35

The failure of axon regeneration in the injured spinal cord is largely caused by the growth-inhibitory environment of the adult CNS that is especially prominent in white matter. The inhibitors of CNS regeneration can be classified into three main categories: (1) inhibitors associated with the glial scar that forms after injury, (2) myelin-associated inhibitors, and (3) inhibitors of the “guidance type.” There are many types of ligands and receptor complexes that convey signals from the multiple growth-inhibitory proteins (Fig. 35.1). However, despite this diversity, inside neuronal cells, most if not all inhibitory signaling complexes converge on the Rho pathway.

Inhibitors Associated with the Glial Scar

The glial reaction to CNS injury occurs almost immediately as a mechanism to protect healthy CNS tissue from inflammatory damage and to repair the blood–brain barrier.¹ The glial scar forms both a physical and a chemical barrier that is composed of astrocytes, microglia, macrophages, oligodendrocyte precursor cells, and an extra-cellular matrix of secreted basal membrane components.^1,2 Astrocytes undergo reactive gliosis and are a major contributor to the inhibitory nature of the glial scar by releasing chondroitin sulfate proteoglycans (CSPGs), which are potent inhibitors of axon growth.¹ Recently, the receptor for CSPG inhibitory signaling has been identified as protein tyrosine phosphatase (PTPs), and PTPs gene disruption promotes axon regeneration.^3,4 It is known that inactivating Rho will overcome growth inhibition on CSPG substrates,⁵ but the full signaling cascades still need to be elaborated.

Myelin-Associated Inhibitors

Myelin Inhibitors

Many potent growth-inhibitory proteins exist in myelin, and the myelin debris in the environment of the injured CNS is a formidable barrier to regeneration The best-characterized myelin-derived growth-inhibitory proteins are myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte myelin glycoprotein (OMgp),⁶ although other inhibitors, such as versican,⁷ are also present in myelin. MAG was the first myelin inhibitor identified^8,9 after the Schwab group first characterized CNS inhibitory activity.¹⁰ Nogo was next identified through the use of IN-1 antibody to identify the antigen peptide sequence.^11–13 OMgp is another myelin-derived inhibitor identified^14,15 and may play a role in preventing collateral sprouting and determining the spacing of the nodes of Ranvier.¹⁶ MAG, Nogo, and OMgp all have in common the ability to share interaction with the Nogo receptor complex.⁶

Nogo Receptors

The receptors for the various myelin-derived growth-inhibitory proteins have taken longer to identify and new receptors are still being added to the list. The difficulty in identifying receptors is that MAG, Nogo, and OMgp all signal to receptor complexes on the neuronal membrane that may have different components on different types of neuronal cells. MAG, Nogo, and OMgp signal through a receptor complex composed of Nogo-66 receptor (NgR1)17 or paired immunoglobulin-like receptor B (PirB),¹⁸ leucine rich repeat and Ig domain containing 1 (LINGO1),¹⁹ and either p75 neurotrophin receptor (p75 NTR)²⁰ or tumor necrosis factor receptor superfamily, member 19 (TROY/TNSRF19).^21,22

NgR1 is localized to the axolemma in many classes of CNS neurons, and because it lacks an intracellular domain, additional co-receptors transduce the signal following ligand binding.¹⁷ The p75NTR co-receptor can physically interact with NgR1 to mediate intracellular signaling,²⁰ although in many neurons TROY takes the place of p75 NTR. The p75NTR is known to signal directly to Rho,²⁰ and TROY also signals to Rho.^21,22 The other components of the NgR receptor complex are LINGO1, a transmembrane protein,¹⁹ and PirB.¹⁸ PirB is expressed in a subset of neurons in the brain, and interfering with PirB activity, either genetically or with a function-blocking antibody, reduces neurite outgrowth inhibition in response to Nogo-66, MAG, OMgp, and myelin.¹⁸

Gangliosides and NgR2

Other receptors may be involved in mediating some of the extracellular inhibitory signals by myelin-derived inhibitors. Two additional human homologues for NgR1 have been identified: NgR2 and NgR3.^23,24 NgR2 mRNA levels have been detected in neurons in the adult mouse brain that project to the spinal cord.²⁵ Neither NgR2 nor NgR3 can bind Nogo-66, but NgR2 has been demonstrated to bind MAG.²⁶ The gangliosides GT1b and GD1a act as receptors for MAG that signal to Rho.^20,26,27 However, although MAG is not thought to be a very important growth-inhibitory protein, it may have an important role in promoting resistance to axonal injury and disease.²⁸

Guidance-Type Growth Inhibitors: Eph/Ephrins, Netrins, and Semaphorins

Chemorepulsive axon guidance molecules are known to be important in the development of the nervous system, and now it is clear that many of the developmentally expressed proteins act as inhibitors in CNS injury by signaling to Rho. Three classes of proteins have been implicated in inhibiting nerve regeneration: Ephrins/Eph receptors, netrins, and semaphorins.

Ephrins and Eph proteins are part of the large receptor tyrosine kinase family capable of bidirectional signaling between neurons and oligodendrocytes (Fig. 35.1). Both Ephs and Ephrins have been localized to CNS tissue following a spinal cord injury (SCI),^29,30 and Ephrin-B3 is persistently expressed in oligodendrocytes, where it has strong axon growth-inhibitory activity.³¹ Studies with knockout mice show that Ephrin-B3 and Eph4 play a role in limiting axonal regeneration and functional recovery following SCI,³¹ and Rho is activated by inhibitory Ephrin signaling.³²

Netrins are a family of proteins that play an important role in development of spinal cord, and Netrin-1 is expressed in the neurons and oligodendrocytes of the adult spinal cord.³³ Netrin-1 is a bifunctional ligand that can function as a chemoattractant or chemorepellent depending on the receptor type it interacts with. Netrin-1 inhibits axon growth,³⁴ and netrins affect Rho signaling.³⁵

The semaphorin family has both soluble and transmembrane-bound members that mediate repulsion in development, and these proteins are re-expressed in CNS injury.36,37 Sema4D is an oligodendrocyte transmembrane protein that inhibits axon growth and is transiently up-regulated following CNS injury,³⁸ as is Sema3A, which is a soluble inhibitory protein.³⁹ Sema5A induces growth cone collapse, and blocking Sema5A with a function-blocking antibody neutralizes these effects.⁴⁰ Semaphorins inhibit axon growth via the activation of Rho.^41,42 Therefore, inactivation of Rho should be effective to overcome many of the guidance-type of axon growth inhibitors.

Growth Inhibition: All Roads Lead to Rho

The challenge for translational medicine to treat SCI is to find a strategy that blocks all the CNS growth-inhibitory activity. The growing body of evidence indicates that Rho regulates the neuronal response to diverse growth-inhibitory proteins. Therefore, Rho is a potentially very powerful target to promote repair after SCI.

Rho guanidine triphosphate (GTPases) are a family of highly related proteins that are present in all cells as important signaling switches. GTPases have two conformations: a guanidine diphosphate (GDP)-bound inactive state and a GTP-bound active state. Treating neurons with C3 transferase to inactivate Rho was first demonstrated by us as an effective way to overcome growth inhibition on myelin.⁴³ C3 transferase is a bacterial protein that adenosine diphosphate (ADP) ribosylates Rho to keep it in its inactive state. Rho kinase interacts with Rho, and inhibition of Rho kinase with Y-27632 has similar effects—both C3 and Rho kinase inhibitors promote growth on inhibitory substrates in vitro and in vivo.^43–57 More-over, both compounds override growth inhibition by myelin as well as by the CSPGs.⁵ Newer evidence also indicates that many of the guidance-type inhibitors signal to Rho. The in vitro studies have been followed by a wealth of studies on animal models of SCI and regeneration (Table 35.1). The general finding is that inactivating Rho after SCI has beneficial effects on tissue sparing and functional recovery, and anatomical studies indicate that inactivation of Rho stimulates axon regeneration and prevents apoptotic cell death (Table 35.1).

We are now beginning to understand the molecular mechanisms that explain why Rho is such an important target to block growth inhibition and promote regeneration. Rho activation signals through downstream effectors to modulate the cytoskeleton and influence growth cone behavior. Rho kinase is activated in response to Rho activation, leading to phosphorylation of myosin light chain II^58,59 that in turn regulates myosin in motility. The actin depolymerizing factor/cofilin family is also important in cytoskeleton rearrangements in the growth cone, and these proteins are also regulated by Rho.⁶⁰ Collapsing response mediator protein (CRMP) is implicated in the signaling cascade activated in response to myelin-associated inhibitors and CSPGs and can physically interact with Rho to mediate growth inhibition.^61,62 The current findings suggest that inhibitory signaling from myelin inhibitors and CSPGs converge on Rho-CRMP4 to cause cytoskeletal rearrangements.

Translational Medicine

The in vitro and in vivo demonstration that C3 transferase could specifically inactivate Rho to overcome growth inhibition suggested it could be a good drug candidate. As a biologic drug, breakdown products are amino acids, so there is less risk of a phase 3 failure due to toxicity. With a view to translating early preclinical findings on Rho inactivation to clinical testing, it was necessary to (1) develop a method to obtain highly purified active protein and standardize the enzymatic activity of different purification batches, (2) develop a suitable delivery method and determine effective dose, and (3) complete proof-of-concept and confirm delivery method. We have developed a drug candidate, called BA-210, by modifying the properties of C3 transferase to enhance its ability to penetrate cells. BA-210 has been given the trademark name of Cethrin (Alseres Pharmaceuticals). The steps that were needed to bring BA-210 to clinical trial are shown in Fig. 35.2.

Chemistry, Manufacturing, and Control

With any drug, and especially with a protein drug, a critical component is to scale up production and purification procedures and develop assays to characterize purity, potency, and reproducibility. Before this step, we ensured that we had the best drug candidate possible by engineering several cell-permeable versions of C3 and comparing their biological activity.44 We also optimized expression systems for synthesis and the method of purification.⁴⁵

To use a drug in a clinical trial, chemistry, manufacturing and control (CMC) must be performed according to good manufacturing practices (GMPs) (Fig. 35.2). Although a discussion of GMPs is beyond this chapter, it is important to state that the earlier the procedures needed for GMPs are developed, the better, and the importance of rigorous characterization of a drug candidate as early as possible cannot be overstated. There are many reasons for putting an early effort into quality control: (1) Impurities can have unwanted biological effects and influence preclinical findings. For example, endotoxins in protein drugs are likely to cause variability in SCI experiments because of their effect on inflammation. (2) Assays required to monitor potency, purity, and reproducibility are not trivial and may require a significant development effort. (3) Thinking about formulation is a critical component with respect to drug delivery and drug stability in storage.

In the development of BA-210, we made different variants along the way^44,45 (Table 35.1). BA-210 manufactured at large scale is functionally interchangeable with its predecessors, having the same or better enzymatic and biological activities but fewer impurities, and a standardized enzymatic activity. An example of a change made during the CMC development process can be seen in the removal of a single cysteine that was located in a nonfunctional region of the protein. This was done after it was noted that the C3 variants had a tendency to aggregate during scale-up purification.⁶³

Fig. 35.2 Diagram to illustrate the development path for BA-210 from proof-of-concept (POC) research to investigational new drug (IND) application. After the decision to go forward from POC data, BA-210 was synthesized following chemistry, manufacture, and control (CMC) guidelines. This well-characterized drug was used for further dose-validation and delivery studies, for good laboratory practice (GLP)-compliant safety studies, and to finalize GLP-compliant drug purity and activity assays. A good manufacturing practice (GMP) compliant batch of drug was synthesized for the clinical trials.

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