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RIBONUCLEASE ENRICHED LACTOFERRIN R ELF All Natural Multi Patented Clinically Validated Canine Bone Joint Health Technology OPTIMIZED BONE TURNOVER REGULATED INFLAMMATION ENHANCED FRACTURE HEALING

ABOUT THIS R ELF WHITE PAPER Ribonuclease enriched Lactoferrin R ELF is a class of multi functional all natural milk protein complexes with several beneficial effects on bone physiology The individual roles of lactoferrin LF a metal binding protein and ribonuclease RNase an angiogenic protein have been well documented in medical literature over the past three decades Recent advances in protein engineering and molecular modeling have made it possible to combine RNase and LF into R ELF complexes These bio functional R ELF complexes have demonstrated potent synergistic activity when compared to individual proteins R ELF complexes are known to regulate innate pathways of bone turnover bone remodeling joint inflammation cartilage regulation etc The purpose of this White Paper is to provide clinical data and elucidate the recent advances in R ELF research related to canine bone physiology with emphasis on canine fracture healing after osteotomy procedures Accordingly this R ELF White Paper contains experimental scientific data on i Canine Case Studies Since 2012 N terminus Laboratory has collaborated with veterinary hospitals to measure the post operative effects of R ELF on the recovery and overall fracture healing of canine patients Hundreds of dogs that underwent osteotomy procedures had their recovery rates monitored by veterinarians through radiographic images This document cites THREE typical cases representing i CWTO ii TPLO for large dogs and iii TPLO for small dogs The full clinical data is currently being prepared for publication in a peer reviewed veterinary journal ii Human Clinical Trials In 2008 two IRB approved human clinical trials were conducted in collaboration with two major medical universities in Southern California The purpose was to evaluate the effects of R ELF on bone turnover and inflammatory responses The results of these two studies were published in peerreviewed medical journals Osteoporosis International in 2009 and Inflammation Research in 2010 iii R ELF Technology Grid The Patents issued and Patent Applications pending on R ELF spans three levels of intellectual property IP The foundational tier includes 2 patents that describe methods to isolate ultra pure lactoferrin as the R ELF base material The second tier has 2 method patents on how to produce different types of R ELF Angiogenin or ANGex Complexes The final tier of technology transfer consists of 2 patents that describe the preparation of PORTIN compounds for target delivery of R ELF in vivo iv R ELF Safety Regulations LF was granted GRAS Generally Recognized as Safe status by the USFDA LF is widely approved and used in the US European Union and Asia A safety overview is included in this document AUTHOR Dr A S Narain Naidu PhD Medicine FACN FLS Dr Naidu is a medical microbiologist immunologist and the Director of N terminus Research Laboratory He holds more than 35 years of scientific research and experience from academia government and industry Dr Naidu has served as a professor on the faculties of California State University Pomona USA University of North Carolina Chapel Hill USA Lund University Sweden P cs Medical University Hungary and the Institute of Preventive Medicine India He is an elected Fellow or Associate of 18 professional and scientific societies and consultant to public health agencies and major pharmaceutical enterprises Dr Naidu is also a member of the advisory panels for several multinational corporations and an invited speaker at universities worldwide He is the author editor of 4 reference volumes a prolific writer of 30 book chapters and an investigative author of over 100 peer reviewed research publications Dr Naidu s discoveries are the basis for the establishment of 5 start up biotech companies in the United States He holds multiple core patents in health related applications RELF 2

Introduction Dogs typically break their bones due to trauma such as getting hit by a car falling or repetitive activity that places excessive physical force on the bones Certain canine orthopedic procedures such as Tibial Plateau Leveling Osteotomies TPLO also require surgically cutting and or altering the bone structure In dogs the majority of the traumatic bone fractures involve the hind legs femur fractures can represent up to 45 of all fracture cases The second most common long bone fracture in dogs is the tibia followed by the radius and ulna It is common for dogs to fracture both the radius and ulna in traumatic accidents such as car accidents or falls Humeral fractures account for 10 of all limb fractures Fractures disrupt circulation in the bone and lead to necrosis and hypoxia of adjacent bone tissue Normal bone will respond to a fracture with undergoing an orderly regeneration of its tissue matrix When the anatomical positions are also corrected the bone will be able to regain all of its mechanical properties Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity Skeletal development includes the coordination of multiple events such as migration differentiation and activation of multiple cell types and tissues Reestablishment of circulation is an early event in fracture healing 1 The development of a microvasculature and micro circulation is critical for the homeostasis and regeneration of living bone without which the tissue would simply degenerate and die 2 The strength of bone is defined both by its quality as well as density Vascular tissue plays a major role in the formation of new bone and maintenance of skeletal strength 3 It has also been shown that changes in blood supply to bone tissue lead to deterioration of bone health and can cause skeleto muscular diseases 4 Formation of new capillaries or angiogenesis within the bone tissue facilitates the supply of nutrients and removal of waste products This process also promotes healing of fractures and stimulates the remodeling and regeneration of bones 5 1 Glowacki J 1998 Angiogenesis in fracture repair Clin Orthop and Related Research 355S S82 S89 2 Schmid J et al 1997 The significance of angiogenesis in guided bone regeneration A case report of a rabbit experiment Clin Oral Implants Res 8 244 248 3 Brandi ML Collin Osdoby P 2006 Vascular biology and the skeleton J Bone Miner Res 21 183 192 4 Alagiakrishnan K et al 2003 Role of vascular factors in osteoporosis The J Gerontol Series A Biological Sciences and Medical Sciences 58A M362 M366 5 Carano RAD Filvaroff E 2003 Angiogenesis and bone repair Drug Discov Today 8 980 989 AUTHOR Dr Frank Borostyankoi DVM DACVS Dr Borostyankoi is a veterinarian practicing in Southern California with special interest in small animal orthopedics minimally invasive surgery and post surgical rehabilitation He is a Diplomate of the American College of Veterinary Surgeons With a referral base of close to 3000 veterinarians he performs hundreds of orthopedic surgeries for small animals every year Due to his vast experience Dr Borostyankoi has become a respected consultant in small animal orthopedics for the Southern California veterinary community His research experience with surgical methods has contributed to the development and advancement of better orthopedic methods and technology He has invented and improved several orthopedic implants He greatly contributes to the continuous education of fellow veterinarians and lectures in the US and abroad 3 Nterminus

Lactoferrin LF and Canine Bone Physiology LF Ribbon Structure LF regulates various cell types involved in skeleto muscular regeneration LF stimulates the proliferation of osteoblasts bone forming cells and chondrocytes cartilage cells LF inhibits osteoclastogenesis reducing the number of cells that can actively resorb bone thus producing a greater overall increase in bone volume Lactoferrin LF is an 80 kDa metal binding glycoprotein found in exocrine secretions The ability of LF to bind to large quantities of iron provides protection against pathogens and their metabolites by enhancing phagocytosis cell adherence and controlling the release of pro inflammatory cytokines LF is a primary constituent of immune homeostasis functioning to reduce oxidative stress at the molecular level and thus controlling excess inflammatory response 6 LF levels in the blood are normally low 0 2 0 6 g mL but at sites of inflammation are significantly elevated 200 g mL due to its release from neutrophils Elevated LF in the synovium is primarily due to anti inflammatory response LF regulates synovial iron load and has been shown to reduce inflammation in bone joints Iron can suppress bone remodeling by reducing osteoblast formation and new bone synthesis 7 LF has been shown to regulate various cell types involved in skeleto muscular regeneration It stimulates the proliferation and differentiation of osteoblasts the bone forming cells 8 LF also inhibits osteoclastogenesis and reduces the number of cells that actively resorb bone thus producing an overall increase in bone volume 9 LF is a positive regulator of bone with an important role in growth and healing There is a growing interest in the potential use of LF for the improvement of bone health In recent studies dietary supplementation with LF demonstrated improved bone mineral density and bone strength LF appears to be a promising candidate for the development of anabolic therapeutic agents for canine bone health 10 LF Documented Roles in Bone Joint Physiology Regulates synovial Fe II loading to reduce inflammation in bone joints Binds to zinc and regulates MMPs from degrading bone cartilage Modulates immune responses and prevents development of arthritis Stimulates osteoblasts and regulates normal bone growth formation Inhibits osteoclasts and regulates excessive bone resorption Promotes strontium deposition in bone and as mineral transport protein Evade infections at fracture site as a broad spectrum antimicrobial agent 6 Naidu AS 2000 Lactoferrin In Natural Food Antimicrobial Systems ed AS Naidu pp 17 102 Boca Raton CRC Press ISBN 0 8493 2047 X 7 Weinberg ED 2006 Iron loading a risk factor for osteoporosis Biometals 19 6 633 635 8 Naot D et al 2005 Lactoferrin a novel bone growth factor Clin Med Res 3 93 101 9 Lorget F et al 2002 Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity Biochem Biophys Res Commun 296 2 261 266 10 Naidu AS 2009 Bioreplenishment for Bone Health California Bio Rep Media ISBN 978 0982445105 RELF 4

Ribonuclease RNase Angiogenesis and Fracture Healing Blood vessels play a pivotal role in skeletal homeostasis and bone repair therefore angiogenesis is important for successful bone remodeling 11 12 Vascularization of the growth plate region is critical for two fundamental processes that determine the rate of bone growth i Chondrogenesis cartilage production and ii Osteogenesis bone formation Precise coupling of these two processes is crucial during periods of rapid bone growth or fracture repair RNase or angiogenin is a 14 kDa basic heparin binding protein which is a member of the pancreatic RNase superfamily It is a key mediator in nearly all phases of angiogenesis which makes it an important precursor in skeleto muscular development 13 Angiogenesis is a strictly regulated multi step process that occurs during normal bone remodeling and regeneration pregnancy and tissue repair conditions such as fractures and wound healing 14 15 Bone tissue engineering in particular has benefited from the application of pro angiogenic strategies The need for an adequate vascular supply increases during healing along with the challenges associated with vascularization of scaffolds implanted in vivo The lack of a functional vascular supply can hamper the whole range of clinical applications of successful bone tissue engineering strategies especially in canine bone grafts These grafts depend on post implant vascularization accordingly RNase could be promising in the establishment of a microcirculation in the engineered constructs RNase Multi functional Role in Bone Joint Physiology RNase Ribbon Structure Angiogenesis is a strictly regulated multi step process that occurs during normal bone remodeling and regeneration pregnancy and tissue repair conditions such as fractures and wound healing Accelerates bone mineralization bone regenerationand remodeling Inhibits osteoclastic bone resorption Scavenges free radicals as an antioxidant and protects bone integrity Binds to endothelial cells and stimulates cell migration and invasion Promotes cell proliferation differentiation and mediates cell adhesion Activates cell associated proteases and induces plasminogen activator Prevents chrondrocyte apoptosis and preserves joint cartilage 11 Ballara SC et al 1999 New vessels new approaches angiogenesis as a therapeutic target in musculoskeletal disorders Int J Exp Pathol 80 235 250 12 Kanczler JM Oreffo RO 2008 Osteogenesis and angiogenesis the potential for engineering bone Eur Cell Mater 15 100 114 13 Strydom DJ 1998 The angiogenins Cell Mol Life Sci 54 811 824 14 Glowacki J 1998 Angiogenesis in fracture repair Clin Orthop and Related Research 355S S82 S89 15 Carano RAD Filvaroff E 2003 Angiogenesis and bone repair Drug Discov Today 8 980 989 5 Nterminus

R ELF AND CANINE FRACTURE HEALING INFLAMMATION I Hematoma Angiogenesis INFLAMMATION II Immuno modulation Granular tissue Ribonuclease enriched Lactoferrin R ELF is an all natural clinically validated and multi patented technology developed to improve canine bone and joint health R ELF is a precisely calibrated mixture of two bio functional proteins that help regulate all phases of fracture healing The ability of the LF protein in R ELF complex to bind transport metal co factors such as iron and zinc could facilitate the elimination of harmful free radicals and regulate MMPs that hydrolyze and remove cellular debris from the fracture site LF is a potent modulator of bone turnover that has been demonstrated to increase bone growth by 200 and down regulate bone resorption by 50 RNase the immobilized enzyme in R ELF complex is critical for revascularization of regenerated tissue and in amplifying inflammatory cell mediators The combination of LF and RNase in R ELF complex elicits greater synergistic activity compared to the individual proteins The following sections in this white paper i elucidate the effects of R ELF on phases of fracture healing ii review radiographic evaluations of canine osteotomy cases and iii cover bone turnover and inflammation profiles in human clinical trials Fracture healing involves a complex series of coordinated cellular and molecular processes leading to removal of contaminating material angiogenesis validating reinstatement of disrupted microcirculation and restoration of bone continuity by activation proliferation and chemotaxis of bone progenitors from surrounding periosteum and endosteum Regulation of the various cells that orchestrate the fracture healing process depends on the biological effect evoked by angiogenin RNase and other growth factors cytokines R ELF seems to be a potent regulator of canine bone turnover and fracture healing There are 3 distinct phases of fracture healing i Inflammation ii Repair and iii Remodeling i Inflammatory Phase The Inflammatory Phase begins immediately after the initial disruption of bone and surrounding soft tissues and persists until the formation of cartilage or bone This phase lasts for 3 4 days and potentially longer depending on the degree of fracture Hematoma sets the stage for the repair phase by releasing growth factors which stimulate angiogenesis and bone formation As an inflammatory mediator R ELF could stimulate angiogenesis and also signal early bone resorption by osteoclasts and proliferation of osteoprogenitor cells Mast cells containing vaso active substances such as R ELF help in the formation of new vessels Within hours a transient extra osseous blood supply emerges from surrounding soft tissues revascularizing the hypoxic fracture site Mononuclear phagocytes delivered by these new vessels assist in the removal of necrotic bone and aid in callus formation RELF 6

ii Repair Phase During the Repair Phase osteogenesis continues and a callus is formed to bridge the fracture site At the end of this phase bone union is achieved but the structure of the fracture site differs from that of the original bone The time required to achieve union varies based on fracture configuration and location status of the adjacent soft tissues as well as animal characteristics species age health status concurrent injuries diseases Eventually the injured bone regains enough strength and rigidity to allow low impact exercise REPAIR Soft Callus Hard Callus iii Remodeling Phase During the final Remodeling Phase the large callus is reduced to the size of actual bone at the fracture site The woven primary bone is replaced with secondary lamellar bone This process may take months or even up to a year or more The balanced action of osteoclastic resorption and osteoblastic deposition can be regulated by R ELF In spontaneous healing of a fracture progression from soft to hard callus depends upon an adequate blood supply and a gradual increase in stability at the fracture site Most fractures if left completely alone would probably heal however due to any delayed union or malunion the bone might lose its function Successful healing of a fracture is not only determined by complete bony union on X rays but also by the functional use of the limb thereafter R ELF has been shown to promote functional fracture healing while concurrently negating some of the problems of prolonged treatment such as decreased vascularization and poor bone turnover Bone grafts benefit from pro angiogenic strategies especially with adequate vascular supply from RNase during implantation and healing R ELF has been demonstrated to increase new bone formation and optimize bone resorption Also R ELF is shown to improve anti inflammatory response that could help fracture healing process REMODELING Ossification Lamellar Bone In summary R ELF is a multi functional all natural protein complex that promotes optimum bone turnover modulates the inflammatory response and regulates angiogenesis in canine physiology These bio activities are critical for canine bone tissue engineering R ELF could play a pivotal role in bringing out successful outcomes with various bone corrective procedures such as orthopedic surgeries bone tissue grafts and implants Furthermore R ELF could serve as a potent nutraceutical agent for a healing and or aging canines that need bone and joint healthcare 7 Nterminus

CANINE OSTEOTOMY CASE STUDY I R ELF and Closing Wedge Tibial Osteotomy CWTO INE AN OSTEOT O M Y C Rupture of the cranial cruciate ligament can be partial or complete and the cartilages in the knee can be damaged because of the instability that results Commonly used tibial bone cutting techniques include Closing Wedge Tibial Osteotomy CWTO and Tibial Plateau Leveling Osteotomy TPLO among other procedures CWTO is often used in small dogs and in dogs with steep tibial plateaus While TPLO is used in larger breed dogs if they are notably bouncy or if they have small tibial crests an anatomical feature of the tibia The tibial plateau is the top of the tibia and forms the floor of the knee joint This usually has a down slope of about 22 degrees In some breeds like West Highland White Terriers the slope can be 40 degrees or more CWTO procedure reduces this down slope to something in the region of 5 degrees This allows dogs to use the knee much more comfortably despite the ruptured cruciate ligament In the CWTO procedure two cuts are made in the tibia so that a wedge of bone can be removed A wire is placed at the front of the bone between two bone tunnels and by tightening it the wedge shaped gap in the bone can be compressed This reduces the slope of the tibial plateau The bone is then stabilized with one or more bone plates and screws Post operative X rays are taken to confirm satisfactory healing of the bone CA SE STUDY History Diagnosis The patient was evaluated for right hind leg lameness persisting for 4 weeks The patient reportedly jumped out of owner s car and had been limping ever since A family veterinary exam localized the lameness to a loose right stifle joint and sent the patient for orthopedic evaluation and surgery to a specialty clinic The patient was ambulatory on the physical exam with a moderate lameness on the right rear leg Palpation of the stifle joint revealed a mild joint effusion with a severe drawer motion in the stifle consistent with a complete rupture of the cranial cruciate ligament The referring veterinarian s radiographs revealed a non displaced proximal fibula fracture Patient also had a likely pre existent Medial Patella Luxation MPL a common developmental disorder in small dogs Surgical Procedure A Closing Wedge Tibial Osteotomy CWTO was performed on the right rear leg to correct the cranial cruciate ligament deficient stifle The procedure also included a MPL repair with a femoral trochlear resection and tibial crest transposition The tibial crest was affixed in its new position with two K wires The routine surgical procedure is designed to reduce the tibial plateau slope by removing a wedge shaped piece of bone from the proximal tibia and to fix the MPL condition at the same time The created wedge shaped bone defect then is collapsed and held together with an orthopedic wire and bone plate The size of the wedge is determined by the Tibial Plateau Angle TPA The surgery resulted in a significant cortical alignment deficiency with aligning the cranial cortex Post Operative Care Effects of R ELF Post surgical recommendations included antibiotics NSAIDs and activity restriction Additionally R ELF 160 mg day was orally administered as bone and joint supplement The veterinary reported that expected bone healing time for a clinically normal dog such as this patient is normally 8 weeks with continued activity restriction However an expedited 7 week bone healing was observed from radiographic evidence with the R ELF intake accordingly the patient was released with no restrictions at the 7 week check up The radiographs also showed complete bone remodeling of the previously created caudal cortical misalignment this improvement is usually not observed until 12 weeks R ELF demonstrated canine fracture healing bone reunion 30 faster R ELF shortened post osteotomy recovery time in dogs by 3 4 weeks RELF 8

Patient Cairn Terrier Spayed Female Age Body Weight 2 year 21 lb Clinical Condition Rupture of Cranial Cruciate Ligament Medial Patella Luxation Surgical Procedure Closing Wedge Tibial Osteotomy CWTO PRE PROCEDURE DAY 0 WEEK 7 9 Nterminus

CANINE OSTEOTOMY CASE STUDY II R ELF and Tibial Plateau Leveling Osteotomy TPLO Large Dogs A ruptured cranial cruciate ligament is one of the most common orthopedic injuries in dogs It is estimated that 1 million dogs suffer from this injury every year in the USA alone TPLO has become the standard procedure to fix such injuries due to its estimated 90 successful outcome The procedure entails cutting the tibia bone under the knee joint and changing the position of the top segment to other ligaments to take over for the ruptured ligament The ligament is stabilized with a special bone plate and screws Part of the success of this procedure depends on a proper bone healing If the bone is not healed there is a risk that the surgically created position may fall out of alignment therefore dogs need to be limited in their activity during the healing period This is achieved initially with cage confinement and later with indoor confinement and controlled leash walks History Diagnosis The patient had a history of limping on right rear leg for 4 weeks During the physical examination the patient walked with a narrow gait on the rear legs with moderate right rear leg lameness A palpable joint effusion was observed in the right stifle joint and flexing of the knee seemed painful The stifle was slightly unstable on palpation with a small degree of drawer motion in the joint Both hips had decreased range of motion with minimal discomfort on extension Based on the radiographs bilateral hip dysplasia and right anterior cruciate ligament trauma partial tear was diagnosed Surgical Procedure A Tibial Plateau Leveling Osteotomy TPLO was performed on the patient The stifle was opened up with a mini arthrotomy to remove the damaged portion of the ligament and to release the medial meniscus The proximal tibia was cut with a 21 mm TPLO blade to mobilize the proximal tibia The tibial plateau was rotated 7 mm to eliminate the tibial thrust The segment was stabilized with a 3 5 mm Universal TPLO plate 6 x 3 5 mm cortical screws and a 0 062 inch Kirschner wire INE N A OSTEOT O Y CA SE STUDY RELF 10 M C Post Operative Care Effects of R ELF Post surgical care included the usual 4 weeks of cage confinement and an additional 4 weeks of indoor confinement with controlled leash walks In addition the patient was orally administered a daily dose 240 mg of R ELF Follow up radiography Day 0 Week 4 and Week 9 showed rapid bone healing The patient was released from the confinement and restricted activity to full activity at the 9 week check up At the 9 week evaluation the patient was fully active on her operated leg with no sign of lameness The veterinary surgeon reported that the operated leg could be not differentiated from the intact leg just by observation without checking the chart or touching the legs The radiographic images showed complete bone healing and remodeling In over 100 vet supervised clinical cases post surgical R ELF supplementation seemed to help canines achieve satisfactory bone healing turnover within 7 8 weeks instead of the previously recorded 12 week recovery In several veterinary clinics dogs were released out of confinement taken off exercise restrictions and returned to full function within 8 weeks This significantly decreased the post operative recovery time of the patient Note In this case study the client has missed the 8 week recheck appointment and the evaluation was postponed to 9th week

Patient German Shepherd Spayed Female Age Body Weight 4 year 59 lb Clinical Condition Bilateral Hip Dysplasia Right Anterior Cruciate Ligament Trauma Surgical Procedure Tibial Plateau Leveling Osteotomy TPLO PRE PROCEDURE DAY 0 WEEK 4 WEEK 9 11 Nterminus

CANINE OSTEOTOMY CASE STUDY III R ELF and Tibial Plateau Leveling Osteotomy TPLO Small Dogs The TPLO procedure was initially developed for knee repair of larger dogs to stabilize the stifle joint during weight bearing and compensate for the loss of the cranial cruciate ligament The outcomes of TPLO were so successful on large dogs that this procedure eventually extended to most cruciate ligament repair on all size and breeds dogs such as this Case Study 3 patient a small sized dog with 9 5 kg bodyweight History Diagnosis Patient had a history of non weight bearing lameness of the right rear leg During the physical exam the dog was walking with moderate right rear leg lameness There was a palpable joint effusion present in the right stifle joint and flexing the knee was painful The stifle was partly unstable on palpation with a drawer motion in the joint Veterinary diagnosis indicated a rupture in the right anterior cruciate ligament and an unstable knee joint Surgical Procedure A right Tibial Plateau Leveling Osteotomy TPLO was performed by opening of the stifle with a mini arthrotomy to remove the remnants of the damaged ligament and to release the medial meniscus The proximal tibia was cut with a 12 mm TPLO blade to mobilize the joint surface the tibial plateau The proximal segment was rotated 4 5 mm to eliminate the tibial thrust The segment was stabilized with a 2 0 mm Cadmus stile TPLO plate 6 x 2 0 mm cortical screws and a 0 045 inch Kirschner wire INE AN OSTEOT O Y CA SE STUDY RELF 12 M C Post Operative Care Effects of R ELF During post surgical care on top of the usual 4 weeks of cage confinement and an additional 8 weeks of indoor confinement with leash walks the patient was orally administered R ELF 160 mg on a daily basis The routine X ray checkups Day 0 Week 4 and Week8 indicated expedited bone healing The patient was released from confinement and restricted activity to full activity 4 weeks sooner than expected in clinically similar cases where dogs wer By the 8 week recheck the bone was completely healed and the dog regained full activity

Patient Bichon Frise Neutered Male Age Body Weight 7 year 21 lb Clinical Condition Rupture of the Right Anterior Cruciate Ligament Unstable Knee Joint Surgical Procedure Tibial Plateau Leveling Osteotomy TPLO DAY 0 WEEK 4 WEEK 8 13 Nterminus

HUMAN CLINICAL TRIAL I R ELF and Bone Turnover in Post Menopausal Women SUMMARY Osteoporosis a major health issue among post menopausal women causes increased bone resorption and reduced bone formation A reduction in angiogenesis could also contribute to this imbalance Current treatments such as hormone replacement therapy and bisphosphonates have drawbacks of severe side effects Milk ribonuclease RNase is known to promote angiogenesis and lactoferrin LF to stimulate bone formation by osteoblasts The effect of RNase enriched lactoferrin R ELF supplement on the bone health of postmenopausal women was examined Methods A total of 35 healthy post menopausal women aged 45 to 60 years were randomized into placebo or R ELF supplement groups The bone health status was monitored by assessing bone resorption markers serum N telopeptides sNTx and urine deoxypyridinoline uDpd cross links and serum bone formation markers bone specific alkaline phosphatase sBAP and osteocalcin sOC AUTHORS Bharadwaj S Naidu AGT Betagiri G Prasadarao NV Naidu AS ARTICLE Milk ribonuclease enriched lactoferrin induces positive effects on bone turnover markers in postmenopausal women JOURNAL Osteroporos Int 20 1603 1611 2009 Results R ELF supplementation demonstrated a decrease in urine Dpd levels by 14 19 increase for placebo and serum NTx maintained at 24 of the baseline 41 for placebo while serum BAP and OC levels showed a 45 and 16 elevation 25 and 5 for placebo Conclusions R ELF supplementation showed a statistically significant reduction in bone resorption and increase in osteoblastic bone formation to restore the balance of bone turnover within a short period Background Bones undergo a continuous remodeling process through repeated cycles of destruction and rebuilding In healthy young adults the amount of new bone formation approximately balances the amount of bone resorption As the age increases however the balance shifts to favor bone resorption Current efforts to treat bone diseases have primarily concentrated on the development of drugs to block bone resorption which decrease the formation or activity of osteoclasts Present treatment options for postmenopausal osteoporosis include hormone replacement therapy HRT and bisphosphonates HRT is known to reduce osteoclast activity but is prone to adverse effects such as increased risk of breast cancer 16 17 Bisphosphonates have a risk of development of esophageal ulcers 18 Therefore it is imperative to explore and develop strategies for enhancing the bone formation and simultaneously prevent bone loss without side effects Angiogenesis or formation of new capillaries within bone tissue could facilitate supply of nutrients and removal of waste metabolites and facilitates healing of fractures remodeling and regeneration 19 20 16 Herrington DM Howard TD 2003 HRT and heart disease from presumed benefit to potential harm N Engl J Med 349 5519 17 Collins JA et al 2005 Breast cancer risk with postmenopausal hormonal treatment Hum Reprod Update 11 545 560 18 Makins R Ballinger A 2003 Gastrointestinal side effects of drugs Expert Opin Drug Safety 2 421 429 19 Glowacki J 1998 Angiogenesis in fracture repair Clin Orthop 355S S82 S89 20 Carano RAD Filvaroff E 2003 Angiogenesis and bone repair Drug Discov Today 8 980 989 RELF 14

Study Design FIGURE 1 Women n 35 included into the study were assigned to placebo group or R ELF group randomly Subjects n 15 in the placebo group and were supplemented with 100 RDA Recommended Daily Allowance of calcium in tablet form Subjects of the R ELF group n 20 were administered with two R ELF capsules of 125 mg each along with 100 RDA of calcium administered orally from Day 1 to Day 180 Venous Blood by standard venipuncture and urine samples were collected from each subject on Day 0 baseline before supplementation Day 15 Day 30 Day 60 Day 90 and Day180 of the study Standard systolic diastolic blood pressures as well as the body weight were also monitored at baseline and the aforementioned days R ELF and Bone Resorption Markers The median sNTx and uDpd changed gradually to a peak level for both placebo as well as R ELF groups as shown in Figure 1 AB The resorption markers for each observational day were also normally distributed P 0 34 0 99 sNTx for the placebo group increased from 15 8 2 6 nM BCE Bone Collagen Equivalents on Day 0 to 22 1 1 7 nM BCE on Day180 while R ELF group displayed a relatively smaller rise 12 9 3 3 to 16 1 2 0 nM BCE The change in bone turnover markers calculated as a percent of the corresponding baseline levels are represented in Figure 2 AB Median sNTx for the placebo group showed an increase of 40 1 in 180 days reflecting significant bone resorption while the R ELF group showed a relatively smaller rise of 24 5 during the same period P

FIGURE 3 An interesting reversal of trend was observed with median uDpd levels although the placebo group showed an increase from 6 6 0 8 to 7 7 0 7 by Day 180 the R ELF group decreased from 8 2 0 6 to 6 9 0 6 nM Dpd mM Creatinine The overall rise of 17 8 indicated a significant level of bone resorption in the placebo group In contrast Dpd levels of the R ELF group showed a reduction in resorption by 10 within 30 days and continued to fall to 14 6 by the end of the study P 0 0021 95 CI The R ELF group achieved 80 of peak change in Dpd levels in about 30 days compared to 95 days taken by the placebo group A R ELF and Bone Formation Markers The two markers of bone formation sBAP and sOC were determined at the same pre defined intervals during the study Figure 3 AB shows the measured variation of median sBAP and sOC levels for both groups As with the bone resorption markers the sBAP and sOC data sets for each observational day were normally distributed P 0 39 0 98 The variation of sBAP and sOC from their respective baseline levels is depicted in Figure 4 AB Median sBAP gradually increased to a peak level for both the groups 25 0 1 3 to 31 4 3 0 U L for placebo and 19 7 2 4 to 28 1 3 1 U L for the R ELF group Although median levels for R ELF group are lower than those for the placebo group the change from baseline was better for the R ELF group The increase in 42 7 for R ELF group compared to 25 7 for the placebo was also statistically significant P

Bone formation markers were observed to be highly correlated with bone resorption markers for both groups with Pearson correlation coefficient r values in the range 0 58 0 93 for the placebo and 0 66 0 90 for the R ELF group respectively The correlation plots along with the statistical parameters are shown in Figure 5 The positive correlation observed for placebo group is generally improved with R ELF supplementation as seen from the higher r and smaller P values for sNTx with sBAP as well as sOC In case of uDpd the positive correlation seen with the placebo group is changed to negative for the R ELF group r changing from 0 93 to 0 87 for sBAP and from 0 83 to 0 66 for sOC respectively This change reflects the effect of R ELF supplementation to reduce resorption markers while increasing the formation markers to restore balance of bone turnover FIGURE 5 Correlation plots of bone formation markers vs bone resorption markers Top row corresponds to the placebo group and the bottom row to the R ELF group The Pearson correlation coefficient r and significance P are shown for each plot Bone formation osteoblastic activity increased by 200 almost doubled with R ELF intake compared to the placebo A significant reduction 50 in bone resorption osteoclastic activity was measured in the R ELF group R ELF restored the innate balance of bone turnover within 2 to 4 weeks of supplementation 17 Nterminus

HUMAN CLINICAL TRIAL II R ELF and Improvement of Inflammatory Responses OBJECTIVE AND DESIGN A six month randomized clinical study was conducted to evaluate the effect of a ribonuclease enriched lactoferrin R ELF supplement on the circulating cytokine levels and bone health of post menopausal women Subjects Healthy postmenopausal women n 35 aged 4560 years were randomized into placebo and R ELF groups Treatment R ELF group was supplemented with R ELF 2 x 125 mg day and calcium 100 RDA while the placebo group received only the calcium supplement Serum levels of receptor activator for NF B ligand RANKL C reactive protein CRP and various pro and anti inflammatory cytokines were determined by ELISA Results Pro inflammatory cytokines IL 6 and TNF decreased significantly 44 and 10 respectively while anti inflammatory IL 10 increased 140 with R ELF supplementation RANKL and CRP were modestly reduced 50 relative to the placebo while RANKL elevated initially AUTHORS Bharadwaj S Naidu AGT Betagiri G Prasadarao NV Naidu AS ARTICLE Inflammatory responses improve with milk ribonuclease enriched lactoferrin supplementation in postmenopausal women JOURNAL J Inflamm Res 59 11 971 978 2010 Conclusions R ELF supplementation showed beneficial effects towards improvement of inflammatory status Background The immune and the skeletal systems share several regulatory factors such as cytokines transcription factors and receptors The immune cells and osteoclasts are derived from the same hematopoietic precursor cells which originate in bone marrow and interact with bone cells 21 22 Inflammation is the primary defense mechanism of the body characterized by a high alert state of the immune system triggered by the release of several proinflammatory cytokines It also affects the processes of bone resorption and formation due to the presence of pathways that are common to both bone cell maturation and inflammation Pro inflammatory cytokine levels are known to rise during aging and stress 23 Elevated levels of tumor necrosis factor TNF interleukin IL 6 andIL1 receptors suggest subtle changes in the immune system 24 Thus the body is in a state of mild continuous inflammation characterized by a rise in the levels of acute phase proteins such as C Reactive Protein CRP a wellknown bio marker for inflammation In healthy elderly individuals higher serum CRP levels were associated with high bone turnover rate resulting in low bone mineral density 25 21 Arron JR Choi Y 2000 Bone versus immune system Nature 408 535 536 22 Goldring SR 2003 Inflammatory mediators as essential elements in bone remodeling Calcif Tissue Int 73 97 100 23 De Martinis M et al 2005 Inflammation ageing and lifelong antigenic load as major determinants of ageing rate and longevity FEBS Lett 579 2035 9 24 Bruunsgaard H 2006 The clinical impact of systemic low level inflammation in elderly populations With special reference to cardiovascular disease dementia and mortality Dan Med Bull 53 285 309 25 Koh JM et al 2005 Higher circulating hsCRP levels are associated with lower bone mineral density in healthy pre and postmenopausal women evidence for a link between systemic inflammation and osteoporosis Osteoporos Int 89 735 42 RELF 18

Study Design R ELF is a ribonuclease angiogenin enriched LF either co isolated from bovine milk 50 50 ratio wt wt or both proteins admixed to obtain required ratios US Patents 7601689 8003603 Thirty five women included in the study were randomly assigned to one of the two groups placebo group or R ELF group Fifteen subjects assigned to the placebo group were supplemented with 100 RDA Recommended Daily Allowance of calcium in a tablet form Whereas 20 subjects of the R ELF group were given with two R ELF capsules of 125 mg each along with 100 RDA of calcium administered orally from Day 1 to Day 180 Venous blood samples were collected by standard venipuncture technique from each subject on Day 0 baseline before starting the supplement Day 30 Day 90 and Day 180 of the study Inflammatory cytokines and markers The levels of inflammatory cytokines IFN IL 6 IL 12 p40 TNF IL 1 IL 10 and TGF were determined by enzyme linked immunosorbent assay ELISA using respective monoclonal antibodies High purity C Reactive Protein CRP and anti CRP rabbit polyclonal antibody were obtained from Calbiochem EMD San Diego CA and anti RANKL rabbit polyclonal antibody from Abcam Inc Cambridge MA CRP assay had a sensitivity of 8 pg mL and detection limit of 1 ng mL Statistical analysis Cytokine data from each day was analyzed for measures of central tendency deviation and distribution of data Data were considered outliers if they were 1 5 times the inter quartile range IQR above the third quartile or below the first quartile Outliers were discarded from datasets for statistical tests of significance In view of the small size of placebo and R ELF groups median and standard error of the mean were used as the preferred measures of central tendency for this modest set of data The KolmogorovSmirnov test KS test was used as a test for normal distribution of data within each set Student s unpaired two sample t test was used for comparison of the mean observed change in markers for placebo and R ELF data sets to establish the effect of R ELF supplementation OriginPro Ver 8 OriginLab MA USA software was used for data analysis 19 Nterminus

Pro Inflammatory Cytokine Levels Improved with R ELF Intake The median IFN ranged from 14 4 1 7 to 18 3 2 9 pg mL for the placebo group while the values increased from 28 6 13 7 to 32 4 12 8 pg mL for the R ELF group and the difference between them was statistically significant P 0 0007 for median IFN placebo vs R ELF IFN levels for the R ELF group decreased 6 6 within thirty days but increased considerably 42 for the placebo By the end of study although IFN levels for the R ELF group increased 13 5 the increment was smaller compared to the placebo 27 Figure 6A Median IL 1 levels ranged from 10 2 3 8 to 15 0 10 2 pg mL for the placebo and from 22 6 10 9 pg mL to 23 3 19 9 pg mL for the R ELF group P 0 0131 for median IL1 placebo vs R ELF A large decrease in IL 1 levels was observed initially but the trend is reversed by Day 90 and turned out significantly positive 46 5 for the placebo while it remained close to the baseline 2 8 for the R ELF group Figure 6B TNF levels ranged from 239 3 15 8 on Day 0 to 238 3 69 5 pg mL for placebo while a decrease from 279 7 31 0 to 251 4 43 2 pg mL P 0 0341 for median TNF placebo vs R ELF was observed with R ELF supplementation A large initial decline in TNF levels 27 4 for R ELF vs 15 5 for placebo was sustained until Day 180 for the R ELF group 10 1 but not with the placebo 0 4 Figure 6C Furthermore median IL 6 levels slightly decreased for the placebo from 2 1 1 5 on Day 0 to 2 0 2 1 pg mL by the end of study while IL 6 decreased from 5 5 3 9 to 3 0 4 1 pg mL for the R ELF group As shown in Figure 6D by the end of study the decrease in IL 6 was significant P 0 0338 for median IL 6 placebo vs R ELF for the R ELF group 44 1 compared to the placebo 4 3 Levels of IL 12 p40 also referred to as IL 12 were similar for both groups placebo ranging from 68 5 1 6 to 90 2 1 6 pg mL and R ELF group from 73 1 4 0 to a slightly higher 111 1 5 6 pg mL R ELF supplementation significantly raised IL 12 levels by 51 9 by Day 180 compared to placebo with an increase of 31 8 from their respective baseline levels P 0 0005 for mean IL 12 placebo vs R ELF Figure 6E These results demonstrate that supplementation of R ELF induced down regulation of pro inflammatory TNF and IL 6 and a moderate rise in IL 1 and IFN levels within 6 months FIGURE 6 Effect of R ELF on ProInflammatory Cytokines Median change in the serum levels of pro inflammatory cytokines IFN panel A IL 1 panel B TNF panel C IL 6 panel D and IL 12 panel E in subjects from placebo open bars and R ELF filled bars supplemented groups RELF 20

R ELF Effects on Anti Inflammatory Cytokines Levels of IL 10 for the placebo were close to the baseline median value of 17 4 2 7 until Day 180 16 3 3 6 pg mL while the levels increased from 7 7 9 2 to 18 8 4 2 pg mL for the R ELF group A major positive change was observed in IL 10 with R ELF supplementation 150 compared to placebo 6 2 P 0 0235 for change in median IL 10 placebo vs R ELF by the end of study Figure 7A Median TGF levels slightly decreased for both the groups 786 3 73 1 to 761 5 85 1 pg mL for the placebo compared to 739 4 58 9 to 727 6 51 9 pg mL for the R ELF P 0 0072 for median TGF placebo vs R ELF This decline was smaller for the R ELF group 2 than the placebo 3 Figure 7B FIGURE 7 Variations in Serum Levels of Anti Inflammatory Cytokines with R ELF supplementation Median change in the serum levels of anti inflammatory cytokines IL 10 panel A and TGF 1 panel B in subjects from placebo open bars and R ELF filled bars supplemented groups These results indicate a relatively positive change in anti inflammatory cytokines with R ELF supplementation compared to calcium supplementation alone The correlation between pro and anti inflammatory cytokines was analyzed and the results are presented in Table 3 TABLE 3 Correlation between Pro inflammatory and Anti inflammatory Cytokines Cytokine Day 0 IL 10 Day 180 TGF IL 10 TGF nc 0 97

The correlation matrix for placebo and R ELF groups are generally maintained from Day 0 to Day 180 and improved with R ELF supplementation Most cytokine correlations also remained statistically significant at 95 confidence interval The correlation of IL 6 versus IL 10 improved with R ELF supplementation In general TNF IL 12 p40 and TGF did not show any correlation neither with the progress of study nor with supplementation IL 10 was highly correlated with IFN and IL 6 at the beginning of the study for both groups which improved by the end of the study Interestingly IFN was not correlated with IL 10 at baseline but demonstrated a high degree of correlation after R ELF supplementation These positive correlations suggested a moderate elevation of anti inflammatory cytokines that could reduce pro inflammatory response Inverse correlation is a favorable outcome of reduction in pro inflammatory and elevation of anti inflammatory cytokines in which could diminish inflammation An improved negative correlation was observed between IL 6 and TGF with R ELF supplementation although not statistically significant R ELF Reduced the Levels of CRP and RANKL RANKL is a well established marker for the onset of osteoporosis in postmenopausal women RANKL a mediator of osteoclast formation indicated a sharp rise by Day 30 followed by a steady decline for both placebo and R ELF groups Figure 8A However in response to R ELF supplementation there was a larger and rapid decline by 34 8 60 6 on Day 30 to 25 8 on Day 90 compared to 11 6 increase 35 0 on Day 30 to 46 6 on Day 90 with calcium supplementation alone By the end of the study RANKL levels were further decreased to 8 2 for R ELF group and remained at 22 5 for placebo P 0 0202 for median change in RANKL placebo vs R ELF CRP levels showed a sharp decrease for both the groups by Day 30 but the trend reversed and CRP levels increased to baseline by the end of the study Figure 8B A relatively large reduction in CRP levels was observed for R ELF group 55 9 by Day 30 which sustained for a long time 49 8 by Day 90 P 0 0286 for mean change in CRP placebo vs R ELF On the other hand placebo group regained CRP levels rapidly 40 6 to 23 1 of its baseline CRP level in sixty days These results suggest an improvement in the inflammatory status and reduced bone resorption in postmenopausal women with R ELF supplementation FIGURE 8 Changes in Serum Levels of RANKL and CRP with R ELF Supplementation Median change in the serum levels of RANKL a marker of osteoclast formation panel A and CRP an inflammatory marker panel B in subjects from placebo open bars and R ELF filled bars groups R ELF was shown to regulate both pro inflammatory and anti inflammatory responses critical for fracture healing and optimal joint function RELF 22

R ELF TECHNOLOGY GRID Patents and Intellectual Properties IP The R ELF Technology Grid is landscaped on THREE levels of Intellectual Property IP Since R ELF is based on lactoferrin LF and ribonuclease RNase two glycoprotein fractions from milk their purity and structure function related attributes define the potential bio activity of the final R ELF complex Level 1 Ultra Pure Lactoferrin LF TCR Technology Due to microbial and endotoxin contaminants most commercial LF preparations are not suitable for making protein complexes It is also important to ensure that LF protein is not denatured and the bio burden or other chemical impurities do not interfere in the LF RNase interaction to form stable complexes To circumvent these issues LF TCR was developed using a novel decontamination technology consisting of food grade surfactants antioxidants and polyphenols The compounds utilized in the multi tier TCR process are also known to provide additional nutraceutical benefits The multi functional in vitro performance of LF TCR is summarized in 2 US patents 7125963 7326775 In conclusion LF TCR is a functionally enhanced ultra pure lactose free milk protein produced by an innovative protein engineering technology Level 2 R ELF or ANGex Technology Canine bone physiology especially fracture healing requires an amplified inflammatory response and an enhanced bone turnover for callus formation as well as bone reunion The LF protein is shown to support these functions Formation of new capillaries and revascularization of the bone matrix is essential for regaining total bone function since bone is the central site for synthesis and transport of blood cells RNase mediated angiogenesis could help reach this functional target R ELF technology was developed US Patents 7601689 8003603 to immobilize RNase on milk lactoferrin substrate via noncovalent interactions and conjugation methods including biotinavidin affinity binding and disulfide bonding techniques Level 3 Neo PORTIN Technology Bone fracture healing is an energy consuming process with a high metabolic demand The activation of immune cells via inflammatory response and elevated bone turnover needs ATP input Furthermore target delivery of minerals and cellular building blocks requires transport mechanisms and target delivery systems A combination of CoQ10 ubiquinone for ATP synthesis and LF the innate metal binding transport protein with special cellular receptors complexed with RNase may help these cellular energy dependant functions A technology to trigger and release bio energy US Patent 7956031 and an R ELF CoQ10 composition for target transport US Patent 8021659 has been developed to promote canine bone health 23 Nterminus

R ELF TECHNOLOGY GRID Ultra pure Lactoferrin LF TCR Technology US Patent 7125963 Issued Oct 24 2006 Treatments for contaminant reduction in lactoferrin preparations and lactoferrin containing compositions A method of preparing an ultra cleansed lactoferrin preparation termed treatment for contaminant reduction TCR is provided which includes the steps of treating commercial lactoferrin preparations with at least one each of surfactants antioxidants and polyphenols to form purified lactoferrin LF TCR and drying the LF TCR Additionally a therapeutic lactoferrin composition is provided which contains LF TCR and optionally surfactants antioxidants polyphenols tissue membrane diffusion facilitating agents and anionic compounds The therapeutic lactoferrin composition can additionally contain bioactive agents dietary supplements nutraceuticals functional foods prophylactic agents therapeutic agents and probiotic lactic acid bacteria US Patent 7326775 Issued Feb 05 2008 Several lactoferrin LF based dietary supplements are currently available in health food markets worldwide A majority of such products are derived from partially isolated enriched LF fractions from colostrum or whey concentrates Bulk isolation of LF directly from milk is limited and relatively an expensive process High purity LF preparations commercially exist however products from such protein materials are cost prohibitive and fall short of consumer acceptance without valid functional assurance The nutraceutical benefits of LF as a dietary supplement for human or animal health application requires an innovative technology compatible with large scale manufacturing practices Such technology transfer should ensure the highest standards of product safety quality assurance and delivery of an optimal dosage for an effective functional outcome Furthermore the microbiological and toxicological quality issues compromise the in vivo performance standards of LF as a potent dietary supplement The following QA QC issues are critical for the development of LF as a canine bone health supplement LF TCR is an ultra purified activated milk glycoprotein which is functionally superior than most commercial LF from milk whey or other sources It takes four liters of bovine milk to produce a single 80 mg dose of ultra pure LF RELF 24

FIGURE 9 Based on the types and levels of contaminants as well as the microbiological quality assurances implemented with cGMPs in the commercial scale manufacturing of LF a multi tier TCR process has been developed using natural substrates as decontaminant agents Figure 9 This patented process extends the scope of TCR process in combination with synergistic compositions to enhance multifunctionality of LF LF TCR is a powerful physiological system to complex or transport other bioactive compounds The TCR process could be used as a stand alone technology or integrated with different lab scale pilot scale or commercial scale technologies practiced in the isolation and purification of LF The TCR process includes the creation of a surfactant tier analogous to the physiological gastric detergents which selectively disrupt the cell membranes of contaminant microbes Natural and or food grade surfactants for use in the present invention include plant derived saponins food grade polysorbates and bile salts This tier also utilizes carbonate or bicarbonate anions at specific ratios in combination with natural antioxidants such as vitamins A C or E to enhance the anion dependent LF bioactivity For the purpose of restoring the anion dependent bioactivity of commercial LF methods related to generating carbonated aqueous systems or anaerobic encapsulations are also suitable Finally the ultra cleansing TCR process uses effective and permissible amounts of food grade polyphenols to neutralize endotoxin contaminants in commercial LF preparations Polyphenols of particular use for this purpose include oleoresins aquaresins oleuropeins terpenes flavonoids and biliproteins Lactoferrin TCR TABLE 4 Enhanced Multi functional Performance of LF TCR FUNCTIONAL ACTIVITY LF Whey LF Milk LF TCR 48 h Salmonella Typhimurium 12 5 h 22 9 h 48 h ANTIOXIDANT ACTIVITY Total Antioxidant Status FRAP Value at 24 h BACTERIOSTASIS ACTIVITY PREBIOTIC ACTIVITY Lactobacillus spp n 13 96 147 200 Bifidobacterium spp n 3 110 146 213 Lactococcus lactis 105 120 186 Streptococcus thermophilus 92 116 192 The multi functional in vitro performance of LF TCR is summarized in Table 4 In conclusion LF TCR is a functionally enhanced bio active molecule produced by an innovative patented protein engineering technology US Patents 7125963 7326775 25 Nterminus

R ELF TECHNOLOGY GRID R ELF or ANGexTechnology US Patent 7601689 Issued Oct 13 2009 Angiogenin complexes ANGex and uses thereof Stabilized angiogenin compositions and methods of preparing a stabilized ANG compositions by non covalent immobilization on a naturally occurring substrate such as a protein lipid nucleic acid or nucleotide substrate are disclosed R ELF complexes are formed with RNase Angiogenin or ANG and a second protein such as lactoferrin LF based on functional association or a synergy that may enhance the biological function of the complex R ELF protein complexes can be formed by physical charge and or chemical interactions RNase ANG and a protein substrate such as lactoferrin LF may be complexed together directly or they may be complexed by means of an appropriate bi functional reagent A non covalent complex may be formed by means of electrostatic interactions which may be enhanced by inclusion of appropriate buffers and or salts US Patent 8003603 Issued Aug 23 2011 The formation of a R ELF complex may be confirmed by using co immuno precipitation techniques In preferred embodiments the protein substrate is from the group including but not limited to transport proteins subepithelial matrix proteins and antimicrobial proteins Transport proteins include but are not limited to lactoferrin transferrin ovotransferrin conalbumin ceruloplasmin metallo thionein and transfer factors Subepithelial matrix proteins include but are not limited to fibronectin fibrinogen laminin vitronectin osteopontin native collagens and denatured collagen gelatin Antimicrobial proteins include but are not limited to peroxidases lacto myelo and salivary forms and lysozyme Both LF and RNase co exist in milk and other biological fluids as key regulatory molecules with specific functions in various physiological pathways From a bone metabolism standpoint both proteins elicit potent effects individually as anabolic and anti resorptive agents during aging process disease and repair conditions For the first time a synergistic effect of these two bio active molecules mixed together as an R ELF complex to advance bone health was discovered and patented R ELF complexes or ANGex have higher protein stability and have been shown to play a multi functional role in bone regeneration joint lubrication bone turnover and fracture repair RELF 26

FIGURE 10 Figure 10 ANGex Detection by Chromatography Cationexchange chromatogram is shown for the separation of ANGex from free ANG using NaCl gradient of 20 100 B in 20 min Free native LF 1 mg mL elutes as a single peak at retention time of 84 6 min Curve 1 The retention time of this peak increases upon ANGex formation with 1 mg mL Curve 2 and 4 mg mL Curve 3 of ANG and LF 1 mg mL in solution Elution of ANGex was monitored by the absorbance at 214 nm using a UV vis detector The structure function enhancement of R ELF complex was initially evaluated for antioxidant activity by kinetic ferric reducing antioxidant power FRAP assay 26 The antioxidant efficiency AE was measured as change in absorbance with the concentration of R ELF formed at different RNase LF ratios When individually tested free RNase showed an AE value of 0 6 Abs mM However when mixed with 25 M of LF the AE value increased to 0 7 indicating a synergistic potentiation in the antioxidant power After 24 h the maximum AE value attained individually for RNase and LF were 0 64 and 0 87 respectively however after the two proteins were mixed the resulting R ELF complex exhibited an AE value of 1 18 which is a 35 and 84 increase from that of LF and RNase respectively FIGURE 11 Specific ratios and molecular stoichiometry between LF and RNase are critical in the functional design of the R ELF complex US Patents 7601689 8003603 The synergistic potentiation in antioxidant capacity of the R ELF complex indicated a re arrangement of molecular structure function of the two regulatory proteins Further in vitro studies also revealed a synergistic enhancement in antimicrobial and enzymic activities of R ELF complex The molecular and functional design interplay of these two key regulatory proteins in the R ELF complex has been established in randomized placebo controlled human clinical trials 26 Benzie IFF Strain JJ 1999 Ferric reducing antioxidant power assay Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration Methods in Enzymol Oxidants and Antioxidants Part A 299 15 27 Figure 11 Antioxidant Activity of ANG LF and ANGex Antioxidant activity was determined by FRAP assay with Vitamin C TROLOX and FeSO4 as standards by following the increase in absorbance at 593 nm with time Top panel shows the FRAP reaction kinetics data for LF ANG and ANGex The concentrations of the three standards LF ANG and ANGex were 10 mg ml 125 M for LF 694 M for ANG Bottom panel compares the antioxidant efficiency r as measured by the change in absorbance with the concentration of ANGex Lines represent ANGex formed at varying concentrations of ANG with 0 2 6 and 10 mg mL of LF ANGex formed with 2 mg mL of LF exhibits the highest antioxidant efficiency with r 0 046 27 Nterminus

R ELF TECHNOLOGY GRID R ELF and Neo PORTIN Technology US Patent 7956031 Issued Jun 07 2011 Metallo lactoferrin coenzyme compositions for trigger and release of bioenergy Formulations are provided for the trigger and release of bioenergy The formulations generally include a trigger complex an elemental complex and a coenzyme vitamin B complex The trigger complex is high in fiber and includes at least one metal binding protein in an alkaline buffer system The elemental complex includes one or more trace element as a suitable salt The coenzyme vitamin B complex includes one or more coenzyme coenzyme precursor and or B vitamin The compositions can be administered orally in a variety of forms This invention describes methods to prepare specific combinations of LF TCR CoQ10 mixtures to trigger the release of bio energy bio E in the form of adenosine triphosphate ATP Additionally the invention discloses compositions of functional delivery systems to recreate physiological proton gradients for rapid activation and release of cellular and extracellular ATP Coenzyme Q10 CoQ 10 is a fundamental molecule for production of cellular energy in most living organisms Although found in all human cells CoQ 10 occurs at relatively elevated concentrations in bone cells with high energy requirements such as osteoblasts osteoclasts and chondrocytes The total body content of CoQ 10 has been estimated at 0 5 1 5 g Normal blood levels range from 0 7 1 0 g mL Folkers 1996 Fracture healing is an intensive metabolic process with high energy ATP consumption This CoQ10 Lactoferrin based technology supports canine fracture healing by providing bone cells and inflammatory cell mediators with direct ATP release A vital role in the production of cellular energy combined with its potent antioxidant activity makes CoQ 10 an essential bone health supplement Furthermore its multi functional properties including vitamin like adjuvant activity protection against agerelated degeneration support of homeostasis prophylactic and therapeutic effects against several diseases makes CoQ 10 an important nutraceutical agent Embodiments of the invention provide formulations that provide effective assimilation of CoQ 10 while supporting angiogenesis and vascular generation particularly in bone fracture healing 27 Folkers K 1996 Relevance of the biosynthesis of coenzyme Q10 and the four bases of DNA as a rationale for the molecular causes of cancer and a therapy Biochem Biophys Res Commun 224 358 61 Neo PORTIN with CoQ10 helps support the metabolic energy demand of bone cells during bone turnover and fracture healing RELF 28

US Patent 8021659 Issued Sep 20 2011 Coenzyme Q10 lactoferrin and angiogenin compositions and uses thereof Methods of enhancing the bio availability of coenzyme Q10 and methods of supporting the cardiovascular system to accommodate the increase in cellular energy synthesis as a result of the bio availability of coenzyme Q10 are described Compositions which include coenzyme Q10 lactoferrin and or angiogenin are described for use in the related methods for multi functional health applications Bio availability of bone minerals receptor mediated endocytic release of cations anions and GAG molecules such as chondroitin glucosamine and hyaluronic acid require energy ATP dependant active transport in vivo This physiological function could be achieved with R ELF complex with calibrated LF RNase wt wt ratios from 50 1 to 9 1 admixed with CoQ 10 R ELF could perform an efficient target delivery function with specific transport mechanism s in vivo There are three forms of LF proteins based on iron saturation status apo LF ironfree mono ferric form one ferric ion and holo LF binds two Fe3 ions The ability to bind ferric iron with high affinity KD approximately 10 20 M and retain it at low pH gives LF its antioxidant and antimicrobial properties The ability to keep iron bound at low pH has in vivo significance especially at sites of inflammation where the pH drops below 4 5 due to neutrophil degranulation Besides iron LF is capable of binding a large amount of other compounds and substances such as glycosaminoglycans GAGs chondroitin glucosamine hyaluronic acid DNA or other metal ions including manganese Mn3 cobalt Co3 copper Cu2 zinc Zn2 etc Apart from CO32 LF can also bind a variety of other anions such as oxalates carboxylates and others Accordingly LF could affect bone metabolism with transport and distribution of various minerals anionic and cationic substances into the extra cellular matrix ECM R ELF with its mineral transport activity may help promote calcium deposition in the bone Osteoblast mediated Ca II deposition in the ECM is a critical step in bone tissue generation CH3 CH3 0 0 0 0 CH3 6 10 CH3 H Co enzyme Q10 Neo PORTIN with metal transport protein LF TCR provides an efficient target delivery system for minerals and nutrients that are essential for bone physiology 29 Nterminus

R ELF COMPLIANCE AND SAFETY R ELF Regulatory Compliance QA QC and Safety Lactoferrin LF is a bio active milk protein therefore the source of the dairy plays a critical role in the largescale production of LF for nutraceutical applications The LF used in the R ELF and Neo PORTIN complexes is isolated from cow s milk certified by the New Zealand Food Safety Authority NZFSA The operation of farm dairies manufacture storage and transport of dairy products is approved by the New Zealand Ministry of Agriculture and Forestry MAF following a stringent Bio Security Monitoring Program The dairy milk is processed in an ISO 9001 2000 facility that meets the requirements set out in the internationally recognized standard LF is isolated from fresh milk through patented serial fractionation and chromatographic techniques to remove milk sugars i e lactose milk fats and other milk proteins Most commercially available LF is obtained through high temperature spray drying process which can denature heat sensitive LF protein In contrast LF TCR utilizes a patented freeze drying technique that preserves LF protein structure and function Purified milk LF is subjected to a series of QA QC tests to ensure the quality of the final protein product These tests include Chemical Analysis for protein content levels of heavy metals if any water activity etc Microbiological Analysis for total bio burden coliform counts etc The LF protein is also subjected to functional tests such as iron binding activity prior to QA QC release LF base material is further tested for functional properties such as antioxidant antimicrobial and prebiotic activities see LF TCR patents before integrating into R ELF ANGex or Neo PORTIN complexes Kosher Certified GMO Certified Lactose Free Hormone Free Antibiotic Free RELF 30

In the United States milk derived LF has been granted GRAS Generally Recognized As Safe status by the U S Food and Drug Administration US FDA under 21 CFR 179 36 f In the European Union milk derived LF is permitted by the European Food Safety Authority EFSA under EU Council Directive 83 417 EEC The EFSA Panel on Dietetic Products Nutrition and Allergies NDA endorsed LF with no safety issues in a range of matrices including food supplements infant formulas dietetic food for special medical purposes and sports nutrition After evaluation of animal and in vitro data the NDA found no safety issues at proposed intakes ranging from 667 mg 100 g of baby foods and foods intended for children aged 1 3 years to 4000 mg 100 g for energy bars for sportsmen and women In reaching this decision the NDA Panel considered that LF up to the highest dose 2000 mg kg bw per day tested in the sub chronic rat study did not show adverse effects In Japan LF is specified in the List of Existing Food Additives which is a list of the permitted natural additives set by the Japanese Ministry of Health Labor and Welfare JMHLW In South Korea LF concentrates are listed as Authorized Natural Additives In Taiwan LF may be used in special nutritional foods under the following condition only for supplementing foods with an insufficient nutritional content and may be used in appropriate amounts according to actual requirements LF is considered safe and has been approved worldwide for various human and animal health applications 31 Nterminus

ABOUT US N terminus a division of en N tech Inc is a pioneer in the research of bio active molecules and a global leader in successful technology transfers that impact areas of dietary supplements food safety veterinary and human medicine N terminus is internationally recognized for its cutting edge research and intellectual properties on protective and therapeutic aspects of natural bioactive molecules N terminus applies its scientific expertise in the discovery and development of novel technologies in veterinary and human health N terminus is committed to establish and maintain the reputation of highest quality and reliability with health technologies that it offers and to advance the field applications of this emerging multifunctional molecule through education research and publication N terminus was started in the year 2000 and employs a team of professors and medical scientists from the fields of microbiology immunology biochemistry and physiology Since its formation this researc facility has been playing an active role in educating the world about the research and health implications of natural bio active molecules Based in Southern California N terminus collaborates with several academic and corporate research institutions worldwide www nterminus com DISLCLAIMER This white paper has been prepared by N terminus Research Laboratory hereafter N terminus to provide scientific and educational information about R ELF complex This document is neither intended to be used to market or advertise a product containing R ELF nor to make labeling or health claims Companies who sell goods containing R ELF are responsible for ensuring that their uses and claims are in compliance with the legal and regulatory requirements relevant to their markets Although N terminus has used diligent care to ensure that the information provided herein is accurate and up to date no representation or warranty of the accuracy reliability or completeness of the information is made The content of this document is subject to change without further notice Copyright N terminus Research Laboratory 2015 All rights reserved