ramco Laboratories, Inc., Stafford, TX). Thiobarbituric acid reactive substances (TBARS) for lipid peroxidation determination was assessed, within 30 days, using a malondialdehyde (MDA) colorimetric assay (gCayman Chemical, Ann Arbor, Ml).

[081] Protein carbonyls (PCOs) were measured in serum based on a modified assay by Colombo et al. 2016.

Acute serum iron, transferrin saturation and NTBI responses in 8-hr period

[082] On the first day of the study (n=17) and after the 3-week washout period (n=16), fasted baseline blood draws were collected before the individuals were randomly assigned to consume either Ultimine™ product or FeS0₄ pills containing 65 mg of iron each with a semi-purified meal (egg albumin, corn syrup solids, corn oil and vanilla extract). Blood samples were drawn at 0 (time of supplementation), 1 , 2, 3, 4, 6 and 8 hours following the consumption of the meal. The following parameters were measured at each time point: serum iron, TIBC, NTBI, % TS. At hours 3 and 6, all subjects consumed unfortified white bread, cheese and butter; and an apple, respectively. The NTBI was determined as previously described (Jakeman et.al. 2001 ).

Side effects questionnaire

[083] Gastrointestinal side effects questionnaire (GISQ) is a modified assessment tool based on a previously reported oral iron supplement questionnaire by Pereira et.al. 2014. The GISQ covers gastrointestinal related side effects commonly reported with oral FeS0₄ supplementation. The following side effects: nausea, heartburn, abdominal discomfort, headaches, fatigue, diarrhea and constipation, were reported by the subjects. The severity of the symptoms was ranked on a 7-point Likert scale (0=absent, 1 =somewhat mild, 2=mild, 3=somewhat moderate, 4=moderate, 5=somewhat severe, 6=severe). Frequency of weekly side effects was collected as the number of side effects reported for two randomly selected days and one weekend day over the 9-week study. From the six side effects reported, the most common ones associated with abdominal discomfort were nausea, diarrhea and constipation (Schmulson and Drossman 2017). The latter were combined to assess overall gastrointestinal effects of the iron supplements.

Statistical Analysis

[084] All analyses were performed using SAS 9.4 (version 9.4, 2018, SAS Institute Inc., Cary, NC). Changes in serum iron, %TS and NTBI from baseline to 8 h after administration of one acute dose of 65 mg iron from either FeS0₄ and Ultimine™ product were analyzed using repeated measures regression models. Changes from baseline for normally and non- normally distributed data were report as (mean + SEM) and geometric means (95% Cls), respectively. Effects of the treatments were compared on the change using repeated measures analyses of variance (ANOVA) with Tukey multiple comparisons to test the difference between least square (LS) means. A total of 16 subjects were included in all biochemical and questionnaire analyses, whereas 15 subjects were included in serum iron, NTBI, and %TS analyses, since one subject had difficulty with multiple blood draws.

[085] Data for the side effects were obtained from the online survey of the GISQ exported from Qualtrics™ into excel. The severity of the side effects was recoded as previously mentioned. To record the frequency of side effects, a dichotomous variable from the 7-point Likert scale as follows: 0=absent and 1 =present (somewhat mild, mild, somewhat moderate, moderate, somewhat severe, and severe) was used. Following the 3-week

supplementation, the frequency of weekly side effects was aggregated to obtain the total reported side effects for each supplemental period. The models included fixed effects for treatment, period and sequence; they also included random effects for subjects nested within sequence. Descriptive statistics were presented as frequencies for the side effects. Differences between treatments in the frequency of reported side effects were specified using SAS PROC GLIMMIX. One subject dropped out following FeS0₄ and placebo treatments, due to reported side effects of gastrointestinal discomfort; therefore, a total of 16 subjects were used for the side effects analysis. For all statistical analysis, P< 0.05 was considered statistically significant. RESULTS AND DISCUSSION

Subject Characteristics

[086] General baseline anthropometric and biochemical characteristics of subjects are presented in Table 1 . Mean age of participants in the study was 21 y. Average BMI was 22.9 kg/m² and was within normal range (19.5-28.9 kg/m²). At screening, all participants had hemoglobin levels within normal levels (>12 g/dL). Although one of the subjects had borderline elevated (at the beginning of the study) serum ferritin (40.4 pg/L), this individual had 37.4. pg/L at the screening test. Another subject did have an elevated baseline CRP concentration (1 1 .4 mg/L), but serum ferritin was 24.4 pg/L. Hepcidin concentrations was high for one subject (12.61 ng/L) but the CRP and ferritin levels were within the acceptable levels of 0.33 mg/L and 26.5 pg/L, respectively.

Table 1 : General baseline characteristics and biochemical indicators of all subjects (n=16)

¹ Mean (±SD) ²Geometric means [95%confidence intervals]

Acute serum iron, %TS and NTBI responses in 8-hr period

[087] Figure 3a shows mean changes in serum iron from the 8-hr acute supplementation test. The changes in serum iron peaked at 4 hours with FeS0₄ (27.8 ± 3.6 mM), but reached its highest concentration around 3 hours; quicker than the Ultimine™ product, which also peaked at 4 hours (8.3 ± 1.6 mM). Serum iron concentrations decreased after 4 hours for both treatments, but neither treatment returned to baseline. The lower percent of serum iron change observed with the Ultimine™ product is in alignment with a previous study (Reddy et al 2018) showing that the iron within the Ultimine™ product is released slowly and is highly absorbed (Figure 3b).

[088] The Ultimine™ product showed serum iron release that are effective in restoring iron deficiency, such as heme iron, iron polymaltose (IP), iron caseinates, and sodium iron ethylenediaminetetraacetic acid (NaFeEDTA), but do not cause a surge in serum iron after oral consumption (Schumann et al 2012). Changes in serum iron throughout 4.5 h post supplementation with oral IP and NaFeEDTA in men and women had slow iron release in spite of the known efficacy of both compounds in reducing iron deficiency. After completing these studies, Schumann et.al. 2013 concluded that IP and NAFeEDTA are bioavailable compounds that are“slow release” into the serum and result in highly significant

suppression of the appearance of NTBI in circulation (Schumann et al 2013).

[089] The Ultimine™ product has similar slow-release properties of the above synthetic compounds (Figure 4a, 4b). Serum iron levels measured throughout 4 hours after supplementation- showed a slow-release pattern for the Ultimine™ product. In this study, the highest absorption of iron from the Ultimine™ product occurred at 4 hours and started to go down around the 8-hour time point (Figure 3a). Furthermore, when the serum iron changes are visually compared between the two studies (Figure 4a, 4b), a lack of dose response for the Ultimine™ product is apparent, further supporting the slow release mechanism of iron-enriched Aspergillus oryzae product.

[090] The percent and change of transferrin saturation (TS) by the Ultimine™ product as compared to FeS0₄ are shown in Figures 5a and 5b. As stated earlier, normal % TS levels for women are <45. After 2 hours, the FeS0₄ reached 54% TS and a maximum of 65 % TS after 3 hours. The values remained above normal levels for the rest of the test. In contrast, the Ultimine™ product %TS levels never exceeded normal levels (Figure 5a). [091] More importantly highly significant correlations were found between both increases in serum iron (Figure 7a) and TS and NTBI (Figure 7b) production, supporting a direct association of dietary iron influx and NTBI production.

[092] Similar increases in serum NTBI was correlated with increase in %TS after oral intake of 60 mg elemental iron from ferrous sulfate with a meal (Brittenham et al 2014). Furthermore, the authors reported that the NTBI formation occurred even in the presence of unsaturated transferrin, suggesting NTBI may be formed when the rate of iron influx exceeds the rate of uptake of iron by transferrin, regardless of the saturation level of transferrin. The NTBI results from this study follow the same pattern; exhibiting a significant production of free iron in the FeS0₄ group compared to none for the Ultimine™ product. From the published literature, it seems plausible that iron surge entering plasma surpasses the rate of iron uptake by plasma transferrin, even at normal TS (Flutchinson et al 2004; Brittenham et al 2014).

[093] Production of NTBI post iron supplementation has become a major concern after studies have shown increased susceptibility to bacteria and parasite infections (Sazawal et al 2006; Prentice et al 2017). Furthermore, chronic high levels of transferrin saturation have been associated with premature aging and mortality (Mainous 2004).

Iron status, inflammatory markers, safety and oxidative stress biochemical indicators

[094] Table 2 shows the changes from baseline after 3 weeks of supplementation for iron status, inflammatory markers, oxidative stress and safety indicators. The Ultimine™ product demonstrated a tendency to increase serum iron and serum ferritin levels, but the results were not statistically significant.

Table 2. Change from baseline to 3 weeks with supplementation of ULT, FeS0₄ and placebo^* (n=16)

¹ Mean ±SEM of change from baseline to final for each treatment period. Different letters indicate statistical significance at alpha =0.05 (P<0.05) using One-way ANOVA and Tukey multiple comparison test.

[095] Having higher serum iron with the Ultimine™ product compared to FeS0₄ after 3 weeks of consumption suggests that the serum iron in the Ultimine™ product group had not returned to baseline, after 10-hours of overnight fast (Table 2), in contrast to placebo and FeS0₄.

[096] Peris Stain and DAB/H202 iron intensification confirmed that more than 90% of the iron is inside the iron-enriched A. oryzae mycelia, which is mainly composed of protein and complex carbohydrates. Thus, it is possible that the iron from this complex matrix is digested over a longer period of time compared to FeS0₄ and the digested iron may be directly taken up into enterocytes by an absorption pathway different from that of nonheme iron. Nearly five decades ago a study showed a delayed peak of circulating iron with hemoglobin iron compared to FeS0₄ because of its slow absorption, and its alternative heme-absorption pathway (Callender et al 1957). New evidence indicate that heme-iron absorption may be saturable because of the lack of dose-response observed after 15 mg Fe doses (Pizarro et al 2003). The observation that iron from the Ultimine™ product did not exhibit a dose response in a previous serum iron study and the slow mechanism of release observed in this trial, may support a heme-like alternative absorption pathway. Interestingly, the saturability of heme-iron is thought to be a protective mechanism from acute iron overdose.

[097] Also interesting is the significant finding that the Ultimine™ product was able to maintain a higher TIBC after 3 weeks of supplementation (Table 2) compared to FeS0₄, reducing the potential for oxidative stress longer. The observation that TIBC was not restored after 10-hours overnight fast with the FeS0₄ treatment raises questions about clearing time of NTBI from the blood. In this study, NTBI was not back to baseline after 8 hours of acute supplementation (Figure 6).

[098] The above finding, the normal %TS status and lack of production of NTBI (below limit of detection) throughout the supplementation studies by the Ultimine™ product has not been reported before for any iron-enriched fungus.

[099] Other iron status indicators were not significantly changed throughout the study probably due to the short supplementation period (Table 2). A minimum of four weeks may be required to get measurable changes in hemoglobin and hematocrit levels in a healthy population.

[0100] There were no significant changes in safety, oxidative and inflammatory biomarkers among all treatments (Table 2), with the exception of ALT and eGFR. Alanine

Aminotransferase increased (P<0.05) in the placebo treatment compared to the Ultimine™ product, suggesting a potential protective effect of the Ultimine™ product on the liver.

Flowever, this effect is not biologically significant.

[0101 ] Glomerular filtration rate (eGFR) was increased with the Ultimine™ product treatment (P<0.05) versus placebo (Table 2). Flowever, there was not significantly different between the Ultimine™ product and FeS0_4.

Gastrointestinal Side Effects

[0102] There was a tendency for a higher compliance (P=0.055) with the Ultimine™ product (97.3%) compared to FeS0₄ (93%), based on returned pills. The frequency of reported side effects by subjects for nausea, abdominal discomfort, diarrhea and constipation were higher with FeS0₄ than the Ultimine™ product, but not significant (Figure 8). The lack of significant differences amongst treatments on side effects may be explained by the qualitative nature of the test requiring higher power in this study. The study was design (n=16) to achieve differences in NTBI (P<0.05).

[0103] When the combined frequency of nausea, diarrhea and constipation data were analyzed across 3-week supplementation period, both the Ultimine™ product and placebo had significantly lower (P=0.004 and P=0.0004, respectively) side effects on week three compared to FeS0₄ (Figure 9). No differences in side effects were measured between Ultimine™ product and Placebo at week three. Furthermore, severe abdominal discomfort was reported with subjects taking FeS0₄.

[0104] The natural encapsulation of the organic iron by Aspergillus oryzae, causing slower digestion and uptake of the iron may potentially reduce the liberation of free reactive iron in the gut, consequently producing less side effects and inflammation. Because this was a cross over design, it is possible that after the FeS0₄ treatment a longer washout/placebo period was needed to reduce inflammation caused by it, explaining the high frequency of side effects reported with placebo and the Ultimine™ product during week one and week two.

CONCLUSIONS

[0105] The results of this randomized, double blinded, cross-over clinical study show for the first time that the Ultimine™ product whole food fermented iron improves iron status of healthy young women without causing acute iron overload and production of NTBI. This study also shows that the stored iron in koji is“slow release” while maintaining normal ferritin, hemoglobin and hematocrit levels.

[0106] Overall, all the safety, oxidative stress and inflammation biomarkers measured support the Ultimine™ product as a safer alternative to FeS0₄ in maintaining normal iron status in humans without the production of harmful reactive free iron and with fewer gastrointestinal side effects.

[0107] The results show that an iron supplement comprising fungal biomass harvested from filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass can be used to treat iron deficiency or iron deficiency anemia in a subject where normal transferrin saturation capacity is maintained in the subject during treatment, and iron levels in the subject are increased without formation of non-transferrin bound iron during treatment. [0108] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

References

Hutchinson C, Al-Ashgar W, Liu DY, Hider RC, Powell JJ, Geissler CA. Oral ferrous sulphate leads to a marked increase in pro-oxidant nontransferrin-bound iron. European journal of clinical investigation 2004;34(1 1 ):782-4.

Mainous AG, 3rd, Gill JM, Carek PJ. Elevated serum transferrin saturation and mortality. Annals of family medicine 2004;2(2):133-8.

Brittenham GM, Andersson M, Egli I, Foman JT, Zeder C, Westerman ME, Hurrell RF. Circulating non-transferrin-bound iron after oral administration of supplemental and fortification doses of iron to healthy women: a randomized study. The American Journal of Clinical Nutrition 2014;100(3):813-20.

Sazawal S, Black RE, Ramsan M, Chwaya HM, Stoltzfus RJ, Dutta A, Dhingra U, Kabole I, Deb S, Othman MK, et al. Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet 2006;367(9505) :133-43.

Reddy MB, Armah SM, Stewart JW, O'Brien KO. Iron absorption from iron-enriched

Aspergillus oryzae is similar to ferrous sulfate in healthy female subjects Current

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ethylenediaminetetraacetic acid (NaFeEDTA), results in a substantial increase of non- transferrin-bound iron in healthy iron-adequate men. Food Nutr Bull 2012;33(2):128-36. Schumann K, Solomons NW, Orozco M, Romero-Abal ME, Weiss G. Differences in circulating non-transferrin-bound iron after oral administration of ferrous sulfate, sodium iron EDTA, or iron polymaltose in women with marginal iron stores. Food Nutr Bull

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Claims

Claims:

1. A method for treating iron deficiency and iron deficiency anemia in a subject, the method comprising providing to the subject an iron supplement or product comprising fungal biomass harvested from cultured filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass, wherein normal transferrin saturation capacity is maintained in the subject during treatment.

2. The method according to claim 1 wherein iron levels in the subject are increased without formation of non-transferrin bound iron during treatment.

3. The method according to claim 1 or 2 wherein the iron supplement or product contains biomass from Aspergillus oryzae.

4. The method according to any one of claims 1 to 3 wherein the subject is provided 1 to 195 mg iron on a daily basis.

5. The method according to claim 4 wherein the subject is provided 65 mg iron on a daily basis.

6. The method according to any one of claims 1 to 5 wherein the treatment is maintained for at least 3 weeks.

7. The method according to claim 6 wherein the treatment is maintained for at least a

month, for at least 3 months, for at least 6 months, or for at least 1 year.

8. The method according to any one of claims 1 to 7 wherein iron supplement or product is in the form of a tablet, capsule, gummy, caplet, powder, granule, ampoule, vial, medicinal food, ready-to-use solution or suspension, drink, medicinal food, a food in the form of a drink, powder, soup, cereal, bread, cookies, meat analogue, ready-to-eat meals or lyophilized material.

9. The method according to claim 8 wherein the iron supplement or product is a capsule formulated for daily consumption.

10. The method according to claim 8 wherein the iron supplement or product is a medicinal food containing fungal biomass with accumulated organic iron.

1 1. The method according to claim 10 wherein the medicinal food in the form of a drink, ready-to-use solution or suspension, powder or meal

12. Use of an iron supplement comprising fungal biomass harvested from filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass to treat iron deficiency or iron deficiency anemia to a subject, wherein normal transferrin saturation capacity is maintained in the subject during treatment, and wherein iron levels in the subject are increased without formation of non-transferrin bound iron during treatment.

13. Use of an iron supplement comprising fungal biomass harvested from filamentous fungi Aspergillus oryzae or Aspergillus niger having organic iron accumulated in the fungal biomass to provide iron supplementation to a subject, wherein normal transferrin saturation capacity is maintained in the subject during supplementation, and wherein iron levels in the subject are increased without formation of non-transferrin bound iron during supplementation.