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Kytogenics Pharmaceuticals, Inc.

Kytogenics_The Technology

Description

Kytogenics has developed two technologies based on modified chitinous derivatives. The Mucoadhesive Cell Control Technology (MACC) has outstanding properties as discussed below and is mainly but not exclusively used for the development of medical devices.  It is based on the mucoadhesive polysaccharide N-O-carboxymethylchitosan (NOCC).

The Tight Junction Opener Technology (TIJO) uses the ability of some special chitinous derivatives to open the tight junctions between cells, thus enhancing the permeation of drugs through mucosal tissues. The technology has potential for the delivery of peptides, proteins and other macromolecules as well as for cancer and small molecule drugs.

Tight Junction Opening Technology (TIJO) for the Novel Delivery of Drugs

Kytogenics has developed technology based on chitin, chitosan and their derivatives for drug delivery and medical device development. One of the novel technologies is based on the modification of chitosan to give highly electronegative charges to the chitosan based derivatives.  This sulfated derivatives of chitosan are the basis of the TIJO technology which has been used in drug delivery with exciting results.

Mechanism of Action 

Tight junctions are cell to cell adhesions found in epithelial and endothelial cell layers. When closed, these junctions stitch together the cells prohibiting extracellular transport of water, solutes, drugs and other active ingredients.Tight junctions consist of integral membrane proteins, claudins, occloudins and junctional adhesion proteins.  Figure 1 is a schematic of two epithelial cells showing the tight junction between the cells and the position of the tight junction proteins.  Figure 2 is an electromicrograph of two in vivo cells showing the tight junction between the cells and the tight junction proteins.  Several Universities, including the University of Leiden have shown that the tight junctions can be opened by electronegative forces applied to the tight junction proteins mentioned above.

Figure 1

Kytogenics graphic

Figure 2

Kytogenics Graphic

Proof of Concept

Delivery of drugs in vitro has been accomplished by preparing confluent monolayers of Caco-2 cells and determining the permeation by placing the active ingredient on the donor side and sampling for the active ingredient on the receptor side.  Another approach used to determine if the tight junctions have been opened is to determine transepithelial electrical resistance across the Caco-2 cell monolayer.  Figure 3 shows a scanning micrograph of a confluent Caco-2 cell monolayer with red dextran placed on the donor side.  The fluorescent red on the dextran allows for visualization.  On the control portion of the graph it is seen that the red marker has not penetrated through the Caco-2 cell monolayer.  In the TIJO portion, where the red dextran was placed in the donor phase together with the sulfated NOCC, the micrograph shows a clear indication of the movement of dextran through the tight junctions between the Caco-2 cells, proving that paracellular transport of a 10,000 molecular weight molecule is possible.

Figure 3

Kytogenics Graphic

The Mucoadhesive Cell Control Technology (MACC) for Medical Devices  

Kytogenics has been developing technologies based on chitin and chitosan for the last ten years.  Since chitin and chitosan are not water soluble, the company invented and patented the derivative N,O-carboxymethylchitosan (NOCC) which is a water soluble compound with unique properties.  The NOCC polymer, see chemical structure below, is the basis of Kytogenics' mucoadhesive control technology.

Kytogenics graphic

Figure 4 summarizes the unique properties of NOCC. These highly unique properties allow for the development of many medical devices, such as solutions and gels for post-surgical adhesion prevention which is in pivotal clinical trials (see Appendix A for in-vivo and in-vitro results) wound dressing products, bone grafts, synovial fluid supplementation gels, tendon and dura wraps.  Due to its exceptional mucoadhesive properties to soft tissues as well as bone, NOCC can also be used in the delivery of drugs including macromolecules.

Figure 4

Unique Properties of MACC Technology

•Raw material derived from the exoskeleton of shrimp
•Viscoelastic, lubricious and highly hydrophilic
•Biocompatible and bioresorbable
•Excellent bioadhesive properties to both hard and soft tissue
•Inhibits protein adsorption and attachment of cells
•Derivatizable for optimal drug delivery
•Flexibility for manufacturing a broad range of physical forms beads:
  • Sponges
  • Films
  • Foams
  • Hydrogels
  • Solutions

Competitive Advantages

NOCC has properties in common with other polysaccharide polymers that are found in the fluid that bathes many cells in the human body. Many of the properties of NOCC, however, are uniquely different from other polysaccharides:

NOCC possesses both the amine and carboxyl groups of amino acids and proteins but on a polysaccharide backbone. The presence of these substituents makes NOCC bioadhesive to both hard (bone and teeth) and soft tissues (mucosal membranes).

NOCC carries a sufficient negative charge per repeating sugar molecule so that the attachment of certain cells to surfaces that have been coated with NOCC is prevented or inhibited.

The adsorption of proteins to NOCC-coated substrates is also inhibited.
NOCC can be readily modified by simple chemical means due to the presence of reactive amine and carboxyl groups along the polymer chain. One modification involves cross-linking NOCC to obtain higher molecular weight polymers and hydrogels that persist in vivo for extended periods of time.

Therapeutic agents (drugs, proteins, etc.) can also be easily coupled to NOCC via these same sites.

NOCC readily forms films, sponges, foams, beads, hydrogels, pastes and viscoelastic solutions.

Comparative

Comparison of NOCC Technology and HA

Kytogenics' technology is often compared to that of HA, a naturally occurring biopolymer found in the body that extensively coats, protects, cushions and lubricates soft tissue. Since a number of Kytogenics' competitors use HA as their core technology, it is important to provide a comparison of the key characteristics of NOCC and HA.

The NOCC polymer has certain properties in common with the polysaccharide HA. HA is found in a number of different areas of the body, such as the fluid that bathes the cells of connective tissues, in the synovial fluid, and the fluids of the eye. HA is a long chain polymer made of two repeating sugars; one of these sugars is the same as the sugar found in chitin, which has no electrical charge, and is the parent compound of NOCC. The second sugar of HA has a carboxyl group that imparts a negative charge to the polymer.

In scientific terms, as NOCC is synthesized, essentially two changes occur to the sugars of chitin: 1) the group that is bound to a nitrogen center is removed to yield a free amine group, which allows Kytogenics to further modify NOCC; and, 2) carboxyl groups are added to impart a negative charge to NOCC. The carboxyl group is important because it aids in the solubility of NOCC as well as HA. Since NOCC carries both the amine and carboxyl groups, whereas HA carries only the carboxyl group, NOCC is able to perform in many beneficial ways that HA can not.

All amino acids and proteins have both amine and carboxyl groups. NOCC is unique because it also has both amine and carboxyl groups, which are a part of all living systems on its polysaccharide chain. Furthermore, NOCC is readily modified by the simple chemical methods that have been developed over the years for the alteration of proteins, which gives it a distinct competitive advantage. For example, therapeutic agents - such as drugs and peptides, etc. - can be coupled to NOCC or NOCC can be easily cross-linked in aqueous solutions to yield hydrogels, which is a key component of the NOCC anti-adhesion device. This is critical because Kytogenics believes the formulation of the NOCC gel allows it to remain longer in the body, thereby helping to prevent adhesions. The coupling of cancer drugs to NOCC could provide a significant and very useful advancement because it will improve the therapeutic effectiveness and reduce the toxicological profile of the cancer drug. In the case of peptides, covalently bonding them to NOCC will substantially improve their stability in vivo, which will increase their effectiveness. Kytogenics knows of no other bio-polymers carrying both amine and carboxyl groups that are as easily modified as NOCC.

NOCC, in addition, has certain properties that are clearly different from those of HA. For example, when NOCC is prepared, the level of addition of carboxyl groups exceeds the number of carboxyl groups on HA by almost twice as much. Since the carboxyl groups carry a negative charge in vivo, NOCC is more highly charged than HA. This additional negative charge is believed to be responsible for inhibiting the attachment of certain cells to the NOCC coated surfaces and tissues. HA does not exhibit this inhibitory effect of the attachment of certain cells which is critical because the cells that are prevented from attaching are the ones that are involved in the formation of scars and adhesions. Kytogenics has also found that NOCC coated surfaces prevent the attachment of proteins which should prove useful, for example, in treating contact lenses to prevent protein buildup.

NOCC is an exceptional bio-polymer because of its chemical structure. Since NOCC is a new material to the body, it is able to remain in the body for a longer period of time than HA before it is degraded and resorbed. HA, on the other hand, is resorbed quicker because the body has control mechanisms in place to maintain certain levels of HA. In the medical profession, it is generally believed that adhesion formation is initiated within 2-3 days following injury. Because of the chemical structures of HA and NOCC, HA is generally degraded in 1-2 days, whereas the NOCC gel can stay in the body for more than twice as long. Kytogenics has found that NOCC can stay in the body for 7-9 days. The table below summarizes the distinct differences between NOCC and HA and NOCC's main competitive advantages.

Comparisons of the Key Characteristics of NOCC and HA

Key Characteristics
NOCC
HA
Bio-compatible
Yes
Yes
Bio-resorbable
Yes
Yes
Polysaccharide
Yes
Yes
Carboxyl Group
Yes
Yes
# 1) Amine Group
Yes
No
# 2) Large negative charge
Yes
No
# 3) Inhibition of attachment of cells*
Yes
No

# 4)Inhibition of attachment of proteins

Yes
No
# 5) Simple chemical modification
Yes
No

* cells involved in inflammation leading to adhesion formation.

The table above lists the comparisons of the key characteristics that NOCC possesses, and the last five are characteristics that HA does not possess. The importance of these additional five key characteristics cannot be overstated, since they will allow NOCC to address the following sizable and broad markets.

Exceptional Safety Profile

Extensive toxicological studies have been performed on NOCC with no toxic effects attributed to the NOCC compound. Figure 5 shows the list of toxicological studies performed.  Although NOCC has electronegative properties, our studies have shown that it is not adequate to open the tight junctions so as to significantly increase drug permeability.  Kytogenics developed additional derivatives with higher electronegative properties by attaching sulfate groups to NOCC, and chitosan.  These electronegative properties have allowed for substantial increase in tissue permeability.

Figure 5

TOXICOLOGY REPORTS

  • 6184-101 14-Day Feeding Study in Rats
  • TI264-504 ISO Acute Systemic Toxicity Study in the Mouse (Liquid/Chemical)
  • T1251-804 ISO Acute Intracutaneous Reactivity Study in the Rabbit (Chemical Solution)
  • T1251-804/S ISO Acute Intracutaneous Reactivity Study in the Rabbit With Histopathology
  • MG065-110 Cytotoxicity Study Using the ISO Agarose Overlay Method
  • MG019-221 Genotoxicity: Salmonella Typhimurium Reverse Mutation Study
  • MG016-110 Genotoxicity:  Chromosomal Aberration Study in Mammalian Cells
  • TS200-901/S Subchronic Intraperitoneal Toxicity Study in the Rat (14 days)
  • T1250-807/ TH035-800 ISO Muscle Implantation Study in the Rabbit With Histopathology (1 Week)
  • T0212-500 Mouse Bone Marrow Micronucleus Test
  • T1261-303 ISO Sensitized Study in the Guinea Pig (Maximization Method, Chemical Solution)
  • TA088-200 T1261-306Determination of Clotting Time Using the Lee-White Method
  • MG074-200 Hemolysis Study – IN VITRO Procedure (Direct Contact Method)
  • C0019-000 USP Physicochemical Study
  • TU013-800 USP Intracutaneous Toxicity Study in the Rabbit (Extracts)
  • TU012-500 USP Systemic Toxicity Study in the Mouse (Extracts)
  • TU014-807 USP Muscle Implantation Study in the Rabbit (7 days)
  • C0019-000 USP Physicochemical Study
  • TU013-800 USP Intracutaneous Toxicity Study in the Rabbit (Extracts)
  • TU012-500 USP Systemic Toxicity Study in the Mouse (Extracts)
  • TU014-807 USP Muscle Implantation Study in the Rabbit (1 week)
  • Rat Hypersensitivity Study
  • Histological Pathology from Intraperitoneal NOCC
  • Intradermal Inflammation Caused by Plane Formation with NOCC in Comparison to Plane Formation With Sterile Air
  • Evaluation of NOCC in an Animal Model for Intraabdominal Sepsis
  • Study No RT-01 Protocol 2903-002 Intraperitoneal Developmental Toxicity Study of NOCC in Rabbits
  • Study No RT-02 Protocol 2903-003Intrapertioneal Injection Combined Fertility and Developmental Toxicity Study of NOCC in Female Rats
  • Expert Review of Argus Research Study 2903-002:  Intraperitoneal Developmental Toxicity Study of NOCC in Rabbits
  • Study No RT-03 Protocol 2903-005 Intraperitoneal Developmental Toxicity Study of NOCC in Rabbits