The Evolution of Chondroitin Sulfate

Volume editor NICOLA VOLPI THE EVOLUTION OF CHONDROITIN SULFATE

NON-ANIMAL CHONDROITIN SULFATE The information, guidance and advice contained in this book are based upon the research and the personal and professional experiences of the author. They are intended to be used by medical, scientific or health-care professionals and are not intended as a substitute for consulting with a health care professional. All matters pertaining to your physical health should be supervised by a health care professional. Every possible effort has been made to ensure that the information contained in this book is complete and accurate at the time of going to press. However, neither the publisher nor the author cannot accept responsibility for any errors or omissions caused. No responsibility for loss or damage occasioned to any person acting, or refraining from action, as a result of the material in this publication can be accepted by the editor, publisher or the author. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written consent of the copyright owner.

With a special thanks to Ms Lorena Carboni

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INDEX

INTRODUCTION (BACKGROUND) 4

1. JOINT HEALTH AND CHONDROITIN SULFATE 6 1.1. Joint structure 7 1.2. Osteoarthritis 9 1.2.1. Etiology of osteoarthritis 11 1.2.2. Role of the chondrocyte in cartilage destruction 12 1.2.3. Role of biomechanics and load 13 1.2.4. Mediators of inflammation in osteoarthritis 15 1.3. Considerations & thoughts 17 1.4. Chondroitin sulfate and its role in the management of osteoarthritis 19 1.4.1. Biological functions of chondroitin sulfate 22 1.4.2. Preclinical studies of chondroitin sulfate 24 1.4.2.1. Intestinal absorption of chondroitin sulfate 24 1.4.2.2. Bioavailability, distribution and target tissue orientation of chondroitin sulfate (animal and human studies) 26 1.4.3. Mode of action of chondroitin sulfate in vitro and in vivo models 26 1.4.3.1. Anti-inflammatory effect of chondroitin sulfate 26 1.4.3.2. Other effects of chondroitin sulfate 28 1.4.4. Efficacy of chondroitin sulfate 30 1.4.4.1. Pre-clinical efficacy in animal models 30 1.4.4.2. Establishment of dose regimen 31 1.4.4.3. Clinical efficacy and safety of chondroitin sulfate in humans 32

2 THE EVOLUTION OF CHONDROITIN SULFATE

2. SAFETY CONCERNS OF ANIMAL-DERIVED CHONDROITIN SULFATE 36 2.1. Animal chondroitins sulfate safety concerns 37 2.2. Heterogeneity of chondroitin sulfate in animal tissues 39 2.2.1. Natural contaminations 41 2.2.1.1. Natural polysaccharides 41 2.2.1.2. Virus or prions (or infective agents) 42 2.2.1.3. Natural polymers: proteins and nucleic acids 43 2.3. Adulterations 45 2.4. Uncontrolled supply chain and risks 48 2.5. Analytical controls 49 2.6. Conclusions 51

3. MYTHOCONDRO ®, THE CHONDROITIN SULFATE OF NON-ANIMAL ORIGIN 52 3.1. State of the art 53 3.2. The evolution of the fermentation-derived production process 54 3.3. Mythocondro® structural features and physical-chemical properties 56 3.4. Biological activity of Mythocondro® 58 3.4.1. Toxicological studies 58 3.4.2. Permeability 59 3.4.3. Bioavailability 60 3.4.4. Anti-arthritic effect 62 3.4.5. Why Mythocondro ® is better than animal chondroitin sulfate? 64

4. REFERENCE 66

GLOSSARY

3 INTRODUCTION (BACKGROUND)

According to EULAR (the European League Against Rheumatism) [1,2] , OARSI (Osteoarthritis Research Society International) [3] and World Health Organi - zation (WHO) [4] , the musculoskeletal or rheumatic diseases are the major cause of morbidity throughout the world, having a substantial influence on health and quality of life, also causing an enormous burden of cost on health systems. Osteoarthritis (OA) is an important example of the more than 150 conditions and syndromes with the common denominators of pain and inflammation. 40% of people over the age of 70 years suffer from OA of the knee, 80% of patients with OA have some degrees of limitation of movement, and 25% cannot perform their major activities of daily living [4] .

Most people do not develop symptoms of OA until significant joint damage has occurred, commonly after age 50-60 years, but there is radiographic evidence for OA in a significant percent of women beginning in the early 40’s [5] . However, this stage already represents a situation where cartilage is heavily damaged leading to a narrowing of the joint space.

Over the last years, there has been an increasing interest on individuals with ageing and their quality of life along with increased healthcare costs for the demographic shift towards higher fractions of elderly people [6] . Today, along with traditional use of drugs to treat the pain of OA, great re - sults are associated with administering chondroprotective bioactive (macro)molecules in the early stages of OA, in order to delay progress and/or to reduce symptoms.

Chondroitin sulfate (CS), a basic natural component of cartilage and syn - ovial fluid, is one of the most effective Symptomatic Slow-Acting Drugs for Osteoarthritis (SYSADOAs) and it is a S/DMOAD (structure/disease modify - ing anti-osteoarthritis drug) used to provide relief from the pain and inflam - mation associated with joint degeneration. Its supplementation helps to regenerate the joint structure, leading to reduced pain and increased mo - bility of the affected joint [7] . The clinical benefits of CS have been established by a large number of human trials and offers an excellent safety and toler - ability profile which allows long term administration for OA and joint health.

4 THE EVOLUTION OF CHONDROITIN SULFATE

However, it should be pointed out that all studies have been carried out on quality-controlled products of CS. The respective results can in no way be translated to the heterogeneity of products available over the counter.

CS supplied to the market is extracted from cartilaginous feedstocks using organic solvents, with purification processes that may cause chemical degra - dation/desulfation and loss of activity along with an uncontrolled and unre - liable supply chain of CS raw materials. This poses serious concerns about the quality and the safety of the ingredient.

As a matter of fact, several published studies have reported poor quality of CS present in nutraceuticals [8,9] . Finally, today, a new and pioneering non- animal CS characterized by high purity and obtained through a fermenta - tion-derived manufacturing process is available and promise to resolve the long-standing acknowledged problem of poor quality and potential safety issues of animal-derived CS.

Mythocondro ® is a revolutionary ingredient, patented and developed by Gnosis using a pharmaceutical approach, to ensure strict quality standards that promises to change completely the CS industry, providing a reliable and reproducible source of CS which definitely solves concerns related to animal derived CS: clinically tested, well characterized and environmentally friendly, it is the first CS suitable for vegetarians and free from restrictions of use related to religious and supply issues.

This book gives an extensive overview of the aetiopathogenesis of OA , of the current database of CS in the treatment of OA and the relevant descrip - tion and clinical evidence of this new non-animal, fermentation derived CS with relevant effectiveness in joint disease.

5 JOINT HEALTH AND CHONDROITIN SULFATE

6 1.1 JOINT STRUCTURE

Understanding of joint dysfunction requires some knowledge of normal joint structure, its physiology and the comparison of the diseased state to the physiological situation.

Joints are responsible for movement and stability of the skeleton and are classified based on structure or function. In particular, synovial joints are the only joints that have a space (a synovial cavity filled with fluid) between the adjoining bones and that make possible movements of the opposed articular surfaces without pain, friction with a correct distribution of load across joint tissues. In a healthy joint, the cartilage within the joint serves as a cushion, permitting the bones to rotate, glide and roll upon each other smoothly and easily during activities like walking [10-12] .

Articular cartilage tissue varies in thickness, cell density, matrix composition, and me - chanical properties within the same joint, among joints and among species [13] . Nonethe - less, it consists of the same components and has the same general structure.

It is composed of a dense extracellular matrix (ECM) with a sparse distribution of highly specialized cells called chondrocytes. The ECM is principally composed of water, collagen and proteoglycans (PGs) with other non-collagenous proteins and glycoproteins present in lesser amounts [14] (Figure 1).

Figure 1. To form articular cartilage, chondrocytes organize collagen, proteoglycans and other components into a unique, highly ordered structure.

7 THE EVOLUTION OF CHONDROITIN SULFATE

Water contributes up to 80% of the wet weight of articular cartilage and its interaction with the matrix macromolecules significantly influences the me - chanical properties of the tissue.

PGs are heavily glycosylated structures. In articular cartilage, they represent the second-largest group of macromolecules in the ECM and account for 10% to 15% of the wet weight. PGs consist of a protein core with one or more linear glycosaminoglycan (GAG) chains (CS, keratan sulfate and others) covalently attached [14] (Figure 2).

Maintenance of the articular surface requires turnover of the matrix macro - molecules (continual replacement of degraded matrix components) and probably alteration in the matrix macromolecular framework in response to joint use [14] .

Figure 2. Structure of a proteoglycan with its CS and keratan sulfate chains and the for - mation of a macromolecular complex associated with hyaluronic acid.

8 1.2 OSTEOARTHRITIS

During the normal biological process of ageing, multiple factors may impact on joint structure (including genetic, developmental, metabolic and traumatic events [15] ) determining an imbalance between the normal coupling of degra - dation of joint and the synthesis of articular cartilage, ECM and subchondral bone by chondrocytes (Figure 3). Moreover, a loss of chondrocytes due to an increased susceptibility to cell death appears to be important as well [16-18] .

These changes are directly responsible to the development of a typical age- related disorder, the degenerative joint disease also known as OA.

In OA, the gradual loss of matrix PGs and subsequent loss of mechanical in - tegrity is often a slow process believed to take place over decades.

Human articular cartilage is continuously remodeled as a consequence of ana - bolic and catabolic processes. The chondrocytes in normal adult cartilage main - tain a balance between synthesis and degradation of ECM components. In OA, the metabolic activity of the chondrocytes is shifted towards a state where new

Figure 3. The correct definition of OA is a chronic (long-term) condition “characterized by focal areas of loss of articular cartilage within the synovial joints, associated with hyper - trophy of the bone (osteophytes and subchondral bone sclerosis) and thickening of the capsule. In this sense it is a reaction of the synovial joints to injury” [4] .

9 THE EVOLUTION OF CHONDROITIN SULFATE

matrix synthesis is outweighed by breakdown of matrix constituents. The result is degeneration and gradual loss of articular cartilage [10] .

The avascular nature of cartilage leaves the chondrocyte as the only mecha - nism of matrix repair and the isolated nature of the cell severely limits its re - pair capabilities. The breakdown of the tightly regulated structure of the ECM leads to a weakening of the mechanical properties of the matrix and can progress to the endpoint of total failure.

The pathology of OA involves the whole joint in a disease process that in - cludes focal and progressive hyaline articular cartilage loss with concomitant changes in the bone underneath the cartilage, including development of mar - ginal outgrowths, osteophytes and increased thickness of subchondral bone.

OA can affect all joints of the body but not all localizations have the same con - sequences for the individual. Some joints are more prone to be affected by OA than others (Figure 4). Exact conditions for the individual joints are sparse, mostly

Figure 4. Common joints affected by OA .

10 THE EVOLUTION OF CHONDROITIN SULFATE non-representative and differ considerably depending on definition (radiographic vs. symptomatic), age group, sample size, ethnicity and region.

Severe OA of the large joints like the hip or the knee will lead to more dramatic effects such as immobility of the individual and loss of working resources to society [18] .

1.2.1 Etiology of osteoarthritis

OA is believed to be caused by a combination of factors. Due to the long period of painless progression, the disease is rarely diagnosed at an early time point. This makes diagnosing the exact cause in specific patients extremely difficult. Injury due to mechanical stress, although poorly understood, is considered to play a predominant role during initial stages of the disease [19-25] . Biochemical and genetic factors are likely to contribute to the further progression.

The role of genetics in the onset of OA is becoming increasingly playing a larger role than first thought. These studies indicate that some forms of OA may have a genetic component of up to 65% [26,27] . An up-regulation of proteinase activ - ities, particularly those of metalloproteinases (MMPs), has been implicated in the disease as the major cause of increased matrix catabolism [28-32] . The degra - dation of structural macromolecules like PGs and collagens leads to depletion of the most important building blocks of the ECM. Under these circumstances, the absence of adequate replenishment becomes a main issue.

The onset of the disease is believed to be a loss of GAGs from the upper layers of the cartilage. This is then followed by a gradual breakdown of collagen fibers with subsequent fibrillation of the cartilage surface. Once this point has been reached the progression of the disease becomes more rapid and irreversible. The cartilage matrix is continually degraded, deep fissures are generated and fi - nally the matrix is removed completely exposing the underlying bone. It has also been demonstrated that there is an increased rate of apoptosis within OA cartilage. As the chondrocyte is an isolated cell, the death of a cell leaves a zone of cartilage which has no means of maintenance.

11 THE EVOLUTION OF CHONDROITIN SULFATE

1.2.2 Role of the chondrocyte in cartilage destruction

Chondrocytes represent the only cell type in hyaline cartilage. These cells are responsible not only for the generation of ECM during growth and develop - ment, but also for the maintenance of tissue homeostasis during adult life. The chondrocyte in mature articular cartilage exhibits virtually no mitotic ac - tivity and a very low rate of matrix synthesis and degradation. However, chon - drocytes even from old individuals are able to respond to given stimuli by showing increased activity.

In early OA, structural changes in the ECM induce chondrocyte proliferation (clonal growth), a stimulated collagen and PG biosynthesis corresponding to a repair attempt, and an increased production of catabolic cytokines and ma - trix-degrading proteinases [28-33] .

The capacity of chondrocytes to degrade constituents of the ECM depends on their ability to synthesize and secrete proteinases (Table 1).

There are more than 20 known MMPs divided into four categories: collage - nases, stromelysins, gelatinases and membrane bound (Table 1).

MMPs Collagenases MMP-1, MMP-8, MMP-13 Gelatinases MMP-2, MMP-9 Stromelysins MMP-3, MMP-7, MMP-10, MMP-11 Membrane type MMP-14, MMP15, MMP16, MMP17 Aggrecanases

Disintegrin and Metalloproteinase type ADAM-TS4, ADAM-TS5

Other proteinases Elastase Cathepsins Cathepsin B, D, G, L Proteinases of the coagulation system tPa, uPA, plasmin Table 1. Proteinases involved in the degradation of cartilage matrix.

12 THE EVOLUTION OF CHONDROITIN SULFATE

This list is representative, not comprehensive.

Under normal conditions, the controlled activity of these enzymes are neces - sary for appropriate morphogenesis and tissue remodeling (Figure 5). Regula - tion of proteinase activity occurs at three different levels: synthesis and secretion, activation of latent enzyme and inactivation by proteinase inhibitors. During degenerative joint diseases, such as OA, the expression and production of proteinases is increased. MMPs are prominent and appear to play several important roles in the pathological destruction of cartilage [28-32,34-37] .

Figure 5. Normal cartilage: balance between biosynthetic and catabolic processes.

1.2.3 Role of biomechanics and load

While the role of mechanical forces on OA has long been speculated, it is only over the last few years that data has appeared supporting this hypoth - esis. The increased incidence of OA in overweight patients may be due to the increased mechanical forces being applied. Body mass index (BMI) and obesity have been shown to be linked to the incidence of OA [22-23] .

13 THE EVOLUTION OF CHONDROITIN SULFATE

In fact, it has been shown that the BMI at 20-29 years was the most predic - tive of future OA incidence [25] , indication that OA progresses over a number of decades.

Joint injury has been shown to be a risk factor for knee OA [22,23] and a num - ber of studies have demonstrated a link between occupations involving heavy lifting or repetitive movements on the incidence of OA [38-42] .

In vitro studies have been used to elucidate the early events after mechanical damage [43-46] . It has been shown in cartilage explants that within hours of an excessive mechanical insult there is a large, enzyme independent, release of GAGs into the surrounding medium [46] and excessive load increases activation and expression of MMPs in explant cultures [47-51] . This would suggest that in - jury modulates the mechanism of GAG turnover [46] . Short term responses to mechanical load and injury include cell death by both necrosis and more commonly apoptosis [44,45] . This loss of cells may have long term effects in the maintenance of the surrounding matrix. Once the disease has progressed OA, chondrocytes have a differential response to mechanical load than nor - mal chondrocytes [52,53] . Whether this altered response is a cause of the dis - ease or a later consequence is still unknown.

The risk factors for OA can be separated in three groups [54] : systemic, intrinsic and extrinsic factors (Table 2).

Systemic Factors Intrinsic factors Extrinsic factors (anatomically or physiologically (somehow acting on a joint) pertain to a joint)

• Higher age • Deformities • Obesity • Female gender • Injuries/damages • Muscle weakness • Race • Malalignment • Repetitive overuse (occupational or sports) • Inheritance • Laxity • Possibly nutritional factors • Proprioceptive deficiencies Table 2. Risk factors for the development of OA .

14 THE EVOLUTION OF CHONDROITIN SULFATE

All these factors may differ in extent from one joint to another. Some, like inheritance, may only multiply a selection of those among in- and extrinsic factors. For women, weight accounts for more OA than any other known factor, for men’s knee OA overweight ranks second after injury as predis - posing factor [55-57] .

1.2.4 Mediators of inflammation in osteoarthritis

It is generally accepted that inflammatory mediators including cytokines, prostanoids and nitric oxide (NO) induce cartilage degradation in disorders such as rheumatoid arthritis (RA) [for review see 58 and 59 ]. In that case, the inflammation primarily takes place in the synovial membrane and the de - struction of cartilage is a secondary event. In synovial cells, reactions towards components released from cartilage into the synovial fluid may contribute to the disease progression [60,61] but the major pathogenic processes are localized within the cartilage itself [62,63] . Chondrocytes in OA affected cartilage display enhanced and coordinated expression of proinflammatory cytokines and the enzyme responsible for NO production [64] . Collectively, evidence is accumu - lating that mediators of inflammation acting in an autocrine/paracrine fashion within the cartilage play a primary role in the pathogenesis of OA [60-65] .

Proinflammatory cytokines include interleukins (IL), tumor necrosis factors, interferons and colony-stimulating factors. Although these factors were iden - tified originally as secreted products of immune cells that modulate the func - tion of other cells of the immune system, many of them have effects on nonimmune cells like fibroblasts and chondrocytes. Among the proinflam - matory cytokines, IL-1 β and TNF- α appear most directly involved in the pathological processes of OA [66] .

Anyway, it appears that cytokine networks with a considerable degree of complexity influence the metabolic state of chondrocytes. Evidences have accumulated that proinflammatory cytokines are important in the pathogen - esis of OA and a large number of in vitro studies have been carried out in

15 THE EVOLUTION OF CHONDROITIN SULFATE

Figure 6. IL-1 induces catabolic processes in inflamed cartilage.

order to examine in detail the influence of these factors on chondrocytes. IL-1 has been found to induce catabolic responses in many different ways. Human articular chondrocytes, when stimulated by IL-1, dramatically in - crease the expression of matrix-degrading proteinases like MMP-1, -2, -3, - 7, -8, -9, -13 [37,67-70] . Moreover, IL-1 strongly inhibits biosynthesis of cartilage PG and collagens [71,72] .

More recently, it has also been discovered that IL-1 may play a role in ana - bolic responses in human knee cartilage from both normal and OA pa - tients [73] . This study showed that IL-1 and TNF- α, both normally associated with inflammatory responses, are able to upregulate the bone morphogenetic protein-2 in a dose dependent fashion and IL-1 is also associated with a re - duction in GAG synthesis (Figure 6).

16 1.3 CONSIDERATION & THOUGHTS

OA is a global issue affecting all known cultures and ethnicities. Detailed infor - mation for incidence and prevalence are sparse, not only because of limitations of available research resources, but also related to methodological and diagnostic difficulties. Not all races are affected equally when differentiating by anatomic criteria. Anyway, OA has a huge incidence and economic impact (Table 3).

Country Cost estimate % of gross domestic product Year

USA US$ 69.8-84.4 billions 0.8-1.0 1997 Australia AUS$ 0.94 billions 0.17 1993-94 Great Britain GB£ 0.22 billions 0.35 1998-99 France EUR 1.5 billions 0.18 1998 Germany US$ 6.7 billions 0.32 1994

Table 3. Perspective of country-specific estimates of OA costs.

African-American women seem to be more prone to OA of the knee than Caucasian women [74] . Paleopathological studies suggest that OA has been with mankind from the beginnings as traces of OA are revealed by many skeletons since Paleolithic times [75-77] .

OA is particularly prevalent among older people, the number of whom is pre - dicted to increase in all countries. Worldwide estimates suggest that 9.6% of men and 18.0% of women from age 60 have symptomatic OA [78] . 40% of people over the age of 70 years suffer from OA of the knee, 80% of patients with OA have some degrees of limitation of movement and 25% cannot per - form their major activities of daily living.

Since OA is a disease whose prevalence increases with age, it will become even more frequent in the future as the proportion of the population above 70 years of age is expected to increase dramatically [79,80] . Moreover, with the observed increase of obesity in western populations an over-proportionate rise in the prevalence of knee, hip and hand OA can be expected [81] .

OA does not per se reduce life expectancy. But comorbidities, predisposing fac -

17 THE EVOLUTION OF CHONDROITIN SULFATE

tors (obesity) and side effects of medications (in particular non-steroidal anti- inflammatory drugs) themselves have an elevated risk of death. In 2000, 13,000 years of life were lost due to premature death because of OA worldwide.

Finding cost effective treatment modalities are among the most important challenges for science and politics in the near future. It is currently uncertain whether measures exist to effectively prevent OA. Great hopes are associated with administering bioac - tive (macro)molecules in the early stages of OA in order to delay progress or to re - duce symptoms. It is not clear yet what economic impact such medication of typically long duration will have. The core question is whether its direct costs will be out - weighed by savings on surgery, analgesics and anti-inflammatory medications.

Current treatments focus mainly on pain relief, although a growing number of ex - perimental treatments are under investigation with the aim of slowing or reversing the progression of the disease (with S/DMOAD). The only reproducibly effective treatment is total replacement of late stage OA joints with prosthetic joints.

More and more data have been cumulated indicating treatment potentials for pain relieve, functional improvement and slowing of destructive processes, most often based on studies applying natural constituents of cartilage to animals and humans.

Among others, the treatment concept using CS in OA appears to be one of the most beneficial strategies both with regard to improvement of symptoms and disease modification. In several published studies, CS has in fact demonstrated to have properties of a so called symptomatic slow acting drug for the treatment of OA (SYSADOA) and to own disease modifying properties in the long-term treatment of OA (so called DMOAD).

Along with these epidemiological and clinical findings research regarding the de - sign of clinical trials has been strengthened. CS in particular was studied exten - sively with regard to pharmacokinetics and mode of action. Several lines of research strongly point to different, perhaps complementary ways how CS appears to re-establish the disturbed metabolism of cartilage in OA.

18 1.4 CHONDROITIN SULFATE AND ITS ROLE IN MANAGEMENT OF OSTEOARTHRITIS

CS is a natural sulfated GAG which play a significant role in biological processes as it is abundantly distributed in humans, other mammals and invertebrates. Based on its structural diversity in chain length and sulfation patterns, CS pro - vides specific biological functions in cell adhesion, morphogenesis, neural net - work formation and cell division.

GAGs are a family of linear, complex, polydisperse natural polysaccharides represented also by hyaluronic acid, keratan sulfate, dermatan sulfate (DS), heparan sulfate/heparin [82-86] . With the exception of keratan sulfate, they are composed of alternating copolymers of uronic acids and amino sugars, and their structures are commonly represented by typical disaccharide sequences. GAGs are usually found covalently linked to protein in the form of PGs and fibrous proteins, including collagen, elastin, fibronectin and laminin having both structural and adhesive functions.

CS, in particular, is composed of alternate sequences of D-glucuronic acid and differently sulfated residues of N-acetyl-D-galactosamine linked by β- [1,3 ] bonds. Depending on the disaccharide nature, CS with different carbohydrate back - bones are known (Figure 7).

R1 = R 2 = R 3 = H: nonsulfated chondroitin R1 = R 2 = SO 3- and R 3 = H: chondroitin-4,6-disulfate, CSE R1 = SO 3- and = R 2 = R 3 = H: chondroitin-4-sulfate, CSA R1 = R 3 = SO 3- and R 2 = H: chondroitin-2,4-disulfate, CSB R2 = SO 3- and = R 1 = R 3 = H: chondroitin-6-sulfate, CSC R1 = R 2 = R 3 and SO 3- : trisulfated chondroitin R2 = R 3 = SO 3- and R 1 = H: chondroitin-2,6-disulfate, CSD Figure 7. Structures of the disaccharides forming chondroitin sulfate. Minor percentages of very rare and peculiar disaccharides may also have a sulfate group in position C3 of the glucuronic acid.

19 THE EVOLUTION OF CHONDROITIN SULFATE

However, even if the known CS samples are mainly composed of various percentages of chondroitin-4-sulfate (CSA) and chondroitin-6-sulfate (CSC), disaccharide units (monosulfated in position 4 and monosulfated in posi - tion 6 of the N-acetyl-D-galactosamine) and disaccharides with a different number and position of sulfate groups can be located, in various percent - ages, within the polysaccharide chains (Figure 7). In fact, different kinds of

Parameters CS Bovine CS Porcine CS Chicken CS Shark CS Raja CS Squid Sturgeon Aorta PGs Fetal bovine muscle, Platelets Human plasma bone ileum, kidney, lung Rabbit bone marrow

Molecular mass MWn (kDa) 12÷17 9÷14 8÷13 25÷40 27÷34 60÷80 25÷30 nd nd nd nd MWw (kDa) 20÷26 14÷20 16÷21 50÷70 50÷70 80÷120 35÷40 nd nd nd ~15 Dispersity 1.8÷2.2 1.4÷1.8 1.6÷2.0 1.0÷2.0 1.2÷2.5 0.8÷1.3 1.0÷1.5 nd nd nd nd Disaccharides ∆Di-0s 6683313 70 nd 0 40÷60 ∆Di-6s 33 14 20 44 39 15 55 95÷100 100 traces 1÷5 ∆Di-4s 61 80 72 32 43 50 38 0÷5 traces >98 60÷40 ∆Di-2,6dis nd nd nd 18 13 0000 00 ∆Di-4,6dis nd nd nd 2122 00 0 00 ∆Di-2,4dis nd nd nd 110 00 0 00 Charge Density 0.90÷0.96 0.92÷0.96 0.90÷0.94 1.15÷1.25 1.08÷1.20 1.00÷1.20 0.90÷0.95 0.98÷1.02 0.98÷1.02 0.98÷1.02 0.40÷0.60 4s/6s ratio 1.50÷2.00 4.50÷7.00 3.00÷4.00 0.45÷0.90 1.00÷1.40 2.50÷4.00 0.40÷0.90 <0.1 <0.1 >45 10÷50 Reference(s) Volpi N. Volpi N. Volpi N. Volpi N. Volpi N. Shetty AK et al. Maccari F, Kapoor R, Phelps CF Sampaio LO, Dietrich CP. Ambrosius M, Ambrosius M, J Pharm J Pharm J Pharm J Pharm J Pharm Carbohydr Res Ferrarini F, Cöster L, J Biol Chem 256, 9205, Kleesiek K, Kleesiek K, Pharmacol 61, Pharmacol 61, Pharmacol 61, Pharmacol 61, Pharmacol 61, 344, 1526, 2009 Volpi N. Fransson LA. 1981 Götting C. J Götting C. J 1271, 2009 1271, 2009 1271, 2009 1271, 2009 1271, 2009 Carbohydr Biochem J 197, 259, Chromatogr A Chromatogr A Res 345, 1981 1201, 54, 1201, 54, 1575, 2010 2008 2008 Volpi N. Volpi N. Volpi N. Volpi N. Volpi N. Theocharis AD, Okayama E, Oguri K, Volpi N, J Pharm Sci J Pharm Sci J Pharm Sci J Pharm Sci J Pharm Sci Theocharis DA, Kondo T, Okayama M. Maccari F. 96, 3168, 96, 3168, 96, 3168, 96, 3168, 96, 3168, De Luca G, Hjerpe A, Blood 72, Clin Chim 2007 2007 2007 2007 2007 Karamanos NK. 745, 1988 Acta 370, Biochimie 84, 667, 196, 2006 2002

Table 4. Main CS disaccharides from various species, organ and tissues.

20 THE EVOLUTION OF CHONDROITIN SULFATE

CS sulfation may be present in the CS backbone in various percentages in relation to specific animal sources (Table 4) [86] .

CS is generally found in all mammalian connective tissues, especially in the cartilage, skin, blood vessels, ligaments, tendons with different kind of sulfation [87] .

Parameters CS Bovine CS Porcine CS Chicken CS Shark CS Raja CS Squid Sturgeon Aorta PGs Fetal bovine muscle, Platelets Human plasma bone ileum, kidney, lung Rabbit bone marrow

Table 4. Main CS disaccharides from various species, organ and tissues.

21 THE EVOLUTION OF CHONDROITIN SULFATE

1.4.1 Biological functions of chondroitin sulfate

The effect of CS in patients with OA is possibly the result of the stimulation of the synthesis of PGs and the decrease in catabolic activity of chondrocytes by inhibiting the synthesis of proteolytic enzymes and other factors that contribute to cartilage matrix damage and cause the death of these cells (Figure 8).

Figure 8. Effect of CS at molecular level on OA chondrocytes and ECM.

In addition to its conventional structural roles in the composition of ECM and formation of organs and tissues, such as cartilages and bones, accumulated evidence implies that CS chains fulfil important biological functions in inflam - mation, cell proliferation, differentiation, migration, tissue morphogenesis, organogenesis, infection and wound repair [88-91] .

These effects are related to the capacity of CS (and CS-PGs) to interact with a wide variety of molecules including (but not limited to) matrix molecules, growth factors, protease inhibitors, cytokines, chemokines, adhesion molecules and pathogen virulence factors via aspecific/specific saccharide domains within the

22 THE EVOLUTION OF CHONDROITIN SULFATE chains. Along with aspecific interactions based on both numerous negative charges related to sulfate and carboxyl groups and to its long chains and on particular functional domain structures which are formed by combinations of the various disaccharide units (see Figure 7), CS may participate in specific bind - ing to bioactive molecules (Table 5).

CS-Binding proteins CS bound Biological effects related to the binding protein

ECM Components Type II collagen CS Primary OA Type V collagen CS Tuberous sclerosis Growth factors FGF-2, -10, -16, -18 CS type E Wound healing, angiogenesis-related diseases KGF/FGF-7 CS Tissue repair, cancer HB-EGF CS Arteriosclerosis, liver cancer, wound healing MK CS type E Cancer PTN/HB-GAM CS type E Cancer PDGF CS Arteriosclerosis, malignant tumor VEGF oversulfated CS Diabetic retinopathy, solid tumor, RA GDNF CS type E Extensive aganglionosis BDNF CS type E Autism Chemokines IFN- γ CS Antiviral and antitumor actions, receptor for INF- γ IL-8 CS Arthritis, sepsis, acute, nephritis, etc. MIP-1 α CS Myeloma bone disease MCP-1 CS Atherosclerosis, multiple sclerosis SLC oversulfated CS Inflammation IP-10 oversulfated CS Inflammation, cancer SDF-1 β oversulfated CS Hematopoiesis PF4 oversulfated CS Hematopoiesis, angiogenesis Cell adesion molecules CD-44 oversulfated CS Inflammation, malignant tumor L-selectin oversulfated CS Leukocyte adhesion deficiency P-selectin oversulfated CS Inflammation, thrombosis Proteases Leukocyte elastase CS Inflammation, lung emphysema, clotting disorders Cathepsin K CS type A Collagenolytic activity Matrix metalloproteinase-2 CS Tumor cell invasion and metastasis Virus protein Glycoprotein C CS type E Herpes simplex virus infection Table 5. Binding of CS to various biomolecules.

23 THE EVOLUTION OF CHONDROITIN SULFATE

1.4.2. Preclinical studies of chondroitin sulfate

The results of the several pharmacokinetic studies in rats, dogs and humans present clear evidence that exogenous CS is absorbed following oral admin - istration as a high molecular mass polysaccharide together with derivatives resulting from a partial depolymerization and/or desulfation of the parent compound. As a consequence, CS has a systemic bioavailability and can exhibit pharma - cological actions in the human target tissues like joint cartilage and synovial fluid.

1.4.2.1 Intestinal absorption of chondroitin sulfate The absorption of sulfated GAGs orally administered remains a controversial issue since longer time, due to the difficulty in accepting that molecules with high molecular mass and charge density can pass the gastric and in - testinal mucosa. In contrast to glucosamine sulfate, for example, that is ab - sorbed easily, CS is a much larger molecule which was long discussed not to be absorbed intact through the intestinal wall and into blood and joints. If the molecule is too large to pass intact through the intestinal wall, bioavailability would be restricted to the intestinal wall cells with which it is in direct contact. As a consequence, the systemic availability would be low in terms of pen - etrating as such into the blood and joints and possibly limiting for the clin - ical efficacy of CS.

Therefore, several studies have dealt with the absorption and metabolic fate of CS, following oral administration in animals and humans using different test methods (Table 6).

These results confirm that CS with high molecular mass can be absorbed orally. The differences in the absorption and bioavailability of the various CS formulations are strongly influenced by the structure and characteristics, such as molecular mass, charge density and clusters of disulfated disaccharides, of the parental molecules.

24 THE EVOLUTION OF CHONDROITIN SULFATE

Model Drug Methodology Effect Reference

Rat Partially Radioactivity A tropism of the radioactivity towards [92] depolymerized GAGs-rich tissues is observed. The presence CS Bovine of CS greater than 4000 Da in plasma, synovia and cartilage after oral and intramuscular administrations may explain its chondroprotective effect Human CS Bovine Uronic acid The absolute bioavailability following [93] determination oral administration is 13.2%. A peak after CS extraction of oligo- and polysaccharides with a molecular weight lower than 5000 Da derived from partial digestion of exogenous CS is also present in plasma Human, CS Bovine Radioactivity After 24 h, radioactivity was higher in [94] Rat and the rat intestine, liver, kidneys, synovial Dog fluid and cartilage than in other tissues. Oral CS administration determined a significant increase of plasma concentration as compared with predose value over a full 24 h period Human CS Bovine CS disaccharides Orally administered CS is absorbed as [95] quantification a high molecular mass polysaccharide together with derivatives resulting from a partial depolymerization and/or desulfation Human CS from shark CS disaccharides CS structure and characteristics, such as [96] quantification molecular mass, charge density and cluster of disulfated disaccharides, strongly influence oral absorption and bioavailability Horse Low molecular CS disaccharides Low molecular weight CS is orally [97] weight CS quantification absorbed Different CS Bovine Radioactivity A small amount of CS may cross the [98] parts of upper intestine intact but in the distal the gastro- GI tract the molecule is effectively intestinal degraded, presumably by the enzymes (GI) tract in the intestinal flora, and adsorbed as in vitro monosaccharides, disaccharides and oligosaccharides

Table 6. Oral absorption of chondroitin sulfate.

25 THE EVOLUTION OF CHONDROITIN SULFATE

1.4.2.2 Bioavailability, distribution and target tissue orientation of chondroitin sulfate (animal and human studies) A suitable systemic bioavailability and even more a preferred distribution to the target site/tissues are very important factors for a sufficient clinical efficacy of CS. Several studies have demonstrated that the substance reaches the joint tissues in appropriate time and concentrations [94] (Figure 9).

Synovial fluid Blood Adipose tissue Joint: cartilage Trachea e

u Eye s s i Muscle T Brain Lung Kidney Liver Small intestine 0 500 1000 1500 2000 dpm x 100/g of tissue

Figure 9. Distribution of radioactivity in rat tissues after oral administration of 3H-CS [94] .

1.4.3 Mode of action of chondroitin sulfate in in vitro and in vivo models

1.4.3.1 Anti-inflammatory effect of chondroitin sulfate Some pharmacological activities of CS in rats compared with the anti-inflam - matory properties of ibuprofen and indomethacin have been reported by Ronca et al. [99] . The results present evidence that CS administered per os has an anti-inflammatory activity comparable to that of ibuprofen and in - domethacin.

26 THE EVOLUTION OF CHONDROITIN SULFATE

CS CS IL-1R Erk1/2 CS + H2O2/O 2 p38MAPK S536 p50 p65 S276 p50 p65 mRNA

↓ MMP-3 Proteolytic ↓ MMP-9 enzymes ↓ MMP-13 ↓ Catepsine B

Proinflammatory ↓ PLA2, COX-2 enzymes ↓ NOS-2 Proinflammatory ↓ TNF- α cytokines ↓ IL-1 β

Figure 10. CS decreases the synthesis of proteolytic and proinflammatory enzymes and of proinflammatory cytokines.

CS was found to decrease significantly the granuloma formation and the cell mi - gration and lysosomal enzyme release in the carrageenan pleurisy. Compared with nonsteroidal anti-inflammatory drugs (indomethacin, ibuprofen), CS appears to be more effective on cellular mechanisms of inflammation than on edema formation itself. These results confirmed earlier data reported by Conte et al. [100] .

To get more information on the mechanisms of the anti-inflammatory effect of CS, the effects of partially depolymerised (3 to 15 kD) and desulfated fragments of the drug on human leukocytes were also investigated by Ronca et al. [99] . CS and its fractions, which inhibit the directional chemotaxis induced by zymosan-activated serum, are able to decrease the phagocytosis and the release of lysozyme induced by zymosan and to protect the plasma membrane from oxygen reactive species.

As previously reported, articular damage and synovitis are secondary to local in - crease of pro-inflammatory cytokines, enzymes with proteolytic activity and en - zymes with pro-inflammatory activity (Figure 10).

27 THE EVOLUTION OF CHONDROITIN SULFATE

Enhanced expression of these proteins in chondrocytes and in synovial mem - brane appears associated to the activation and nuclear translocation of nuclear factor-kB (NF-kB) [101] . CS reduces NF-kB nuclear translocation inducing an anti- inflammatory effect at the chondral and synovial levels.

These mechanisms are important for severity and time course of inflamma - tory reactions giving an insight into the pharmacodynamic mechanisms of the anti-inflammatory and chondroprotective actions of CS, which are the basis for the clinical efficacy of this drug demonstrated in a number of clinical trials in patients with OA (see below).

Finally, it is noteworthy that CS produces no adverse effects on the stomach, platelets and kidneys which is advantageous compared to the usual nons - teroidal anti-inflammatory drugs.

1.4.3.2 Other effects of chondroitin sulfate CS is an inhibitor of extracellular proteases involved in the metabolism of connective tissues and, in addition to its anti-inflammatory effect, CS stimu - lates in vitro PG production by chondrocytes, inhibits cartilage cytokine pro - duction and induces apoptosis of articular chondrocytes. CS also increases the intrinsic viscosity of the synovial liquid and, in bones, it accelerates the mineralization process and repair (Figure 11) [102] . All these data suggest that CS play a role in articular and bone metabolism by controlling carti - laginous matrix integrity and bone mineralization.

Moreover, due to its capacities, there is preliminary evidence showing that in human beings, CS may be of benefit in other diseases where inflamma - tion is an essential marker, such as psoriasis and atherosclerosis. Further - more, the review of the literature suggests that CS might also be of interest for the treatment of other diseases with an inflammatory and/or autoim - mune character, such as inflammatory bowel disease, degenerative dis - eases of the central nervous system and stroke, multiple sclerosis and other autoimmune diseases (Figure 12) [101] .

28 THE EVOLUTION OF CHONDROITIN SULFATE

Inhibitory effects on Anti-inflammatory extracellular proteases effects Decrease in clinical functional symptoms Increase in synovial fluid viscosity: in experimental arthritis viscous barrier formation on the matrix

Pharmacological properties of chondroitin

CHONDROCYTES BONES

Stimulation of the production Increase in the calcium pool of PGs Promotion of in vitro mineralization Reduction of apoptosis Increase in the rate of bone repair Blockade of the TNF- α receptor

Figure 11. Effects of CS on the osteoarticular system.

Alzheimer, disease Amyotrophic lateral sclerosis Atherosclerosis ↓TNF- α Crohn’s disease

↓NF- κΒ ↓IL-1 β Diabetes type I Multiple sclerosis Chondroitin sulfate ↓COX-2 Parkinson’s disease Psoriasis ↓PLA2 Rheumatoid arthritis Systemic lupus erythematosus Ulcerative colitis

Figure 12. CS may be of benefit for multiple autoimmune diseases.

29 THE EVOLUTION OF CHONDROITIN SULFATE

1.4.4 Efficacy of chondroitin sulfate

Pre-clinical and clinical studies have been performed to establish CS dose regimen, safety and efficacy.

Animal models of OA and RA are useful tools for studying joint pathogenic processes. Adjuvant arthritis is one of the most widely used models. Rat adjuvant arthritis is an experimental model of polyarthritis that has been widely used to test numerous anti-arthritis agents either under preclinical or clinical investigation [103] .

Several meta-analyses have also presented a lot of evidence that CS must be considered as a SYSADOA for the treatment of OA. The evalua - tion of clinical trials which were conducted with a sufficient quality have demonstrated that the efficacy of CS treatment is consistent for pain and functional outcomes and have shown in general moderate to large ef - fects for the therapy of OA symptoms. Results concerning the disease- modifying potential of CS (DMOAD) are available but relatively sparse and need further confirmation. Convincing results were obtained from studies using prescription medicines of appropriate quality.

1.4.4.1 Pre-clinical efficacy in animal models While no animal models mimic perfectly the condition of human dis - eases, induced arthritis models are easily reproducible, well defined and have proven to be useful for the development of new therapies for arthri - tis, as exemplified by cytokine blockade therapies. Animal models have been extensively used for pharmaceutical testing, which means that a large amount of data is available for comparison with humans [103] . Be - cause of these factors, the adjuvant arthritis animal model along with col - lagen induced arthritis have been used in several studies to evaluate CS efficacy (Table 7).

30 THE EVOLUTION OF CHONDROITIN SULFATE

Animal model Biological effects Reference

Adjuvant arthritis Inhibition of induced arthritis [104] Chymopapain-induced Significantly higher cartilage proteoglycan [105] cartilage degradation content in CS treated animals Protective effect on the damaged cartilage Type II collagen Reduction of arthritis index and serum [106] induced arthritis anti-collagen II antibody titer in a dose-dependent manner Significant inhibition of hind paw edema, synovitis and destruction of the articular cartilage Type II collagen Significant reduction of hind paw edema, [107] induced arthritis anti-type II collagen antibody and TNF- α Atherosclerosis aggravated CS administration reduced CRP and IL-6 [108] by chronic-antigen-induced CS administration reduced the nuclear arthritis translocation of NF-kB Adjuvant arthritis CS reduced the severity of arthritis and [109] oxidative stress CS improved total antioxidant status and GGT activity CS reduced the production of pro-inflammatory cytokines, CRP, phagocytic activity and the intracellular oxidative burst of neutrophils

Table 7. Animal models and related biological effects of orally administered chondroitin sulfate.

1.4.4.2 Establishment of dose regimen The dose-dependency of the clinical efficacy of CS has been evaluated by a prospective, randomized, double-blind short-term study [110] with the dosages 200 mg, 800 mg, 1200 mg and placebo over a three-month treat - ment period in patients with femoro-tibial OA. This study demonstrated the significant superiority of the two higher doses of CS, namely 800 and 1200 mg, as compared to placebo treatment and to the lower dose of CS. This

31 THE EVOLUTION OF CHONDROITIN SULFATE

difference became evident starting from day 42 and was maintained until the end of the study (day 90). No difference was found between CS 800 mg and CS 1200 mg.

Bourgeois et al. [111] compared the efficacy and tolerability of an oral treat - ment with CS 1200 mg/day, a treatment with CS capsules at a dose of 3 x 400 mg/day versus placebo in patients with mono or bilateral knee OA. As result, the single dose 1200 mg/day did not differ from the results of 3 x 400 mg/day for all clinical parameters taken into consideration.

1.4.4.3. Clinical efficacy and safety of chondroitin sulfate in humans Extensive literature about clinical trials is available on the capacity of CS to act as a SYSADOA and DMOAD in humans affected by OA (Figure 13).

Human clinical trials

21 CS trials 19 CS trials in alone combination 1996-2013 1999-2016

Figure 13. Human clinical trials available on CS.

All obtained results have been analyzed in ten meta-analysis studies (Table 8) [112] .

32 THE EVOLUTION OF CHONDROITIN SULFATE

Year Study Agent Size Results

2000 The efficacy of CS CS 7 trials of 372 patients CS may be useful in the treatment of taking CS were enrolled in OA as a SYSADOA. OA was evaluated in the meta-analysis Further investigations in larger cohorts of patients are needed 2003 To assess the structural CS and An exhaustive systematic This study and symptomatic efficacy Glucosamine (GlcN) research of randomized, demonstrates the of oral CS/GlcN in placebo-controlled clinical indistinguishable knee OA trials published between symptomatic 1980 and 2002. 1775 efficacies for both patients were analyzed in compounds 15 selected studies 2007 To determine the effects CS 20 trials, 3846 patients, Heterogeneity of CS on pain in patients contributed to this among the trials with OA. meta-analysis made initial interpretation of results difficult 2007 To determine the short-term CS (and other 6 In total, 14060 patients CS had maximum pain-relieving effects by pharmacological in 63 trials were evaluated mean efficacies at performing a systematic agents for OA 1-4 weeks review of randomized pain) placebo-controlled trials 2008 A meta-analysis to assess CS A MEDLINE search was CS is effective in the efficacy of CS as an conducted from 1996 patients with OA of S/DMOAD agent for through 2007. There was the knee. CS may knee OA no evidence of heterogeneity have a role as an across the trials S/DMOAD agent in the management of patients with knee OA 2008 A MEDLINE database search CS Five meta-analyses that limited CS has a slight to was conducted for appropriate their analysis to randomized moderate efficacy in meta-analyses published controlled trials comparing CS the symptomatic between 1997 and 2007 with placebo treatment of OA, with an excellent safety profile 2010 The effect of GlcN, CS or their CS and GlcN Direct comparisons within GlcN, CS and their combination on joint pain and trials were combined with combination do not on radiological progression of indirect evidence from other reduce joint pain or have disease in OA of the hip or knee trials by using a Bayesian an impact on narrowing of model that allowed the joint space synthesis of multiple time points (continued)

Table 8. Meta-analysis studies available on CS [112] .

33 THE EVOLUTION OF CHONDROITIN SULFATE

Year Study Agent Size Results

2010 This analysis updated a published CS A meta-analysis of randomized CS is effective in reducing meta-analysis of double-blind controlled trials published in the rate of decline in placebo-controlled randomized clinical peer-reviewed literature and limited minimum joint space trials to assess the efficacy of CS as to randomized clinical trials of width in patients with an S/DMOAD for knee OA 2-year duration. Data were pooled knee OA with no evidence of important heterogeneity 2010 This study assessed the structural CS and GlcN Randomized controlled studies on This meta-analysis shows efficacies of daily GlcN and CS in the effects of long-term daily GlcN that GlcN and CS may patients with knee OA and CS n knee OA patients. Six delay radiological studies involving 1502 cases progression of OA of the were included knee after daily administration for over 2 or 3 years 2012 A meta-analysis to assess the efficacy CS A MEDLINE search was conducted CS is effective on of CS as a SYSADOA in OA of the up to. There was no evidence of symptoms in patients with knee was performed heterogeneity across the trials OA of the knee compared to the placebo

Table 8. Meta-analysis studies available on CS [112] .

The first meta-analysis study was performed in 2000 [113] . Several other meta-analysis of clinical trials have been published since that first report (Table 8). CS, alone or in combination with other agents, was found able to reduce joint pain and to slow down the rate of reduction of joint space in patients suffering of OA. Contrary to these positive reports, data provided by two meta-analysis studies showed a minimal or nonexistent benefit from CS administration [114,115] . However, many criticisms have been addressed to these studies, such as the huge heterogeneity between trials [116] consid - ering that treatment is known to vary in relation to disease severity, as con - firmed by Sawitzke et al. [117] and the high variability in the origin, potency and purity of CS samples used in the evaluated studies [8,9] . Meta-analysis based on no evidence of heterogeneity across the trials showed positive effects of CS as a SYSADOA and S/DMOAD agent (Table 8).

The results of the all quoted clinical studies conducted on a huge number of patients and published in literature under the category of long-term stud -

34 THE EVOLUTION OF CHONDROITIN SULFATE ies (6 to 40 months) confirmed a total absence of toxicity of CS orally ad - ministered at doses of 1 to 2 g/day. Further clinical experiences confirmed the absence of major adverse reactions. Only minor adverse reactions have been reported in the course of these studies generally occurring at the be - ginning of the treatment and generally involving the gastrointestinal tract. A few cases of cutaneous rash were reported but their relation to the treat - ment could not be clearly demonstrated.

35 SAFETY CONCERNS OF ANIMAL -D ERIVED CHONDROITIN SULFATE

36 2.1 ANIMAL CHONDROITIN SULFATE SAFETY CONCERNS

CS, like other natural polysaccharides, is derived from animal sources by ex - traction and purification processes [86] . As a consequence, source material, manufacturing processes, the presence of contaminants and many other fac - tors contribute to the quality, structure and physico-chemical properties of the final product and of the overall biological and pharmacological activities of these agents. As above described, CS has a complex structure that is known to change with the tissue, organ and species [86] . Furthermore, as well known, the biological and pharmacological properties may vary with the struc - ture and the oral absorption may be influenced by the different physico- chemical properties [95,96] . Finally, due to the extractive origin of CS, the presence of virus or prions cannot be excluded [118] , as well as other various natural bioactive (macro)molecules present as contaminants in CS extracts in different amount [8,119] , or voluntary adulteration by similar artificial com - pounds [120] , or a restriction use related to religious issues (Figure 14).

ANIMAL DERIVED CS EXTRACTED IS HETEROGENEOUS AND LARGELY CONTAMINATED

HETEROGENEITY CONTAMINATION

• The animal source used (possible cross-contamination between Natural contamination sources of different origin) Natural polysaccharides

Virus or prions (or infective agents) • The age of the animal Proteins • The manufacturing processes Natural polymers

Variety of extraction and purification techniques can be Adulteration used resulting in different content & composition

Figure 14. Factors responsible for the heterogeneity of structure, properties and purity of animal-derived CS. 37 THE EVOLUTION OF CHONDROITIN SULFATE

Animal-derived CS possess high variability and have different physical- chemical profile according to the various sources [8,86,119] . Moreover, a mix of different animal tissues and sources are possible during extractive pro - cedures producing a final product having varied characteristics and not well identified profile, influencing oral absorption and activity [8,119] .

Quality of different CS preparations are also dependent by extraction and purification processes which may introduce further modifications of the structural characteristics and properties, such as depolymerization, desul - fation and/or chemical modifications. The use of not controlled raw animal material (tissues, bones and cartilages but also soft organs) poses the prob - lem of a final product having a highly variable structure with not well iden - tified origin and poor reproducibility with the consequence of variable grade of purity, biological effects, presence of contaminants, clinical efficacy and safety [8,9,119] .

These aspects pose a serious problem for the final consumer of the phar - maceutical or nutraceutical products that is related to the declaration on the label of the real origin of the active ingredient and to the label traceability of CS.

38 2.2 HETEROGENEITY OF CS IN ANIMAL TISSUES

The industrial production of CS uses animal tissue sources as raw material, derived from different species of animals. Currently it generally relies on bovine, porcine, chicken or cartilaginous fish such as sharks and skate by- products, in particular cartilage, as raw material [8,119] . More important, a mix of all these sources is possible producing a CS final product having mixed characteristics and not well identified activities.

Although all animal CS have an identical backbone, several studies indicate that the chemical composition (sulfation, overall charge density, nature of PGs) and quality of CS changes with age of the animal, tissue, organ and it is specific for each species (see Tables 4 and 9 for example).

Moreover, as a result of the biosynthetic processes related to specific tissues and species, CS with different grades of polymerization may be biosynthe - sized producing macromolecules having various molecular weight, type and grade of sulfation, and polydispersity (Table 9).

Animal extractive CS has been shown to present a high content of not well

Parameters CS Bovine CS Porcine CS Chicken CS Shark CS Raja

Molecular mass (kDa) Mn 10,0÷15,0 9,0÷13,0 7,8÷12,8 20,5÷26,0 27,0÷34,0 Mw 20,0÷26,0 13,7÷18,7 15,6÷20,6 64,2÷70,2 50,0÷60,0 Dispersity 1.80÷2.20 1.35÷1.65 1.60÷2.00 2.50÷3.10 1.50÷2.50 Disaccharides ΔDi-0s 6.0 6.0 8.0 3.0 3.0 ΔDi-6s 33.0 14.0 20.0 50.0 39 ΔDi-4s 61.0 80.0 72.0 29.0 43 ΔDi-2,6dis Trace nd nd 15.0 13.0 ΔDi-4,6dis Trace nd nd 2.0 1.0 ΔDi-2,4dis Trace nd nd 1.0 1.0 Charge Density 0.90÷0.96 0.92÷0.96 0.90÷0.94 1.10÷1.20 1.08÷1.16 4s/6s ratio 1.50÷2.00 4.50÷7.00 3.00÷4.00 0.45÷0.70 1.00÷1.40 Table 9. General chemical composition and properties of CS from various common sources.

39 THE EVOLUTION OF CHONDROITIN SULFATE

identified proteins, up to 3-6% [8,119,121,122] as well as nucleic acids and their fragments derived from chemical treatment reaching 1-2% [8,119,121,122] and other GAGs extracted during its manufacture, in particular DS and hyaluronic acid [123] and a high percentage of immunogenic keratan sulfate [124-127] (Figure 15).

Due to these structural variations and in addition to the possible presence of specific oligosaccharide sequences as well as the purity of the preparations required for therapy applications or in nutraceuticals, CS may have different properties and effects [8,119] . In fact, different and peculiar activities have been reported depending on the CS structure [for an exhaustive report on CS prop - erties see reference 86 ]. Furthermore, CS is generally orally administered dur - ing the therapy as a nutraceutical supplement and different bioavailability and pharmacokinetic parameters have been reported to change depending on its structural characteristics and origin [95-97] .

Natural Virus or prions Proteins Natural polymers polysaccharides

Highly specific NATURAL analytical approaches CONTAMINATION are necessary

Reduced CS title Possible adverse events

Figure 15. Factors responsible for the heterogeneity of structure, properties and purity of animal-derived CS.

40 THE EVOLUTION OF CHONDROITIN SULFATE

Finally, the use of animal-derived source for producing commercial CS rep - resents a concern also for vegetarians and people with dietary restrictions related to religious and supply issues. Religious reasons and/or the practice of abstaining from the use of animal products have precluded the intro - duction of CS dietary supplements both in key emerging markets (Middle East and Asia) and in consolidated markets (North America and Europe).

2.2.1 NATURAL CONTAMINATIONS

Commercial CS, derived from animal sources, is obtained with long and com - plex procedures of extraction and purification with the aim to attempt elimi - nation of the other (macro)molecules present as natural contaminants [86] . At the same time, contamination of CS may derive from the same processes of extraction and purification.

2.2.1.1 Natural polysaccharides Depending on the purification protocols, CS extracts may have a variable grade of purity due to the presence of not desired side-products [119] .

Contamination by other polysaccharides such as other GAGs is possible along with the presence of nucleic acids, proteins and other (macro)mole - cules. Moreover, not controlled preparative processes may introduce im - portant structural modifications on the CS backbone such as desulfatation, depolymerization, oxidation/reduction and/or introduction of chemical modifications producing a polysaccharide having poor or no biological prop - erties [8,119,125,126] .

In particular, DS with the presence of its building blocks made of iduronic acid and hyaluronic acid in various percentages in raw materials and formu - lations have been detected and declared in scientific papers [123] .

Moreover, keratan sulfate was detected in batches from shark cartilage, averaging 16% of the total GAGs [124,125] . Its unexpected high percentage

41 THE EVOLUTION OF CHONDROITIN SULFATE

compromises the desired amounts of the real ingredient specified on the label claims and forewarns the pharmacopeias to update their mono - graphs.

In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Indeed, keratan sulfate, involved in the structure of cartilage proteoglycan aggrecan, was detected in CS extracts and finished preparations of various origin, ob - viously as CS is extracted from cartilage [126] . A total of 15 samples, including four samples of CS as laboratory reagents, one sample of CS as a food ad - ditive and ten samples of dietary supplements containing CS were exam - ined to detect keratan sulfate in these samples by using immunodiffusion and enzyme-linked immunosorbent assay (ELISA) with anti-keratan sulfate monoclonal antibody. With the exception of three samples of CS as labora - tory reagents, all samples were found to contain varying amounts of keratan sulfate. Finally, keratan sulfate possesses immunogenic capacities able to develop immune reactions [127] .

2.2.1.2 Virus or prions (or infective agents) The animal origin poses potential consumer safety problems associated with the possible presence of pathogen contamination and transmissible infective agents (bacteria residues, viruses and prions) such as those caus - ing spongiform encephalopathy in bovines (BSE), foot-and-mouth disease, influenza spread in birds and other animal diseases [118] (Figure 16).

The presence of prions may be easily demonstrated with a diagnostic test and current available testing is very limited and expensive. Prions adhere very tenaciously to surfaces making them hard to remove and can be re- deposited on other surfaces during processing. Prions are also resistant to protease treatment, certain chemical agents and heat denaturation and represent a major concern for pharmaceutical products derived from ani - mals. Other pathogens are also a concern because of similar attributes as Prions for the potential risk of carry-over into a final purified product.

42 THE EVOLUTION OF CHONDROITIN SULFATE

Creutzfeld-Jacob disease «mad cow disease» Hog cholera Foot-and-mouth disease influenza spread in birds

Application of specific chemical Introduction of specific and steps to destroy and/or selective (and expensive inactivate viruses and/or prions and limited) analytical tests possibly causing chemical to detect the presence of modifications of CS structure viruses/prionic proteins and capacities

Figure 16. Pathogen contamination and transmissible infective agents in animal-derived CS and their effects.

2.2.1.3 Natural polymers: proteins and nucleic acids Animal CS extracts and derived final products for pharmaceutical and/or nutraceutical markets are also contaminated with (macro)molecules pos - sessing similar properties of CS that are co-purified during CS extraction from tissues, such as in particular nucleic acids [123] and proteins. In fact, an - imal CS is contaminated of a variable content of DNA and RNA depending on the purification protocols adopted and on the source [8,119] . Moreover, some of these proteins may have allergenic potential able to develop im - mune reactions (Figure 17).

Finally, we should consider that CS, like other natural sulfated GAGs, is co - valently linked to a core protein to form the PGs. As a consequence, during the extractive procedures involving a proteolytic treatment, polypeptide se - quences still remain linked to the CS backbone in a variable content [86] .

43 THE EVOLUTION OF CHONDROITIN SULFATE

Proteins (Partially degraded) Nucleic acids

Always present in CS preparations in a range Always present in CS of 3-6% w/w preparations due to similar physico-chemical properties

Possible allergenic response able to develop immune reactions: allergenic and/or Positive to CPC titration assay intolerance reactions

Figure 17. Presence of other (macro) molecules in animal-derived CS and their effects.

44 2.3 ADULTERATIONS

Contamination of animal-derived CS may also derive from the voluntary adulter - ation with cheaper polysaccharides and substances that cannot be determined with usual analytical controls utilized in the nutraceutical industry (Figure 18).

Chondroitin is now acknowledged within the nutraceuticals industry as one of the most adulterated supplements in the market, a problem which is wide - spread, deteriorating and on-going. Furthermore, the extent of adulteration is probably underestimated, with much of the adulteration going undetected, encouraged and abetted by the lack of industry-wide validation methods.

The recent application of specific analytical methods such as HPLC and elec - trophoresis on commercially available supplements has allowed to demon - strate not only the poor quality of these finished products in the global market, with a lower concentration of CS than specified in the label, but also the intentional adulteration of them [128-131] .

High content of salts Artificial polymers

Highly specific Not well identified INTENTIONAL analytical approaches ADULTERATION are necessary

Reduced CS title: fraud to clients, traders and consumers. Possible adverse events

Figure 18. Artificially (“FAKE”) adulterated animal-derived CS.

45 THE EVOLUTION OF CHONDROITIN SULFATE

ARTIFICIAL POLYMERS • Carrageenan • Surfactants • Cheaper polysaccharides: glucomannan • Sodium alginate • Propylene glycol alginate sulfate sodium Sodium hexametaphosphate • Alginic sodium diester

Not well identified other adulterans?

Figure 19. Adulteration of animal-derived CS.

The adulteration is pursued with molecules which may influence non-spe - cific wide-used methods of CS quantitation, such as the Cetylpyridinium Chloride (CPC) titration, to give an artificially inflated estimate of CS con - centration [132] .

The materials often used for adulteration may be carrageenan, proteins and surfactants, cheaper polysaccharides (Figure 19). Of the known chondroitin adulterants identified to date, including sodium alginate, propylene glycol al - ginate sulfate sodium, there is also the sodium hexametaphosphate, com - monly used as a detergent or as a water-treatment additive and sold under the commercial name Calgon [132] . These molecules can be separated from CS by their difference in electrophoretic mobility or other specific analytical approaches, but not with common largely used methods [8,119,132] (Figure 20).

One industry trend which drives adulteration is price pressure. The majority (80-90%) of the CS supplied to the US and Europe markets originates from China and intentional adulteration persists due to pricing pressure. This pushes the nutraceutical manufacturer to minimize ingredient costs by using substandard sourcing practices [133] . This aspect related to price pressure is also related to minimize analytical controls just to common non-specific and inexpensive tests (Figure 20). As a consequence of this scenario, some raw

46 THE EVOLUTION OF CHONDROITIN SULFATE

HIGHLY SPECIFIC ANALYTICAL APPROACHES ARE NECESSARY

Gel Electrophoresis according to EU Ph Acetate Cellulose Electrophoresis according to USA Ph Enzymatic Treatment & HPLC/CE HPSEC: Size Exclusion Chromatography

VERY EXPENSIVE Specifically equipped Labs Expensive ACTIVITY Equipments Specific Know-how HIGH COSTS FOR Highly qualified Laboratory technicians CS MANAGEMENT

Figure 20. Analytical procedures and related implications to check the adulterated ani - mal-derived CS. materials and dietary supplement products contain less than the claimed amount of CS and, in some cases, only 5-10%, which is a huge fraud to clients, traders and consumers.

47 2.4 UNCONTROLLED SUPPLY CHAIN AND RISKS

While pharmaceutical supply-chain requires very strict traceability to mini - mize (rather than eliminate) risks of contamination, animal-derived raw materials for nutraceutical market currently derives from not-well identified origin. The source of animals and their geographical origin, the nature of animal material used in manufacture and any procedures in place to avoid cross-contamination with higher risk materials, production process(es) in - cluding the quality assurance system in place to ensure product consis - tency and traceability, are not regulated and often not controlled. Moreover, seasonal, geographical and subspecies variations may alter the product ob - tained from a given animal species. Finally, some countries do not have a strict biosecurity system described as “implementation of practices that cre - ate barriers in order to reduce the risk of the introduction and spread of disease agents”.

Animal-derived CS suppliers have no regulatory requirements to provide materials with batch-to-batch reproducibility and free of contaminants. There are no rules to be transparent about their own processes and supply chains and to provide accurate information. Moreover, the missing guide - lines allow manufacturers of finished products to change often the sources of supply of the CS raw materials for competitive advantages.

Manufacturers should ensure traceability of raw material across the supply chain until the finished product, with in-house analytical evaluation made of each lot supplied in order to guarantee same quality, reproducibility and safety, and avoid also misleading label information on finished products, as already described. The cost of such analytical controls and quality pro - cedures would be so high (see Figure 20) that the dietary supplement manufacturers would not be competitive in the market price with loss of market competitiveness and, for this reason, not generally applied.

On the contrary, manufacturers often “qualify” brokers and suppliers by Self-Audit questionnaires and verification of Certificate of Analysis without testing raw materials or by using less expensive, but not consistent, analyt - ical methods, with serious ineffectiveness to assess critical suppliers.

48 2.5 ANALYTICAL CONTROLS

The industry-wide methods for analysis of CS are titration methods using CPC titration from United States Pharmacopeia (USP). Recent testing on CS dietary supplements has revealed several flaws in this older testing methods. The titration method has relevant limits because can be tricked by the use of expensive natural and/or artificial adulter - ants and cannot distinguish between CS and other material having sim - ilar physico-chemical properties, like nucleic acids, anionic proteins, other polysaccharides, etc.

Moreover, the origin of the CS utilized in a finished product can only be detected by rather expensive and detailed biochemical/structural analyses by determining the disaccharide/oligosaccharide pattern and molecular mass parameters [119] . As a consequence, today it is recog - nized the need to use a combination of specific analytical methods to define the final quality, origin and properties of the product. Such meth - ods include chemical composition analysis, electrophoresis, HPSEC, im - munochemical reactions, selective enzymatic digestion that further requires resolution using sophisticated techniques including HPLC, polyacrylamide-gel electrophoresis (PAGE), FACE, capillary electrophore - sis, mass spectroscopy, and NMR spectroscopy [112,119] . CS profile is readily achieved using well-established laboratory techniques, particu - larly HPLC and electrophoresis. These techniques, applied after enzy - matic digestion of CS into smaller components, can distinguish between CS derived from different animal or marine sources based on disaccharide content, patterns of sulfation and molecular size of the polymer. Anyway, a big problem still remains for the complete identifi - cation of the origin of CS when mixed sources material is used to its production or cross-contamination is possible.

The previous reported information has been confirmed in many studies in which CS finished products have been found of poor quality and not conformed to label declaration. In fact, previous studies reported a quantitative determination of CS in raw materials and various pharma - ceutical preparations, and its content and properties in shark cartilage

49 THE EVOLUTION OF CHONDROITIN SULFATE

powders used as nutraceutical supplements. Several low-quality CS raw materials were identified and the purity and origin of the preparations were found to be inconsistent with the specifications claimed on the product labels in several Countries [128-133] .

50 2.6 CONCLUSIONS

The case study of heparin happened in February 2008 is well known. The China-derived heparin was contaminated by oversulfated CS having a very high charge density quite similar to natural heparin, derived from an un - specific over-sulfation, which provoked hundreds of deaths in Europe and USA when administered iv [134] . This terrible event related to intentional adul - teration of an important natural drug derived from animals induced a strong increase in research for the production of a possible bioengineered he - parin [135] . As for heparin, intentional adulteration of CS is difficult to detect since the tissue supply chain for CS in slaughterhouses generally lacks cur - rent good manufacturing processes (cGMP) oversight.

Furthermore, CS is derived from a variety of animal tissues and these ani - mal source materials can contain infectious agents leading to the potential transmission of viral and prion diseases. This is more important by consid - ering that the mixing of CS obtained from different animal species is highly suspected. Even if specific and high-level analytical methods are generally able to distinguish the various sources, the detection of the presence of small amounts of different raw material source still remains very difficult and a sure challenge.

Moreover, the susceptibility of animal populations to infectious disease, such as porcine epidemics in China, overharvesting, or environmental con - cerns can dramatically reduce the supply animals for CS production. Addi - tionally, product quality can vary with environmental factors and animal subspecies, providing additional difficulties for CS quality and properties standardization.

Finally, commercial suppliers must answer to the increased worldwide de - mand particularly in developing countries. As a consequence, the urgent solution to this scenario is the introduction and utilization in the current market of the non-animal derived source CS Mythocondro ®.

51 MYTHOCONDRO ®: THE CHONDROITIN SULFATE OF NON -A NIMAL ORIGIN

52 3.1 STATE OF THE ART

The potential consumer safety and quality problems previously discussed as - sociated with the use of animal-derived CS, suitable for pharmaceutical and nutraceutical applications, have historically prompted the search for an alter - native source.

An innovative patented production process has been developed in the last years by Gnosis, an Italian-based biotechnology company, leading to an in - novative product closely resembling natural CS, with physico-chemical, struc - tural and functional properties comparable to those of animal-derived products but without any concern related to origin of the production, the pos - sible presence of transmissible infective agents, the contamination and adul - terations typical of animal-derived raw materials.

The new ingredient, branded as Mythocondro ®, is the first CS obtained with a fermentation-based manufacturing process followed by selective sulfation. Mythocondro ® warrants high purity, clear identity profile, batch-to-batch re - producibility, established safety with an extremely low content of proteins and other (macro)molecules, as well as a superior biological activity.

Mythocondro ® is the result of several years of research and development and has generated five international patents based upon scientific advances in the biotechnological areas.

Mythocondro ® has been extensively studied in order to evaluate its charac - teristics, safety profile and efficacy. Pre-clinical and clinical studies have been carried out demonstrating superior biological activity versus animal-derived CS both in vitro and in vivo .

53 3.2 THE EVOLUTION OF THE FERMENTATION-DERIVED PRODUCTION PROCESS

Specific bacterial strains produce capsular polysaccharides showing a strong homology with chondroitin. The study of the DNA region coding for the en - zymes responsible for the biosynthesis of the polysaccharide has been deeply detailed, leading to the selection of non-GMO E. coli strains pro - ducing high levels of the polysaccharide and low levels of impurities. The selected bacterial strain produces a surface capsular polysaccharide named K4, that is identical to unsulfated chondroitin except for carrying fructose residues in the C3 position of the glucuronic acid residues.

The development of selective growing conditions of this bacterial strains able to result in high levels of polysaccharide was a big challenge for Gno - sis, by also considering the goal to use a non-animal origin medium free from the potential variations in the quality of natural medium ingredients.

After the fermentation, the polysaccharide produced in high yields is puri - fied, and hydrolyzed under specific conditions to remove the fructose residues. The recovered chondroitin-like polysaccharide undergoes regios - elective sulfation, modulated in order to obtain a CS (Figure 21) which is mainly monosulfated thus avoiding the formation of polysulfated disaccha - rides which are common in the majority of chemical sulfation processes conferring limited biological activity and safety concerns.

The result of this innovative approach is a non-animal CS with homoge - neous structure and the presence of sulfate groups in defined positions, a

–-– - –-–- CH2OH COO CH2OH CO O CH2OH OH OH OH O O O O O OR O O O O O –-–- –-–- ––– RO CH2OH CO O CH2OH CO O CH2OH O2C O O O O O HNA c OH HNAc OH HNAc O O O O O OH OH O OH OH O OH O O O O O HOC H O HO CH O HO 2 2 RO AcNH HO HO HNAc OH HNAc OH HNAc 20-2 00 CH OH CH OH 2 2 Ac = acet yl , R = SO --- or H OH OH 3

Polym eric Polysac cha ride Chondro iti n Chondro iti n Sul fate

Figure 21. The production of Mythocondro ®.

54 THE EVOLUTION OF CHONDROITIN SULFATE constant charge density and molecular mass parameters overall largely sim - ilar and comparable to animal-derived samples eliminating the safety con - cerns raised by previously described animal-derived.

The non-extractive origin guarantees:

• Homogeneous structure and physico-chemical properties

• High purity

• Very low content of proteins

• No presence of virus and/or prions

• Highly and point-by-point controlled production process for highly controlled and reproducible final product

55 3.3 MYTHOCONDRO ® STRUCTURAL FEATURES AND PHYSICAL-CHEMICAL PROPERTIES

Mythocondro ® has homogeneous structure and physico-chemical properties in particular for the presence of sulfate groups in defined positions, a constant charge density and molecular mass parameters. Moreover, compared to all known animal-derived CS, Mythocondro ® is characterized by an extremely low content of natural contaminants, i.e. proteins, nucleic acids, other poly - saccharides, largely reported to be present in extractive CS strictly due to its chemical nature and purification procedures adopted, as previously largely discussed. As a consequence, Mythocondro ® possesses very high purity level.

Specific and sensitive analytical procedures identify the structure of non-an - imal CS and characterize its properties and purity.

Molecular mass parameters and profile is determined by HPSEC (high-per - formance size-exclusion chromatography) and further by PAGE that con - firmed a medium molecular mass value lower than about 10 kDa, a homogeneous profile and low polydispersity (Figure 22). In fact, the poly - dispersivity in the molecular mass of Mythocondro ®, an important factor for biological actions, is maintained due to the polydisperse nature of the polymer K4 which undergoes hydrolysis and regiosulfation in the manufac - turing process.

HPSEC PAGE

Figure 22. HPSEC and PAGE characterization of Mythocondro ®.

56 THE EVOLUTION OF CHONDROITIN SULFATE

Various kinds of electrophoresis, agarose-gel according to EP (European Phar - macopeia) and acetate of cellulose (ACE) according to USP show a profile with no other possible natural polysaccharide present at LOD (Limit Of De - tection) lower than 0.2% (Figure 23). Moreover, enzymatic HPLC (eHPLC according to AOAC International), 1H-NMR and 13C-NMR defined the struc - tural characteristics of Mythocondro ® (Figure 23).

Currently, USP and EP allow up to 6% and 3% of undefined protein content respectively in their specification while Mythocondro ® has lower than 0.1% of well characterized proteins. Being this parameter possibly linked to allergies onset, Mythocondro ® presents an extremely diminished risk compared to an - imal-derived CS. In fact, depending on the extractive and purification proce - dures adopted to produce CS from animal tissues (and we should keep in mind that more purified is the CS and more purification steps are required and more time-and money-expensive is the final product), up to 6% proteins may be present. On the contrary, Mythocondro ® has been found to contain about <0.1% proteins due to its specific nature and production.

Gel electrophoresis Disaccharide Composition ACE

13C-NMR 1H-NMR

Figure 23. Specific and sensitive analytical procedures characterize the structure of Mythocondro ®.

57 3.4 BIOLOGICAL ACTIVITY OF MYTHOCONDRO ®

3.4.1 Toxicological studies

By using established procedures, E. coli strain was evaluated for any toxi - genic potentials. None of the genes searched for was found to be present in the total DNA extracted from used strain, showing that the strain in ques - tion is genetically unable to produce virulence factors and toxins. Moreover, before removal from the fermenter, the culture is always inactivated by heating.

Furthermore, Mythocondro ® has been positively tested in a validated Safety Assessment Study published on Food and Chemical Toxicology [136] . The ob - jective of this study, performed according to OECD (Organization for Eco - nomic Co-operation and Development) guidelines - in use for pharmaceutical products - was to investigate:

• the long-term repeat dose toxicity (subchronic toxicity study) of the Mythocondro ®, when administered daily for 90-days via oral gavage to Sprague Dawley rats;

• the potential genotoxic effects of Mythocondro ®, investigated in bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strains, in vitro chromosomal aberration assay in CHO and in vitro mutation in mouse lymphoma cells;

• the pharmacokinetics of Mythocondro ®, in a comparative pharmacokinetic single dose, open label, randomized, two-way cross-over study in 24 human subjects with known bovine CS.

The study fully validates the excellent safety profile of the fermentation- based CS Mythocondro ® not revealing any mutagenicity, genotoxicity and with a no-observed-adverse-effect level (NOAEL) of 1000 mg/kg (NOAEL of 1000 mg/kg bw/day). The available evidence suggests that recom - mended daily intake of microbial derived Mythocondro ®, is very safe.

58 THE EVOLUTION OF CHONDROITIN SULFATE

3.4.2 Permeability

One of the major problem associated with oral administration of native ani - mal CS is the low bioavailability. Intestinal animal high molecular weight CS absorption amounts to about 1-5% [95,96] .

In vitro and in vivo clinical studies report that CS is generally hydrolyzed to smaller oligosaccharides by mean of specific enzymes produced by the in - testinal flora [98,137] . By considering the oligosaccharide fraction, the total in - testinal CS uptake amounts to about 20-23% [138] . This documented low bioavailability has limited the CS efficacy as a dietary supplement so far. Mo - lecular weight and the range of polydispersivity may impact on the pharma - cokinetics profile of supplemented CS.

Mythocondro ® shows an improved intestinal epithelial permeability com - pared to animal CS samples due to its lower molecular mass, without the need of further hydrolysis by the intestinal flora.

The variation in molecular masses and sulfation patterns make CS a hetero - geneous molecule. Several studies are available and hypotheses have been made regarding differences in the bioavailability of CS due to its structural variations [92,93,95,96,107,139,140] .

Mechanisms of transportation across the intestinal barrier include transcellular transportation (carrier mediated or active transportation, endocytosis and pinocytosis), paracellular transportation or passive diffusion through the tight junctions. Mode of transportation depends on the physicochemical properties of the compound such as structure, molecular weight or size, charge distribu - tion and hydrophobicity [141] . As identified in the report [141] and recommended by European Center for the Validation of Alternative Methods (ECVAM), ex - amples of in vitro models of intestinal absorption include the human adeno - carcinoma cell line Caco-2. Caco-2 cells are isolated from a primary human colonic tumor. When cultured under standard cell culture conditions, they spontaneously differentiate into polarized enterocytes characterized by apical

59 THE EVOLUTION OF CHONDROITIN SULFATE

and basolateral sides, by the presence of tight junctions and the expression of most of the small intestinal brush border enzymes and transporters.

Mythocondro ® showed a better permeability in the Caco-2 cells model when compared to CS samples of various animal sources, being its permeability coefficient up to 65 times higher (Figure 24).

Figure 24. Coefficients of effective permeability (Peff) for CS samples. Mythocondro ® is significantly higher than the rest of the samples.

3.4.3 Bioavailability

Pharmacokinetic profile of Mythocondro ® versus a sample of bovine extracted CS has been evaluated in healthy volunteers. In this single center, single dose, open-label, randomized, two-way, cross-over study, 24 male and female sub - jects [18-65 years; BMI- 23.67 ± 2.52-] received both test and reference treat - ment and completed the study as per protocol. Single doses of 2400 mg of test or reference product were administered under fasting conditions in two consecutive study periods according to the randomized cross-over design.

The trial and the methodologies for statistical comparison between treatments

60 THE EVOLUTION OF CHONDROITIN SULFATE were designed taking into account the recommendations of the Guideline on the investigation of bioequivalence (CPMP/QWP/EWP/1401/98 Rev. 1, Jan - uary 2010). In pharmacokinetic study in humans, non-animal CS shows higher absorption as compared to CS of animal origin.

Mythocondro ® showed an increased AUC0-24h compared to bovine CS already after 1 h from products administration, and this difference increased up to 24 h. This behavior is evident in Figure 25 where a higher plasmatic concentration of Mythocondro ® is showed and it is evident its capacity to remain higher for a long time in human plasma for more than 24 h if compared to bovine CS.

In particular, 5 h after administration, the plasmatic concentration of Mytho - condro ® remains up to 89% higher than bovine CS (Figure 25). Finally, the quite similar value of C max for both formulations further supports data illus - trated in Figure 25 and demonstrates that the different plasmatic behavior is

Comparative CS bioavailability following a 2400 mg single

dose - AU C0-24h

Figure 25. Plasma CS concentration (mg/mL) vs. time profiles following administration of test and reference formulations to human subjects.

61 THE EVOLUTION OF CHONDROITIN SULFATE

strictly related to the capacity of Mythocondro ® to remain for a longer time in the blood compartment with a higher concentration compared to bovine CS.

The human clinical trial has not only produced data on the safety, but allows to claim a higher bioavailability of Mythocondro ® versus bovine CS of 43%.

3.4.4 Anti-arthritic effect

Adjuvant induced Arthritis (AA) in rats is an animal model of polyarthritis which allows to monitor the disease processes in the acute phase (days 14-21) and in the subchronic phase (day 28) of the disease. The advantages of this model are its many similarities with RA and polyarthritis diseases in man, such as symmetrical joint involvement, persistent joint inflammation, synovial hyper - plasia and a good response to most therapies effective in RA [142] .

AA in rats is widely accepted and considered by the scientific community as a good experimental and preclinical model to test arthritis symptoms and their treatments. AA was induced by a single intradermal injection of Mycobacterium butyricum in incomplete Freund’s adjuvant, a solution of antigen emulsified in mineral oil and used as an immuno-potentiator (booster).

The anti-inflammatory effect of Mythocondro ® (Low-Molecular-Mass CS: LMM-CS) was assessed through the reduction of arthritic score as well as the evaluation of plasmatic levels of pro-inflammatory cytokines IL-1 β and IL-6 as well as by γ-glutamyltransferase (GGT) activity in hind paw joint tissue homogenates and plasmatic C-reactive protein (CRP). Arthritic score is a com - plex index of pathology progression assembling several clinical parameters including periarticular erythema, edema and the local inflammation at the site of injection. The study included healthy animals, untreated arthritic ani - mals and arthritic animals who received 900 mg/kg daily of bovine CS, a high-molecular mass biotechnological CS (HMM-CS) and Mythocondro ® (LMM-CS).

62 THE EVOLUTION OF CHONDROITIN SULFATE

Mythocondro ® significantly reduced the arthritic score by up to about 30% from 14 to 28 days (Figure 26). In contrast, no significant differences were observed for HMM-CS, even if a trend in its capacity to decrease the arthritic score by up to about 11% was observed.

Figure 26. Arthritic score of a pharmaceutical-grade animal CS, a HMM-CS and Mytho - condro ® (LMM-CS) in the adjuvant arthritis animal model.

Additionally, the effect was confirmed by the reduced production of proinflammatory cytokines, GGT and CRP in plasma (Figure 27). Mythocon - dro ® was able to significantly decrease GGT activity by approximately 31% and plasmatic CRP levels by about 9%. Both non-animal CS sam - ples were effective in reducing plasmatic levels of pro-inflammatory cy - tokines. A greater efficacy was also observed for Mythocondro ® compared with the pharmaceutical-grade CS of bovine origin, while the efficacy of the HMM-CS sample was found to be rather similar. The greater effect of Mythocondro ® in reducing arthritic parameters may be related to its lower molecular mass and increased bioavailability with re - spect to HMM-CS and animal CS.

63 THE EVOLUTION OF CHONDROITIN SULFATE

Figure 27. Effect of pharmaceutical grade animal CS, HMM-CS and Mythocondro ® (LMM-CS) on the production of pro-inflammatory cytokines, GGT and CRP in plasma in the adjuvant arthritis animal model.

Mythocondro ® showed to be effective to slow down arthritis development and to reduce disease markers after 28 days of treatment supporting fur - ther clinical phase in humans. This preclinical study was published in 2014 by Bauerova et al. [143] .

3.4.5 Why Mythocondro ® is better than animal chondroitin sulfate?

Some key points illustrated in the following last Table may be considered to compare animal CS, the actual state of art, with Mythocondro ®, opening a new era of CS.

64 THE EVOLUTION OF CHONDROITIN SULFATE

Mythocondro ® Animal CS

Non-Animal CS of Animal CS origin fermentative origin Low molecular weight: 5 - 12 KDa High Molecular weight A controlled, reliable and reproducible Not strictly regulated source of CS with high purity ✖ Not well-identified origin ✖ Highly variable structure ✖ Poor batch-to-batch reproducibility A very low protein content (less than 0.5% High protein level (up to 3% (EP), d.b.). It is usually 0.1% d.b. No presence of and up to 6% (USP) d.b.). other polymers or nucleic acids Not well identified proteins and nucleic acids and their fragments derived from chemical treatment contamination potentially responsible for allergenic reactions in the consumers. Presence of immunogenic keratan sulfate Highly and point-by-point controlled process Process of extraction generally not controlled of production for a highly purified and causing possible modifications of the CS structure, reproducible final product such as desulfatation, depolymerisation, oxidation, etc A substantial absence of unusually sulfated Standard animal CS origin is made up of different and over-sulfated disaccharides. CS (mainly C4S sulfated in position 4 of GalNAc, Mythocondro ® has a very precise sulfation C6S sulfated in position 6 of GalNAc but also pattern variable % of C0S, non-sulfated disaccharide. Variable % of disulfated (and trisulfated) disaccharides Clinically proven superior bioavailability of Lesser bioavailability Mythocondro ® than animal CS due to lower molecular weight and an improved intestinal epithelial permeability Proved anti-inflammatory and protective In general, no evaluation of any biological activity properties in a preclinical arthritis animal model Mythocondro ® is totally safe: no toxicity in The animal origin poses a potential consumer acute and chronic animal models. No prions safety problem associated with the possible or animal viruses can be present due to the presence of transmissible infective agents fermentation origin of the product (bacteria, viruses and prions) Mythocondro ® is suitable for vegetarians Some animal origins are not suitable neither for and all religions as it is of natural origin vegetarians nor for consumers of different religions

65 REFERENCE

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76 GLOSSARY

HPSEC=high-performance size-exclusion AA=adjuvant induced arthritis chromatography ACE=acetate of cellulose IL=interleukins BMI=body mass index LMM-CS=low-molecular-mass-CS BSE=bovine spongiform encephalopathy LOD=limit of detection cGMP=current good manufacturing processes MMPs=metalloproteinases CPC=cetylpyridinium chloride NF-kB=nuclear factor-kB CRP=C-reactive protein NO=nitric oxide CS=chondroitin sulfate NOAEL=no-observed-adverse-effect level CSA=chondroitin-4-sulfate OA=osteoarthritis CSC=chondroitin-6-sulfate OARSI=osteoarthritis research society DMOAD=disease modifying potential of CS international DS=dermatan sulfate OECD=organization for economic co- ECM=extracellular matrix operation and development ECVAM=european center for the validation of PAGE=polyacrylamide-gel electrophoresis alternative methods Peff=effective permeability ELISA=enzyme-linked immunosorbent assay PGs=proteoglycans EP=european pharmacopeia RA=rheumatoid arthritis EULAR=european league against rheumatism S/DMOAD=structure/disease modifying anti- GAG=glycosaminoglycan osteoarthritis drug GGT=gamma-glutamyltransferase SYSADOAs=symptomatic slow-acting drugs for osteoarthritis GlcN=glucosamine USP=united states pharmacopeia HMM-CS=high-molecular mass biotechnological CS WHO=world health organization Nicola Volpi is Associate Professor of Biochemistry at the University of Modena and Reggio Emilia, Italy. He has worked in the field of complex macromolecules, i.e. poly - saccharides, glycosaminoglycans, glycoproteins and pro - teoglycans since 1990, for over 25 years.

Prof. Volpi is one of the worldwide expert in the field of chondroitin sulfate and his studies and researches over the last 25 years have made a great impact on the international scientific community as documented by his many publications, invitations to several national and international congresses in addition to three published international books as Editor. He developed several preparatory and analytical techniques for the study of the structure and properties of this complex biomolecule. His skills in the application of the analytical methodologies essential for the understanding of the structure, properties and quality of chondroitin sulfate and derivatives is docu - mented by his publications of high international level and patents.

Prof. Volpi is currently collaborating with Italian and International research groups with a view to developing new methodologies and applying these approaches to new molecular studies. Furthermore, he also collaborates with Companies to de - velop (macro)molecules having new improved biological and potential pharmaco - logical properties.

Prof. Volpi has been member of the USP (U.S. Pharmacopoeia) Commission in 2000 for “Analytical determination of Chondroitin sulfate sodium”.