The ideal tonometer would be easy to use, portable, reliable, and comfortable for both the patient and physician. It should have low inter‐ and intraoperator variability and provide an IOP reading that is close to the manometric IOP of the individual. To date, no such tonometer exists. Till date, the gold standard for IOP measurements is Goldmann applana‐

[1] Leske MC, Heijl A, Hyman L, Bengtsson B, Dong L, Yang Z, et al. Predictors of long‐term progression in the early manifest glaucoma trial. Ophthalmology. 2007;114:1965–72.

[3] Kass MA, Heuer DK, Higginbotham EJ, Johnson CA, Keltner JL, Miller JP, et al. The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocu‐ lar hypotensive medication delays or prevents the onset of primary open‐angle glau‐

[4] Goel M, Picciani RG, Lee RK, Bhattacharya SK. Aqueous humor dynamics: a review.

[5] Glaucoma. 2007–2008 ed. San Francisco, CA: American Academy of Ophthalmology;

[6] Goldmann H, Schmidt T. Applanation tonometry. Ophthalmologica. 1957;134:221–42

[7] Whitacre MM, Stein RA, Hassanein K. The effect of corneal thickness on applanation

[2] Shiose Y. Intraocular pressure: new perspectives. Surv Ophthalmol. 1990;34:413–35

#### **Newer Intraocular Pressure Measurement Techniques Newer Intraocular Pressure Measurement Techniques**

Maria Letizia Salvetat, Marco Zeppieri and Maria Letizia Salvetat, Marco Zeppieri and Paolo Brusini

Paolo Brusini

[21] Toker MI, Vural A, Erdogan H, Topalkara A, Arici MK. Central corneal thickness and Diaton transpalpebral tonometry. Graefes Arch Clin Exp Ophthalmol. 2008;246:881–9.

[22] Li Y, Shi J, Duan X, Fan F. Transpalpebral measurement of intraocular pressure using the Diaton tonometer versus standard Goldmann applanation tonometry. Graefes Arch Clin

[23] Doherty MD, Carrim ZI, O'Neill DP. Diaton tonometry: an assessment of validity and preference against Goldmann tonometry. Clin Exp Ophthalmol. 2012;40:e171–5.

Exp Ophthalmol. 2010;248:1765–70.

54 Glaucoma - Intraocular Pressure and Aqueous Dynamics

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66260

#### **Abstract**

An elevated intraocular pressure (IOP) has been shown to be one of the major risk factors for glaucoma. It is of utmost importance to obtain accurate and precise IOP when dealing with patients with ocular hypertension and glaucoma, especially patients who have undergone ocular surgery. Goldmann applanation tonometer (GAT) was first introduced in the 1950s and is still currently considered as the gold standard to measure IOP. Although the reproducibility of GAT has shown to be quite good, its accuracy provides several limitations. In particular, IOP measurements taken with GAT have been demonstrated to be influenced by many corneal parameters, including central thickness, curvature, astigmatism and biomechanics. Other disadvantages of GAT include the need for local anesthetic drops, for fluorescein and for a slitlamp. Several different methods have been proposed to overcome the disadvantages found in GAT. The newer devices used as alternative tonometric methods include the iCare rebound tonometer, the BioResonator applanation resonance tonometer, the Pascal dynamic contour tonometer, the ocular response analyzer, the Corvis ST pachy-tonometer and Ocuton S. The precision and accuracy of these alternative tonometric methods in comparison with GAT have been reported and discussed.

**Keywords:** tonometry, intraocular pressure, precision, accuracy, central corneal thickness, corneal biomechanical properties

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

#### **1.1. Importance of the intraocular pressure (IOP) measurement**

Accurate and reproducible IOP evaluation is crucial with regard to the classification, management and follow-up of patients with ocular hypertension and glaucoma, and in patients that have undergone ocular surgical procedures.

Elevated intraocular pressure (IOP) has been shown to be one of the major risk factors for glaucoma [1]. Although the vulnerability of the optic nerve can vary among patients, longitudinal randomized controlled population-based trials have provided strong evidence that the reduction in the mean IOP of only 1 mmHg is significantly effective in preventing the development [2] and in delaying the progression [3] of the glaucomatous damage. Goldmann applanation tonometer (GAT) has been considered as the clinical gold standard for IOP measurement since it was introduced in the 1950s [4]. Although the reproducibility of GAT has shown to be quite good, its accuracy provides several limitations. In particular, IOP measurements taken with GAT have been shown to be influenced by many corneal parameters, including central thickness, curvature, astigmatism and biomechanics. Other disadvantages of GAT include need for local anesthetic drops, fluorescein and a slitlamp. Several different methods have been proposed to overcome the disadvantages found in GAT. The newer alternative tonometric methods include the iCare rebound tonometer, the BioResonator applanation resonance tonometer, the Pascal dynamic contour tonometer, the ocular response analyzer and the Corvis ST pachy-tonometer.

The purpose of this chapter is to discuss the advantages, disadvantages, precision and accuracy of the "newer" alternative tonometric methods.

Newer tonometers covered in this chapter include the following:

#### *I. Rebound tonometry*

Rebound tonometry, also known as "impact" or "dynamic tonometry," was first introduced by Obbink about 60 years ago [5]. In 1997, Kontiola introduced an improved and simpler new rebound tonometer, better known as an induction-based impact tonometer [6], that became commercially available as the iCare tonometer in 2003 (**Figure 1**). The method is based on the use of a moving probe that collides with the eye: the motion parameters of the probe, which vary according to eye pressure, are monitored and used in the calculation of IOP [6].

**Figure 1.** The iCare rebound tonometer.

**1. Introduction**

**1.1. Importance of the intraocular pressure (IOP) measurement**

have undergone ocular surgical procedures.

56 Glaucoma - Intraocular Pressure and Aqueous Dynamics

analyzer and the Corvis ST pachy-tonometer.

of the "newer" alternative tonometric methods.

**iii.** *Pascal dynamic contour tonometer (DCT)*

**iv.** *Ocular response analyzer (ORA)*

**v.** *Corvis ST pachy-tonometer*

**i.** *Rebound tonometry*

**vi.** *Ocuton S*

*I. Rebound tonometry*

Newer tonometers covered in this chapter include the following:

**ii.** *BioResonator applanation resonance tonometer (ART)*

Accurate and reproducible IOP evaluation is crucial with regard to the classification, management and follow-up of patients with ocular hypertension and glaucoma, and in patients that

Elevated intraocular pressure (IOP) has been shown to be one of the major risk factors for glaucoma [1]. Although the vulnerability of the optic nerve can vary among patients, longitudinal randomized controlled population-based trials have provided strong evidence that the reduction in the mean IOP of only 1 mmHg is significantly effective in preventing the development [2] and in delaying the progression [3] of the glaucomatous damage. Goldmann applanation tonometer (GAT) has been considered as the clinical gold standard for IOP measurement since it was introduced in the 1950s [4]. Although the reproducibility of GAT has shown to be quite good, its accuracy provides several limitations. In particular, IOP measurements taken with GAT have been shown to be influenced by many corneal parameters, including central thickness, curvature, astigmatism and biomechanics. Other disadvantages of GAT include need for local anesthetic drops, fluorescein and a slitlamp. Several different methods have been proposed to overcome the disadvantages found in GAT. The newer alternative tonometric methods include the iCare rebound tonometer, the BioResonator applanation resonance tonometer, the Pascal dynamic contour tonometer, the ocular response

The purpose of this chapter is to discuss the advantages, disadvantages, precision and accuracy

Rebound tonometry, also known as "impact" or "dynamic tonometry," was first introduced by Obbink about 60 years ago [5]. In 1997, Kontiola introduced an improved and simpler new rebound tonometer, better known as an induction-based impact tonometer [6], that became commercially available as the iCare tonometer in 2003 (**Figure 1**). The method is based on the use of a moving probe that collides with the eye: the motion parameters of the probe, which

vary according to eye pressure, are monitored and used in the calculation of IOP [6].

The iCare (Tiolat Oy, Helsinki, Finland) is routinely used in clinical practice in several clinics nowadays. The device is handy and light-weighed (250 g) [7]. It is composed of a small disposable thin metal probe (about 5 cm in length and 1 mm in diameter) and a solenoid and magnet housed in a metal case. The tonometer is placed in front of the eye, using the forehead to properly position the tip of the probe about 5 mm from the central cornea. The button is pressed in taking IOP measurements, which activates an electric signal that is sent to the solenoid and magnet to move the probe forward. The tip of the probe hits the cornea, rebounds and induces a voltage in the solenoid, which amplifies the signal to a microprocessor. It is advisable to take at least six readings, so that a mean IOP can be calculated by the built-in software [7].

The iCare PRO is a new version, which provides advantages not offered in the previous model, such as measuring IOP also in a supine position, thus useful in bedridden and surgical patients. The iCare one is a simplified version, which has recently been introduced. It can be used independently by patients for at-home autotonometry [8].

The iCare tonometer has shown good reproducibility [9] and correlation with GAT and other tonometers in healthy and glaucomatous eyes [9], and in eyes after keratoplasty [10]. A tendency of the iCare to overestimate the IOP measurements taken with GAT, with a similar trend at different IOP levels, has been reported by several authors [11, 12].

Although iCare was designed not to be influenced by corneal properties, studies have shown that CCT and other corneal structural characteristics affect IOP readings [9, 10, 13]. The iCare tonometer appeared less influenced by corneal edema when compared to GAT [10, 14].

The main advantages of this tonometric method are that the instrument is small, lightweight and portable, easy to use; slitlamp, local anesthesia and fluorescein are not required; IOP is taken in a comfortable sitting position and with the iCare PRO also in the supine position; the rapid measurement enables monitoring in noncompliant subjects. The probes are disposable, and thus, the risk of microbiological contaminations is avoided.

#### *II. BioResonator applanation resonance tonometer (ART)*

The applanation resonance tonometer (ART) is a new tonometer based on the resonance technique (**Figure 2**) proposed by Eklund et al. in 2003 [15]. The ART estimates IOP by combining simultaneous continuous sampling of both parameters considered in the applanation principle, which include the force needed to applanate the cornea and the corresponding contact area [15]. The current commercial version of ART, known as the BioResonator ART (Medical sensors and Instruments, BioResonator AB, Sweden), has been recently released and is available in a manual and automatic servo-controlled version [16]. The instrument is made up of a sensor and transducer that can measure the contact force in a continuous manner. The resonance sensor is made up of a cylindrical piezoelectric element that has a known resonance frequency. When the sensor is brought in contact with the cornea, the acoustic impedance of the cornea mechanically loads the sensor and modifies the resonance frequency, with a frequency shift, which is proportional to the contact area between sensor and cornea [15]. IOP is calculated from the slope of the relationship between force and frequency evaluated in a specific frequency shift interval corresponding to an interval applanation area between 4.9 and 11.0 mm2 [15]. The sensor module of the ART is attached to a standard slitlamp in a similar position as the GAT probe. The ART probe is disinfected with 70% ethanol before each subject, and local anesthetic drop (0.4% benoxinate hydrochloride) is required before IOP measurements. ART is available in two versions: the manual one (BioResonator ART manual), in which the sensor is manually pushed toward the cornea, and the automatic servo-controlled version (BioResonator ART servo), in which a small motor provides the sensor movement. The device provides the median of repeated IOP values, in addition to a Quality Index (QI >2 should be excluded) that reflects the standard deviation of the data. The ART is self-calibrated.

**Figure 2.** The bioresonator applanation resonance tonometer (ART).

BioResonator ART IOP measurements have been demonstrated to be affected by CCT [15, 17]. The overestimation of the IOP measurements obtained with ART relative to GAT has been demonstrated in previous studies [17].

#### *III. Pascal dynamic contour tonometer (DCT)*

*II. BioResonator applanation resonance tonometer (ART)*

58 Glaucoma - Intraocular Pressure and Aqueous Dynamics

**Figure 2.** The bioresonator applanation resonance tonometer (ART).

demonstrated in previous studies [17].

The applanation resonance tonometer (ART) is a new tonometer based on the resonance technique (**Figure 2**) proposed by Eklund et al. in 2003 [15]. The ART estimates IOP by combining simultaneous continuous sampling of both parameters considered in the applanation principle, which include the force needed to applanate the cornea and the corresponding contact area [15]. The current commercial version of ART, known as the BioResonator ART (Medical sensors and Instruments, BioResonator AB, Sweden), has been recently released and is available in a manual and automatic servo-controlled version [16]. The instrument is made up of a sensor and transducer that can measure the contact force in a continuous manner. The resonance sensor is made up of a cylindrical piezoelectric element that has a known resonance frequency. When the sensor is brought in contact with the cornea, the acoustic impedance of the cornea mechanically loads the sensor and modifies the resonance frequency, with a frequency shift, which is proportional to the contact area between sensor and cornea [15]. IOP is calculated from the slope of the relationship between force and frequency evaluated in a specific frequency shift interval corresponding to an interval applanation area between 4.9 and 11.0 mm2 [15]. The sensor module of the ART is attached to a standard slitlamp in a similar position as the GAT probe. The ART probe is disinfected with 70% ethanol before each subject, and local anesthetic drop (0.4% benoxinate hydrochloride) is required before IOP measurements. ART is available in two versions: the manual one (BioResonator ART manual), in which the sensor is manually pushed toward the cornea, and the automatic servo-controlled version (BioResonator ART servo), in which a small motor provides the sensor movement. The device provides the median of repeated IOP values, in addition to a Quality Index (QI >2 should be

excluded) that reflects the standard deviation of the data. The ART is self-calibrated.

BioResonator ART IOP measurements have been demonstrated to be affected by CCT [15, 17]. The overestimation of the IOP measurements obtained with ART relative to GAT has been The pascal dynamic contour tonometer (DCT) (**Figure 3**), developed by Kaufmann et al. [18], is not based on corneal applanation. It has a concave measuring tip, which is applied on the corneal surface to provide a "contour matching" with the aid of a built-in sensor, which is representative of the pressure inside the eye. The Pascal DCT should be therefore less influenced by corneal properties. Previous studies have shown that there is a good concordance between Pascal DCT and GAT in eyes with normal corneas [19]. In eyes that have undergone laser *in situ* keratomileusis (LASIK), the Pascal DCT appears to provide a more reliable measurement of IOP than GAT, which tends to measure artificially lower IOP values [20].

**Figure 3.** The pascal dynamic contour tonometer (DCT).

The Pascal DCT (SMT Swiss Microtechnology AG, Zurich, Switzerland) [18] is slitlamp mounted and is calibrated automatically. The tonometer has a 10 mm concave radius tip and a built-in 1 mm sensor. The device beeps when the correct position on the corneal surface is obtained. The instrument measures IOP with the use of anesthetic drops in about 5 s and provides digital outcomes in addition to ocular pulse amplitude and quality score (Q that ranges from 1 to 6) data. Acceptable data are defined as Q < 4.

The Pascal DCT has shown high reproducibility [21]. As reported by several authors, the Pascal DCT measurements tend to be higher than GAT readings [22].

The Pascal DCT measurement seems to be not significantly affected by CCT [19]. Moreover, studies conducted on cadaver eyes and in patients who had undergone LASIK showed a significantly lower correlation of CCT with measured IOP values with Pascal DCT than with GAT [18].

#### *IV. Ocular response analyzer (ORA)*

The ocular response analyzer (ORA) (**Figure 4**) was introduced by Luce in 2005 [23]. It is an air-puff tonometer that, in addition to the traditional IOP measurement supplied, further indents the cornea with air pressure and measures the point at which the cornea recovers from applanation. This device is based on the principle that information on corneal biomechanics can be obtained by measuring the corneal deformation in response to an air impulse. The ORA uses an electro-optical infrared system to monitor the 3-mm central cornea [23]. The puff of air moves the cornea in an inward fashion, which is considered as the first inward applanation creating a mild concave form. As the force generated by the air decreases, the surface of the cornea takes on the normal convex form in this second outward applanation phase.

**Figure 4.** The ocular response analyzer (ORA).

The ORA provides interesting information on the biomechanical characteristics of the cornea. The rapid deformation of the cornea during the air impulse absorbs energy that causes a time delay in the occurrence of the applanation events that are the result of viscous damping in the corneal tissue. The difference between two independent pressure values derived from the two applanation events is defined as "hysteresis" (CH). The instruments provide the corneal compensated IOP (IOPcc) that utilizes information of individual corneal biomechanics, and it is reported to be less affected by corneal properties, such as CCT, in comparison with GAT [23].

Previous authors demonstrated that the ORA significantly overestimated IOP compared with GAT, especially at high IOP levels. Moreover, ORA IOP readings have been demonstrated to be affected by CCT [24].

#### *V. Corvis ST*

The Corvis ST (Oculus, Wetzlar, Germany) (**Figure 5**) [25] is a current used instrument that utilizes air-puff noncontact technology with built-in ultra-high-speed Scheimpflug technology. This instrument provides IOP measurements in addition to optical pachymetry data and *in vivo* corneal biomechanical parameters. The device measures corneal biomechanics data based on corneal deformation due to an applied force of air.

**Figure 5.** The Corvis ST pachy-tonometer.

applanation. This device is based on the principle that information on corneal biomechanics can be obtained by measuring the corneal deformation in response to an air impulse. The ORA uses an electro-optical infrared system to monitor the 3-mm central cornea [23]. The puff of air moves the cornea in an inward fashion, which is considered as the first inward applanation creating a mild concave form. As the force generated by the air decreases, the surface of the

The ORA provides interesting information on the biomechanical characteristics of the cornea. The rapid deformation of the cornea during the air impulse absorbs energy that causes a time delay in the occurrence of the applanation events that are the result of viscous damping in the corneal tissue. The difference between two independent pressure values derived from the two applanation events is defined as "hysteresis" (CH). The instruments provide the corneal compensated IOP (IOPcc) that utilizes information of individual corneal biomechanics, and it is reported to be less affected by corneal properties, such as CCT, in compari-

Previous authors demonstrated that the ORA significantly overestimated IOP compared with GAT, especially at high IOP levels. Moreover, ORA IOP readings have been demonstrated to

The Corvis ST (Oculus, Wetzlar, Germany) (**Figure 5**) [25] is a current used instrument that utilizes air-puff noncontact technology with built-in ultra-high-speed Scheimpflug technology. This instrument provides IOP measurements in addition to optical pachymetry data and *in vivo* corneal biomechanical parameters. The device measures corneal biomechanics data based

cornea takes on the normal convex form in this second outward applanation phase.

**Figure 4.** The ocular response analyzer (ORA).

60 Glaucoma - Intraocular Pressure and Aqueous Dynamics

son with GAT [23].

*V. Corvis ST*

be affected by CCT [24].

on corneal deformation due to an applied force of air.

The Corvis ST (Oculus, Wetzlar, Germany) released in 2013 [25] uses a quick burst of air (with a Gaussian distributed intensity and duration of 25 ms) on the center of the cornea that causes the initial inward applanation. The cornea then returns to a normal convex shape as the force of the air decreases to provide outward applanation. The Scheimpflug camera precisely records the corneal movements and records more than 100 images in about a half of a second with a resolution >600 × 400 pixels, for a central corneal surface of about 8.5 mm. The numerous corneal scans without the influence of the air puff are used to provide pachymetry readings. The first applanation phase is used to determine IOP measurements, which is not corrected for CCT and corneal deformation parameters (CDPs). The Corvis ST printout provides IOP and CCT measurements and 10 numerical corneal deformation parameters (CDPs), whose importance remains to be fully elucidated. They have been demonstrated to be highly sensitive to IOP that suggests their variation in relation to normal and pathological short- and long-term IOP fluctuations [26]. On the other hand, they are also known to be affected by IOP-independent variables, including patient age, CCT, corneal hydration, corneal stiffness and boundary conditions related to the sclera and the ocular muscles [26].

The precision of Corvis ST has been demonstrated to be excellent for the IOP and CCT values, but decreased for the CDPs [26]. The Corvis ST showed a tendency to underestimate IOP readings compared to GAT in some studies [25, 26]. IOP readings taken with Corvis ST seem to be significantly affected by CCT [25, 26].

#### *VI. Ocuton S*

The Ocuton S (EPSa GmbH, Saalfeld, Germany) (**Figure 6**) [27] is a handheld self-tonometer that works according to the principle of applanation tonometry established by Goldmann.

**Figure 6.** The Ocuton S self-tonometer.

After applying topical anesthesia, the patient holds the tonometer and applies the measuring prism directly to his cornea. The device provides a digital display of his IOP. Considering that the Ocuton S is based on the applanation principle, it should thus be influenced by the corneal thickness, although it is still unclear whether this influence is of a similar extent to that in Goldmann applanation tonometry [28].

The newer version Ocuton S\*TT-MV automatically verifies the applanated area. Lanfermann and coworkers showed that the Ocuton S\*TT-MV improves measuring accuracy significantly and approaches the accuracy achieved using GAT [29].

There is no "perfect" tonometer till date, and the clinicians must choose a tonometer wisely, balancing precision, accuracy, convenience and cost. IOP is a widely varying physiologic parameter; therefore, a series of IOP measurements should be taken, and the IOP data should only be used in the context of the overall clinical picture.

#### **Conflict of Interest and Source of Funding**

None.

### **Author details**

Maria Letizia Salvetat1\*, Marco Zeppieri2 and Paolo Brusini3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Ophthalmology, Azienda Ospedaliero-Universitaria "Santa Maria degli Angeli", Pordenone, Italy

2 Department of Ophthalmology, Azienda Ospedaliero-Universitaria "Santa Maria della Misericordia", Udine, Italy

3 Glaucoma Unit, "Città di Udine" Health Center, Udine, Italy

#### **References**

**Figure 6.** The Ocuton S self-tonometer.

Goldmann applanation tonometry [28].

62 Glaucoma - Intraocular Pressure and Aqueous Dynamics

and approaches the accuracy achieved using GAT [29].

only be used in the context of the overall clinical picture.

**Conflict of Interest and Source of Funding**

Maria Letizia Salvetat1\*, Marco Zeppieri2

\*Address all correspondence to<EMAIL_ADDRESS>
3 Glaucoma Unit, "Città di Udine" Health Center, Udine, Italy

None.

**Author details**

Angeli", Pordenone, Italy

Misericordia", Udine, Italy

After applying topical anesthesia, the patient holds the tonometer and applies the measuring prism directly to his cornea. The device provides a digital display of his IOP. Considering that the Ocuton S is based on the applanation principle, it should thus be influenced by the corneal thickness, although it is still unclear whether this influence is of a similar extent to that in

The newer version Ocuton S\*TT-MV automatically verifies the applanated area. Lanfermann and coworkers showed that the Ocuton S\*TT-MV improves measuring accuracy significantly

There is no "perfect" tonometer till date, and the clinicians must choose a tonometer wisely, balancing precision, accuracy, convenience and cost. IOP is a widely varying physiologic parameter; therefore, a series of IOP measurements should be taken, and the IOP data should

and Paolo Brusini3

1 Department of Ophthalmology, Azienda Ospedaliero-Universitaria "Santa Maria degli

2 Department of Ophthalmology, Azienda Ospedaliero-Universitaria "Santa Maria della

[27] Draeger J, Schwartz R, Deutsch C, Groenhoff S. Clinical and experimental results with a new fully automatic self-tonometer. Fortschr Ophthalmol 1991;88(3):304–307.

[14] Neuburger M, Maier P, Böhringer D, Reinhard T, Jordan JF. The impact of corneal edema on intraocular pressure measurements using goldmann applanation tonometry, Tono-

[15] Eklund A, Hallberg P, Linden C, Lindahl OA. An applanation resonator sensor for measuring intraocular using combined continuous force and area measurement. Invest

[16] Johannesson G, Hallberg P, Eklund A, Linden C. Introduction and clinical evaluation of servo-controlled applanation resonance tonometry. Acta Ophthalmol 2012;90:677–

[17] Salvetat ML, Zeppieri M, Tosoni C, Brusini P. Repeatability and accuracy of the applanation resonance tonometry in healthy subjects and patients with glaucoma. Acta

[18] Kaufmann C, Bachmann LM, Thiel MA. Intraocular pressure measurements using dynamic contour tonometry after laser *in situ* keratomileusis. Invest Ophthalmol Vis

[19] Pache M, Wilmsmeyer S, Lautebach S, Funk J. Dynamic contour tonometry versus Goldmann applanation tonometry: a comparative study. Graefe's Arch Clin Exp

[20] Park HJ, Uhm KB, Hong C. Reduction in intraocular pressure after laser *in situ*

[21] Wang AS, Alencar LM, Weinreb RN, et al. Repeatability and reproducibility of Goldmann applanation, dynamic contour, and ocular response analyzer tonometry. J

[22] Salvetat ML, Zeppieri M, Tosoni C, Brusini P. Comparison between Pascal dynamic contour tonometry, the TonoPen, and Goldmann applanation tonokmetry in patients

[23] Luce DA. Determining in vivo biomechanical properties of the cornea with an ocular

[24] Matinez-de-la-Casa JM, Garcia-Feijoo J, Fernandez-Vidal A, et al. Ocular Response Analyzer versus Goldmann applanation tonometry for intraocular pressure measure-

[25] Reznicek L, Muth D, Kampik A, et al. Evaluation of a novel Scheimpflug-based noncontact tonometer in healthy subjects and patients with ocular hypertension and

[26] Salvetat ML, Zeppieri M, Tosoni C, Felletti M, Grasso L, Brusini P. Corneal deformation parameters provided by the Corvis-ST Pachy-Tonometer in healthy subjects and

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682.

#### **FM Continuous Monitoring of Intraocular Pressure, an Engineering Perspective FM Continuous Monitoring of Intraocular Pressure, an Engineering Perspective**

Adrian E. Rendon‐Nava, Adrian E. Rendon‐Nava, Alejandro Díaz‐Méndez and Luis Nino‐de‐Rivera

Alejandro Díaz‐Méndez and Luis Nino‐de‐Rivera

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65294

#### **Abstract**

This chapter discusses the problem of continuously monitoring intraocular pressure (IOP) from an engineering perspective. It is aimed to all public in general although we think that medical staff and engineers may benefit the most from it. Although equations are included for engineers to get a glimpse of how the system works, this chapter does not go into great detail in mathematics and physics to make it understandable to medical staff. It provides though references for engineers who wish to get a better understanding of key subjects tackled in this chapter. The chapter is organized as follows: Section 1 introduces intraocular pressure (IOP) and need for its continuous monitoring. Section 2 describes the most recent efforts to develop a continuous IOP monitoring system. Section 3 shows what medical and engineering considerations must be taken into account to effectively measure IOP. Section 4 deals with health issues due to tissue warming and how to prevent them. Section 5 explains how an implant can be fabricated using either passive electronic components or active ones. Finally, Section 6 explains how the pressure sensor and the electronic circuits can be integrated.

**Keywords:** biomedical implants, IOP monitoring, magnetic coupling, wireless cou‐ pling, wireless power transmission

#### **1. Introduction**

Before we can begin to talk about intraocular pressure (IOP) continuous monitoring, let us have a look at some key facts according to the World Health Organization (WHO) [1]:

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Among the three major causes globally that cause visual impairment is glaucoma. Glaucoma can be understood as a group of ocular diseases mainly associated with a rise in intraocular pressure. All these groups of diseases have in common the progressive injury to the optic nerve [2]. If glaucoma is left untreated, it can cause total blindness.

The problem with diseases such as glaucoma is that they present no symptoms, so trying to diagnose it without previously measuring the IOP becomes a real challenge. Actual methods for IOP measurement involve the use of medical equipment called tonometers. Tonometers measure IOP over the cornea but in some cases, where the hardness of the cornea is above normal standards, important measurement errors can be produced which do not allow a correct IOP estimation.

There are nowadays other indirect methods that have been under research in the last years such as multifocal electroretinography to know the effects of IOP rising [3]. Nevertheless, with available tonometry and electroretinography techniques, it is not possible to take measure‐ ments during normal activities of the patient such as the sleep cycle, stage where IOP can be increased in a significant manner. In order to take an IOP measurement with tonometry or multifocal electroretinography methods, the measurement must be taken by qualified personnel which implies that the patient has to be in the hospital facilities.

The development of sensors with the capacity of measuring IOP inside the eyeball is of paramount importance in order to know with all precision not only intraocular pressure values but also IOP variations in daily activities of the patients.

A new measurement instrument is needed that allows medical staff to study the aetiology of diseases such as glaucoma, that is, to provide with a new tool which will enable to know if there is a cause‐effect relationship between daily activities of patients and IOP variations in real time.

Fortunately, we are at a point where technology has evolved in such a way that biomedical implants wirelessly powered are now a reality.

#### **2. State of the art**

There have been many efforts in different parts of the world over time to build an IOP monitoring system. In this section, we only present a few researches to give the reader a broad idea of what has been done in terms of IOP monitoring systems but references [4–8] are included in case the reader wants to know more about researches not mentioned in this chapter. Between the first researches carried out, there is the one done by Tufte et al. when in 1962 in Honeywell, they developed piezoresistive sensors with silicon membranes [9].

In 2000, Mokwa, Schnakenberg and collaborators proposed the design and fabrication of an implantable intraocular system for continuous IOP measurement through OPHTAL project [10]. The system consisted of a pressure sensor connected to the integrated circuits altogether in an artificial contact lens. Three prototypes were fabricated. The last one is shown in **Figure 1**.

**Figure 1.** Third pressure sensor prototype.

**•** There are 285 million people estimated to be visually impaired worldwide, of which 246

Among the three major causes globally that cause visual impairment is glaucoma. Glaucoma can be understood as a group of ocular diseases mainly associated with a rise in intraocular pressure. All these groups of diseases have in common the progressive injury to the optic

The problem with diseases such as glaucoma is that they present no symptoms, so trying to diagnose it without previously measuring the IOP becomes a real challenge. Actual methods for IOP measurement involve the use of medical equipment called tonometers. Tonometers measure IOP over the cornea but in some cases, where the hardness of the cornea is above normal standards, important measurement errors can be produced which do not allow a

There are nowadays other indirect methods that have been under research in the last years such as multifocal electroretinography to know the effects of IOP rising [3]. Nevertheless, with available tonometry and electroretinography techniques, it is not possible to take measure‐ ments during normal activities of the patient such as the sleep cycle, stage where IOP can be increased in a significant manner. In order to take an IOP measurement with tonometry or multifocal electroretinography methods, the measurement must be taken by qualified

The development of sensors with the capacity of measuring IOP inside the eyeball is of paramount importance in order to know with all precision not only intraocular pressure values

A new measurement instrument is needed that allows medical staff to study the aetiology of diseases such as glaucoma, that is, to provide with a new tool which will enable to know if there is a cause‐effect relationship between daily activities of patients and IOP variations in

Fortunately, we are at a point where technology has evolved in such a way that biomedical

There have been many efforts in different parts of the world over time to build an IOP monitoring system. In this section, we only present a few researches to give the reader a broad idea of what has been done in terms of IOP monitoring systems but references [4–8] are included in case the reader wants to know more about researches not mentioned in this chapter.

**•** About 90% of the visually impaired people globally live in low‐income environments.

**•** Eighty per cent of all visual impairment can be prevented or cured.

nerve [2]. If glaucoma is left untreated, it can cause total blindness.

personnel which implies that the patient has to be in the hospital facilities.

but also IOP variations in daily activities of the patients.

implants wirelessly powered are now a reality.

million have low vision.

68 Glaucoma - Intraocular Pressure and Aqueous Dynamics

correct IOP estimation.

real time.

**2. State of the art**

Humayun and collaborators proposed in 2008 a prototype sensor to measure IOP [11]. Two sensor designs were fabricated, one with a variable capacitor and the other one with a variable capacitor and a variable inductor (**Figure 2**).

The pressure sensor response showed a high sensitivity (>7000 ppm/mmHg) in both designs, confirming a resolution of less than 1 mmHg for biomedical applications. The authors also conducted a 6‐month study in animals to verify the implant bio‐stability *in vivo* and no surgical or post‐operation complications were found.

**Figure 2.** Sensor design variations (cross‐sectional view).

**Figure 3.** Sensor device.

In 2011, Melki and collaborators published a study with the purpose of determining the biocompatibility of an IOP sensor in rabbits and comparing IOP measurements from the sensor with other IOP sensor devices [12]. **Figure 3** shows the photograph of the sensor encapsulated in a silicon rubber.

The intraocular sensor was implanted in six New Zealand white rabbits. The upper part of the sensor contains the Application‐Specific Integrated Circuit (ASIC). The external diameter of the coil was of 11.3 mm, the inner diameter was of 7 mm and the thickness was of 0.9 mm.

The animals were observed and examined in intervals of up to 25 months after surgery. From the obtained results, it was found that the sensor had acceptable tolerance by the eye of the rabbit since no evidence of significant inflammation or scar formation was observed in *in vivo* tests. The measurements made with pneumotonometry, tonometry and with the sensor resulted in standard deviations of 2.70, 3.35 and 0.81 mmHg, respectively.

### **3. Merging medicine and engineering**

As seen in the previous section, all efforts to build a continuous IOP monitoring system proposed a wireless power transmission method. This obeys to the fact that having a battery inside the eye to power the biomedical implant can be both bulky and most importantly health threatening to the patient due to the hazardous chemicals it contains.

If we are to develop a continuous IOP monitoring system, we need to approach the problem with a multidisciplinary focus.

#### **3.1. Medical considerations**

**Figure 2.** Sensor design variations (cross‐sectional view).

70 Glaucoma - Intraocular Pressure and Aqueous Dynamics

In 2011, Melki and collaborators published a study with the purpose of determining the biocompatibility of an IOP sensor in rabbits and comparing IOP measurements from the sensor with other IOP sensor devices [12]. **Figure 3** shows the photograph of the sensor encapsulated

The intraocular sensor was implanted in six New Zealand white rabbits. The upper part of the sensor contains the Application‐Specific Integrated Circuit (ASIC). The external diameter of the coil was of 11.3 mm, the inner diameter was of 7 mm and the thickness was of 0.9 mm.

**Figure 3.** Sensor device.

in a silicon rubber.

The human eyeball measures approximately 2.5 cm in diameter so there is a restriction in terms of the maximum area that the implant can have to be successfully implanted in the patient. Among medical factors that come into play for implanting a continuous IOP monitoring system are the following:

#### *3.1.1. Electromagnetic radiation*

At frequencies between 10 MHz and 30 GHz, organic tissue warming is the major effect in electromagnetic energy absorption and temperature increases of more than 1 or 2°C can have adverse health effects [13].

A great number of physiological effects have been observed in cellular studies and with animals when electromagnetic energy is absorbed in levels that cause an increment in body temperature of more than 1 or 2°C [14]. These effects include alterations in neural and neuromuscular functions, increased permeability of the blood‐brain barrier, ocular damage, changes in the immune system associated with stress, haematological changes, reproductive changes and changes in cell morphology.

Experimental data available indicate that human exposition in a resting position to electro‐ magnetic fields for 30 min, which produce a specific absorption rate (SAR) in the whole body of 1–4 W kg−1, will result in an increase of temperature of less than 1°C. Exposition to electro‐ magnetic fields more intense producing SAR values of more than 4 W kg‐1 may overwhelm the thermoregulatory capacity of the body and produce warming tissue harmful levels. **Table 1** shows basic restrictions for exposure to time‐varying electric and magnetic fields for frequen‐ cies up to 10 GHz.

**Table 1.** Basic restrictions for time‐varying electric and magnetic fields exposition for frequencies up to 10 GHz.

#### **3.2. Engineering considerations**

Regarding technological restrictions for the localization of the implant, we can find the following:

Both of the previous considerations are based on the fact that the implant needs to maximize power transfer and magnetic coupling.

IOP measurement requires the size of the implant not to be so large neither so bulky so it can be implanted into the eye of the patient without causing any discomfort. This is the reason why several IOP monitoring systems are based on radiofrequency identification (RFID) technology [15].

Given that power consumption is a key factor in IOP monitoring system design, we may classify electronic implants based on their power consumption briefly as described below.

#### *3.2.1. Active electronic implant*

magnetic fields more intense producing SAR values of more than 4 W kg‐1 may overwhelm the thermoregulatory capacity of the body and produce warming tissue harmful levels. **Table 1** shows basic restrictions for exposure to time‐varying electric and magnetic fields for frequen‐

> ‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 0.4 0.4

‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 0.08 0.08

**Table 1.** Basic restrictions for time‐varying electric and magnetic fields exposition for frequencies up to 10 GHz.

**•** The implant should be placed as near as possible from the exterior of the body.

Regarding technological restrictions for the localization of the implant, we can find the

Both of the previous considerations are based on the fact that the implant needs to maximize

IOP measurement requires the size of the implant not to be so large neither so bulky so it can be implanted into the eye of the patient without causing any discomfort. This is the reason why several IOP monitoring systems are based on radiofrequency identification (RFID)

Given that power consumption is a key factor in IOP monitoring system design, we may classify electronic implants based on their power consumption briefly as described below.

**SAR average for the whole body (W·kg−1)**

**Localized SAR (head and trunk) (W·kg−1)**

‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 10 10

‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 2 2

**Localized SAR (limbs) (W·kg−1)**

‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 20 20

‐‐‐ ‐‐‐ ‐‐‐ ‐‐‐ 4 4

**Frequency range Current density**

10 MHz

10 MHz

**•** The implant must have the maximum area possible.

10 MHz to 10 GHz

10 MHz to 10 GHz

**3.2. Engineering considerations**

power transfer and magnetic coupling.

Up to 1 Hz 1–4 Hz 4 Hz to 1 kHz 1–100 kHz 100 kH to ‐

Up to 1 Hz 1–4 Hz 4 Hz to 1 kHz 1–100 kHz 100 kH to ‐

72 Glaucoma - Intraocular Pressure and Aqueous Dynamics

**for head and trunk (mA·m−2)**

**(rms)**

40 40/*f* 10 *f*/100 *f*/100 ‐‐‐

8 8/*f* 2 *f*/500 *f*/500 ‐‐‐

cies up to 10 GHz.

**Nature of the exposition**

Occupational exposure

General public exposure

following:

technology [15].

An active electronic implant will have active electronic components such as diodes and transistors. By using active electronic components, there is the drawback of higher power consumption. On the other hand, by using transistors and diodes the electronic implant would have the advantage of having more precise and reliable pressure measurements.

#### *3.2.2. Passive electronic implant*

The passive option consists of having pure passive electronic components (resistors, capacitors and inductors). The main advantage of this proposal is that power demand would be much lower than its active counterpart so a possible health risk in the patient due to tissue warming is greatly reduced.

Both types of implants will be discussed in more detail in Section 5.

#### **4. Health issues due to tissue warming**

A vital requirement when designing electronic implants is to limit temperature increment of the implant in order to prevent damaging organic tissue. In general, temperature variation in a body can be described by a partial differential equation of heat conduction which expresses the variation in the temperature of a body with respect of time, but in order for this equation to be used in human tissue, Pennes incorporated a few extra terms into the partial differential heat conduction equation to describe the warming effect of basal metabolism and the influence of blood in temperature regulation of tissue [16]. Pennes bio‐heat equation can be written as follows:

$$C\rho \frac{\partial T}{\partial t} = \nabla \cdot \left(K \nabla T\right) + A - B\left(T - T\_b\right) \tag{1}$$

where *A* is the heat production rate due to metabolic processes per volume unit, *B* is the perfusion constant and *Tb* is the blood temperature (it is assumed as a constant at *Tb* = 37°C).

The bio‐heat equation may be applied to human tissue without any external sources of heat. For our case of study, however, the temperature increase in human tissue is also caused by power dissipation of the electronic implant and by electromagnetic radiation coming from the external device that will be supplying wireless power to the implant.

#### **4.1. Joule heating**

When considering an electronic implant inside the human body, power dissipation by electronic circuitry has to be considered. Assuming that heat dissipation per volume unit of an implant is *Pi* , this can be incorporated in the bio‐heat equation as a source of heat and is expressed as

$$P\_l = V\_l \triangleq I\_l \tag{2}$$

where *Vi* is the biasing voltage of the electronic circuitry and *Ii* is the total electric current circulating through the implant.

#### **4.2. Electromagnetic energy contribution**

If a biological tissue is exposed to electromagnetic radiation, the electric field *E* and the magnetic field *H* penetrate the tissue causing it to warm up due to electromagnetic deposit. On the other hand, SAR is the specific absorption rate and it is defined as the differential with respect of time, of the increment of dissipated energy by a material. The SAR is related with the magnitude of the electric field so, once we know the electric field distribution in the tissue, we can obtain the SAR from which we can obtain the generated heat per volume unit per time unit to include the term to the bio‐heat equation.

In order to have a complete bio‐heat equation, we need to include Joule heating and electro‐ magnetic energy contribution. The complete bio‐heat equation can be expressed as

$$C\rho \frac{\partial T}{\partial t} = \nabla \cdot \left(K \nabla T\right) + A - B\left(T - T\_b\right) + \rho SAR + P\_l \tag{3}$$

Eq. (3) needs to be solved for each particular case of study to know how many degrees of temperature will the tissue increase caused by the implant. For the graphic shown in **Figure 4**, Eq. (3) was solved using Matlab software. **Figure 4** shows a graphic of how an electronic implant heats the tissue of the sclera when exposed to an electromagnetic energy radiation and joule heating. Details of the implant design and characteristics are shown in **Table 2**.

**Figure 4.** Warming graphic of the sclera. Exposition time: 60 s.

**Table 2.** Proposed electrical parameters for the simulated reader coil.

#### **5. Implant fabrication with passive and active electronic circuits**

As stated in Section 3, we can classify electronic implants based on the nature of their compo‐ nents. Next, we describe both options.

#### **5.1. Passive RLC implant**

*P V\*I i ii* = (2)

is the total electric current

is the biasing voltage of the electronic circuitry and *Ii*

If a biological tissue is exposed to electromagnetic radiation, the electric field *E* and the magnetic field *H* penetrate the tissue causing it to warm up due to electromagnetic deposit. On the other hand, SAR is the specific absorption rate and it is defined as the differential with respect of time, of the increment of dissipated energy by a material. The SAR is related with the magnitude of the electric field so, once we know the electric field distribution in the tissue, we can obtain the SAR from which we can obtain the generated heat per volume unit

In order to have a complete bio‐heat equation, we need to include Joule heating and electro‐

Eq. (3) needs to be solved for each particular case of study to know how many degrees of temperature will the tissue increase caused by the implant. For the graphic shown in **Figure 4**, Eq. (3) was solved using Matlab software. **Figure 4** shows a graphic of how an electronic implant heats the tissue of the sclera when exposed to an electromagnetic energy radiation and joule heating. Details of the implant design and characteristics are shown in **Table 2**.

 r

*<sup>t</sup>* (3)

magnetic energy contribution. The complete bio‐heat equation can be expressed as

 ( ) ( ) ¶ =Ñ× Ñ + - - + + ¶ *b i <sup>T</sup> C K T A B T T SAR P*

where *Vi*

circulating through the implant.

**4.2. Electromagnetic energy contribution**

74 Glaucoma - Intraocular Pressure and Aqueous Dynamics

per time unit to include the term to the bio‐heat equation.

r

**Figure 4.** Warming graphic of the sclera. Exposition time: 60 s.

For this kind of approach, an Resistive Inductive Capacitive (RLC) passive circuit is proposed as an IOP sensor like the one shown in **Figure 5**. The capacitor would be a variable capacitor, sensitive to variations in pressure.

**Figure 5.** Electric diagram of the circuit proposed as an IOP sensor.

The resonance frequency of the RLC circuit shown in **Figure 5** is given by Eq. (4) where we can note that if the value of capacitance varies, the resonance frequency of the circuit will change too.

$$f = \frac{1}{2\pi\sqrt{L\_sC\_s}}\tag{4}$$

From electromagnetic theory, we know that if we place two inductors close enough and we make a time‐varying electric current circulate through one of the coils, it will induce a voltage in the other coil. This is the principle by which wireless energy transfer is done. **Figure 6** shows a Maxwell‐Wien circuit which could act as the external device in charge of both delivering power to the implant and reading data from it.

**Figure 6.** Maxwell‐Wien bridge circuit coupled with the coil from the implant.

The implant is shown inside the dotted lines. If the RLC circuit in the implant formed by *Rs*, *Ls* and *Cs* is placed near the inductor *Lx*, a magnetic coupling will be created. By being *Lx* and *Ls* coupled, a change in the capacitance of *Cs* will change the value of the resonance frequency of the RLC circuit of the sensor which will in turn modify the impedance of *Lx* coil.

Eq. (5) shows how the impedance of the external device coil will vary by being coupled with the coil of the implant [17]

#### FM Continuous Monitoring of Intraocular Pressure, an Engineering Perspective http://dx.doi.org/10.5772/65294 77

$$Z\_x = j2\pi f L\_x \left[ 1 + k^2 \frac{\left(\frac{f}{f\_0}\right)^2}{1 - \left(\frac{f}{f\_0}\right)^2 + \frac{1}{Q} j\frac{f}{f\_0}} \right] \tag{5}$$

where *f* is the resonance frequency of the external device, *k* is the coupling factor between both coils, *Q* is given by

$$Q = 2\pi f\_0 L\_s \, / R\_s \tag{6}$$

And *f*0 is the resonance frequency of the implant described previously in Eq. (4).

The main disadvantage for this approach is the lack of precision and accuracy in the meas‐ urements. If we were to obtain precise and accurate measurements with a very small error, then both coils would have to be at the exact same place each time a measurement is to be taken. We could assume that the implant is always fixed so in order to make this kind of approach to work properly, the system design must be done to ensure that the coils will remain in the same position for each measurement and no misalignment will occur.

Another important issue to be noted is the transmission medium between coils. So far, we have considered air as the transmission medium between coils. Much more realistic calculations and simulations may be performed if organic tissue is considered as the transmission medium [18].

#### **5.2. Electronic implant using transistors**

The resonance frequency of the RLC circuit shown in **Figure 5** is given by Eq. (4) where we can note that if the value of capacitance varies, the resonance frequency of the circuit will change

1

*s s*

From electromagnetic theory, we know that if we place two inductors close enough and we make a time‐varying electric current circulate through one of the coils, it will induce a voltage in the other coil. This is the principle by which wireless energy transfer is done. **Figure 6** shows a Maxwell‐Wien circuit which could act as the external device in charge of both delivering

*<sup>f</sup> L C* (4)

2p=

power to the implant and reading data from it.

76 Glaucoma - Intraocular Pressure and Aqueous Dynamics

**Figure 6.** Maxwell‐Wien bridge circuit coupled with the coil from the implant.

the coil of the implant [17]

The implant is shown inside the dotted lines. If the RLC circuit in the implant formed by *Rs*, *Ls* and *Cs* is placed near the inductor *Lx*, a magnetic coupling will be created. By being *Lx* and *Ls* coupled, a change in the capacitance of *Cs* will change the value of the resonance frequency of

Eq. (5) shows how the impedance of the external device coil will vary by being coupled with

the RLC circuit of the sensor which will in turn modify the impedance of *Lx* coil.

too.

Designing an electronic implant using transistors needs special care since they require a much larger amount of power compared to passive RLC implants. Electronic circuit design must be done in such a way that warming of the tissue does not exceed 1°C to avoid organic tissue damage. Tissue warming is the main disadvantage of designing electronic implants using transistors. On the other hand, they are very precise and accurate since measurements depend only on having enough power available in the implant rather than if the coils are misaligned or not.

The active electronic circuit for the implant can be divided into three sub‐modules, which are reviewed below.

#### *5.2.1. Rectifier and regulator circuit*

As mentioned in Section 5.1, a time‐varying electric current circulating through one coil will induce a time‐varying voltage in the other coil. Biasing in electronic circuits, however, requires a direct current (DC) voltage so that is when the rectifier and the regulator circuits come into play.

#### *<IP_ADDRESS>. Rectifier circuit*

A rectifier circuit changes an alternating current into a direct current. It has a small error though, since at the output the voltage has a little ripple. **Figure 7** shows a full‐wave rectifier with P‐type metal‐oxide semiconductor (PMOS) transistors.

**Figure 7.** PMOS rectifier circuit.

#### *<IP_ADDRESS>. Regulator circuit*

To get rid of the small ripple at the output of the rectifier, a regulator circuit is often added.

The aim of the regulator is to deliver a stable power supply voltage for proper operation of the rest of the circuits in the implant. It has a minimum voltage needed at the input below which it will not deliver a constant voltage. If the input voltage is, on the other hand, greater than the minimum, then the regulator will output a constant voltage. This is the circuit responsible for making active electronic implants independent of coils distance. If the distance between coils is enough for the regulator to provide an output, it will provide a steady voltage regardless of how close the coils are. If on the contrary, the distance between coils is not close enough, then the voltage regulator simply will not provide any voltage at all. **Figure 8** depicts a Comple‐ mentary Metal‐Oxide Semiconductor (CMOS) topology for a voltage regulator circuit.

**Figure 8.** Topology of a voltage regulator. Taken with permission from [19].

#### *5.2.2. Signal‐conditioning circuit*

a direct current (DC) voltage so that is when the rectifier and the regulator circuits come into

A rectifier circuit changes an alternating current into a direct current. It has a small error though, since at the output the voltage has a little ripple. **Figure 7** shows a full‐wave rectifier

To get rid of the small ripple at the output of the rectifier, a regulator circuit is often added.

The aim of the regulator is to deliver a stable power supply voltage for proper operation of the rest of the circuits in the implant. It has a minimum voltage needed at the input below which it will not deliver a constant voltage. If the input voltage is, on the other hand, greater than the minimum, then the regulator will output a constant voltage. This is the circuit responsible for making active electronic implants independent of coils distance. If the distance between coils is enough for the regulator to provide an output, it will provide a steady voltage regardless of how close the coils are. If on the contrary, the distance between coils is not close enough, then the voltage regulator simply will not provide any voltage at all. **Figure 8** depicts a Comple‐ mentary Metal‐Oxide Semiconductor (CMOS) topology for a voltage regulator circuit.

with P‐type metal‐oxide semiconductor (PMOS) transistors.

play.

*<IP_ADDRESS>. Rectifier circuit*

78 Glaucoma - Intraocular Pressure and Aqueous Dynamics

**Figure 7.** PMOS rectifier circuit.

*<IP_ADDRESS>. Regulator circuit*

Once we have a stable DC voltage, we can move on to the next sub‐module of the implant. If the sensor is in charge of transforming an analogue physical signal into an electric variable, the signal‐conditioning circuit has the task of taking the analogue electric signal and manip‐ ulate it in such a way that it meets the requirements for the next stage in the system. For our case, the next stage would be the transmission circuit.

**Figure 9.** Cross‐coupled LC VCO topology.

There are many electric circuit designs that can be used as a signal‐conditioning circuit. Here, we take a brief look at a voltage‐controlled oscillator (VCO) [20]. As seen in Section 2, most pressure sensors are variable capacitors which change their value of capacitance according to a change in pressure. On the other hand, a VCO is an oscillator with an oscillating frequency that depends on the value of its capacitor. If we connect the pressure sensor with the VCO, then we would have a signal‐conditioning circuit for IOP monitoring. In **Figure 9**, a cross‐ coupled LC VCO topology is shown.

#### *5.2.3. Transmission circuit*

The transmission circuit sub‐module takes the conditioned signal and transmits it back to the external device. A power amplifier (PA) circuit can achieve the latter. Since we are discussing circuits for an electronic implant, it would be desirable to have a low‐power amplifier to deal with the transmission. There are many PAs from where we can choose; there are linear amplifiers (Classes A, B and AB) or switched‐mode amplifiers (Classes E and F). In **Figure 10**, a class E PA is shown. Switched‐mode amplifiers have a higher efficiency than linear ones (50– 70% in the case of linear vs. a theoretical 100% in switched‐mode amplifiers). Chapter 5 in [21] has a deeper explanation in power amplifiers for biomedical implants.

**Figure 10.** Class E power amplifier. Taken with permission from [22].

#### **6. Sensor and circuit integration**

At this point, the reader may ask: Why do we even need this section since at present, sensors and electronic circuits can be fabricated on the same substrate? And the answer is: Because it depends on what type of application we want to develop.

It is true that nowadays it is possible to fabricate sensors and circuits in the same substrate. The inconvenience with actual fabrication processes is that they use a rigid silicon wafer as a substrate. For many applications, this is enough, but for biomedical applications this approach is not useful. That is why in Section 2 all proposals that have active circuits in their designs (such as the one shown in **Figure 3**) tend to divide the fabrication process of the implant. On one hand, they fabricate the coil in a flexible substrate such as parylene, silicone rubber or polyimide so the implant can adapt to the curved surface of the eye. On the other hand, all electronic circuitry and in general all IOP sensors too are fabricated in a rigid silicon wafer. The final step involves connecting the coil with the sensor and the electronic circuits.

The main drawback from this approach is the potential damage that the silicon wafer can cause to the tissue of the eye. Fortunately, there has been a significant advance in terms of electronic circuit fabrication on a flexible substrate. In **Figure 11**, an electronic circuit fabrication process with Carbon Nanotubes (CNT) on a flexible substrate and photographs of the final chip are shown.

**Figure 11.** Circuit fabrication process on a flexible substrate. (a) Fabrication process of flexible InGaZnO‐CNT CMOS logic circuits. Photographs of flexible CMOS circuits under (b) bending by hand and (c) rolling over a glass bar (∼2.6‐ mm radius). (d) Atomic Force Microscopy (AFM) image of CNT network film for p‐type type thin‐film Transistors (TFT). (e) FEM simulation plot modelling the strain distribution in the InGaZnO channel region under bending (*r* = 2.6 mm). Taken with permission from [23].

#### **7. Summary**

pressure sensors are variable capacitors which change their value of capacitance according to a change in pressure. On the other hand, a VCO is an oscillator with an oscillating frequency that depends on the value of its capacitor. If we connect the pressure sensor with the VCO, then we would have a signal‐conditioning circuit for IOP monitoring. In **Figure 9**, a cross‐

The transmission circuit sub‐module takes the conditioned signal and transmits it back to the external device. A power amplifier (PA) circuit can achieve the latter. Since we are discussing circuits for an electronic implant, it would be desirable to have a low‐power amplifier to deal with the transmission. There are many PAs from where we can choose; there are linear amplifiers (Classes A, B and AB) or switched‐mode amplifiers (Classes E and F). In **Figure 10**, a class E PA is shown. Switched‐mode amplifiers have a higher efficiency than linear ones (50– 70% in the case of linear vs. a theoretical 100% in switched‐mode amplifiers). Chapter 5 in [21]

At this point, the reader may ask: Why do we even need this section since at present, sensors and electronic circuits can be fabricated on the same substrate? And the answer is: Because it

It is true that nowadays it is possible to fabricate sensors and circuits in the same substrate. The inconvenience with actual fabrication processes is that they use a rigid silicon wafer as a substrate. For many applications, this is enough, but for biomedical applications this approach is not useful. That is why in Section 2 all proposals that have active circuits in their designs (such as the one shown in **Figure 3**) tend to divide the fabrication process of the implant. On

has a deeper explanation in power amplifiers for biomedical implants.

**Figure 10.** Class E power amplifier. Taken with permission from [22].

depends on what type of application we want to develop.

**6. Sensor and circuit integration**

coupled LC VCO topology is shown.

80 Glaucoma - Intraocular Pressure and Aqueous Dynamics

*5.2.3. Transmission circuit*

In this chapter, we presented an overview of how an electronic implant can be designed. We showed what has been done in this area over the years and we pointed to a major medical concern that is tissue warming when it comes to implanting the IOP sensor into a patient.

From an engineering perspective and despite the tremendous advance that has been made in this area, there are still key issues that must be tackled in the years to come such as power efficiency in wireless power transfer. Another aspect, not less important, is the possibility to have electronic circuits fabricated in the same flexible substrate along with the pressure sensor. This is an area of recent creation that has though an immense growth potential. Microelectronic devices fabricated in a flexible substrate can have applications not only in intraocular pressure monitoring but in many other biomedical applications. And these circuits can not only have biomedical applications demand but also in other numerous engineering fields such as energy harvesting, domotics and wearable technology (clothing and accessories).

#### **Author details**

Adrian E. Rendon‐Nava1\*, Alejandro Díaz‐Méndez2 and Luis Nino‐de‐Rivera1

#### **References**

[7] Chow, E. Y., Chlebowski, A. L., Irazoqui, P. P. A miniature‐implantable RF‐wireless active glaucoma intraocular pressure monitor. IEEE Trans. Biomed. Circ. Syst. 2010;4(6): 340–349. DOI: 10.1109/TBCAS.2010.2081364.

From an engineering perspective and despite the tremendous advance that has been made in this area, there are still key issues that must be tackled in the years to come such as power efficiency in wireless power transfer. Another aspect, not less important, is the possibility to have electronic circuits fabricated in the same flexible substrate along with the pressure sensor. This is an area of recent creation that has though an immense growth potential. Microelectronic devices fabricated in a flexible substrate can have applications not only in intraocular pressure monitoring but in many other biomedical applications. And these circuits can not only have biomedical applications demand but also in other numerous engineering fields such as energy

and Luis Nino‐de‐Rivera1

harvesting, domotics and wearable technology (clothing and accessories).

2 National Institute of Astrophysics, Optics and Electronics, Puebla, Mexico

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[3] Miguel‐Jiménez, J. M., Ortega, S., Boquete, L., Rodríguez‐Ascariz, J. M., Blanco, R. Multifocal ERG wavelet packet decomposition applied to glaucoma diagnosis. Biomed.

[4] Cooper, R. L., Beale, D. Radio telemetry of intraocular pressure in vitro. Invest.

[5] Rosengren, L., Backlund, Y., Sjostrom, T., Hok, B., Svedbergh, B. A system for wireless intra‐ocular pressure measurements using a silicon micromachined sensor. J. Micro‐

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Adrian E. Rendon‐Nava1\*, Alejandro Díaz‐Méndez2

82 Glaucoma - Intraocular Pressure and Aqueous Dynamics

1 National Polytechnic Institute, Mexico City, Mexico

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\*Address all correspondence to<EMAIL_ADDRESS>
**Author details**

**References**

10.5772/17561. Available from: http://www.intechopen.com/books/current‐trends‐ and‐challenges‐in‐rfid/structural‐design‐of‐cmos‐voltage‐regulator‐for‐implantable‐ devices.

**Managing Intraocular Pressure Dynamics**

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[20] Rendon‐Nava, A. E., Nino‐de‐Rivera, L., Díaz‐Méndez, J. A. A 1‐V supply voltage controlled oscillator for intraocular pressure measurement. Telecommun. Radio Eng.

[21] Siu, C., Iniewski, K. CMOS circuits for wireless medical applications. In: Krzysztof, I., editor. VLSI Circuits for Biomedical Applications. Norwood, MA: Artech House; 2008.

[23] Honda, W., Arie, T., Akita, S., Takei, K. Mechanically flexible and high‐performance CMOS logic circuits. Sci. Rep. 2015;5:15099. DOI: http://dx.doi.org/10.1038/srep15099.

[22] Sokal, A. D. Class E RF power amplifiers. QEX Mag. 2001;(204):9–20.

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2013;72(16):1537–1544.

84 Glaucoma - Intraocular Pressure and Aqueous Dynamics

#### **Managing Intraocular Pressure: Innovation in Glaucoma Management Managing Intraocular Pressure: Innovation in Glaucoma Management**

Anne-Marie Bleau, Beatriz Vargas, Ana Isabel Jiménez and Covadonga Pañeda Anne-Marie Bleau, Beatriz Vargas, Ana Isabel Jiménez and Covadonga Pañeda

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65972

#### **Abstract**

Primary open-angle glaucoma is a progressive ocular neuropathy that if left untreated may lead to blindness. The main risk factor for developing glaucoma is increased intraocular pressure. Intraocular pressure is regulated by the balance of aqueous humour synthesis and secretion into the eye and outflow from the eye; therefore, most therapies for glaucoma seek lowering intraocular pressure to avoid disease progression. There are several types of drugs in the market for the treatment of glaucoma, but there are still unmet needs to be overcome; therefore, significant effort has been put in the last few years to develop new medicines with innovative mechanisms of action as well as devices to improve quality of life in glaucoma patients. The present review offers a thorough revision of the latest advances in the glaucoma therapy field, focusing on innovative approaches, new targets and new mechanisms of action.

**Keywords:** glaucoma, innovation, therapy, oligonucleotides, devices, stem cells, gene therapy

#### **1. Introduction**

Primary open-angle glaucoma (POAG) is a multi-factorial optic neuropathy characterized by retinal ganglion cell degeneration and progressive visual field loss [1]. The underlying molecular changes leading to ocular tissue damage in glaucoma are largely unknown, but it has been shown that reduction in intraocular pressure (IOP) correlates with a decrease in disease progression. As such, treatment in glaucoma is mainly oriented towards reducing IOP to reach a target reduction of approximately 25–30% from the patient's baseline [2]. Pressure in the eye is maintained by balancing the amount of fluid contained within the anterior and

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

posterior chambers; thus, reduction in IOP is achieved either by reducing the amount of aqueous humour (AH) secreted into the eye or by increasing its outflow [1]. AH is produced by the epithelial cells of the ciliary processes through a complex mechanism that involves ultrafiltration, active transport and diffusion; AH is thereafter secreted into the posterior chamber. AH circulates from the posterior chamber around the lens and through the pupil into the anterior chamber and exits the eye trough one of two pathways: the conventional pathway or the uveoscleral route. AH exiting the anterior chamber through the conventional route crosses the trabecular meshwork (TM) to reach the Schlemm's canal (SC) located at the limbus. Contraction of the ciliary muscle causes the TM to expand and SC to open resulting in increased outflow through this route. The main source of outflow resistance through this route is the extracellular matrix of the TM and the inner wall endothelium of the SC. Through the unconventional or uveoscleral route, AH flows from the iris angle through the anterior face of ciliary muscle into the connective tissue located between the muscle bundles to finally reach the suprachoroidal space. The fluid is thereafter drained through the sclera or the perivascular spaces into the episcleral tissue where it enters the venous circulation. In contrast to the conventional route, the main source of AH outflow resistance is the ciliary body [3].

The eye is a contained organ that is partially isolated from the rest of the body; this isolation provides a certain immune privilege and limits the amount of compound needed to perform proof-of concept studies. In addition, the eye has a sophisticated structure with many different cell types and specialized barriers making it an ideal organ for studying delivery of larger compounds. As such, the eye has often been used to study new mechanisms of action and to obtain initial data for new compounds in development. Therefore, it is not surprising that most innovative new classes of drugs have, at some point, been tested in the eye, among these new classes of drugs are aptamers, antisense oligonucleotides (ASO), short interfering RNAs (siRNA), antibodies, stem cells, gene therapy and different types of delivery approaches and devices.

Here, we will review innovative programs developing drugs for the treatment of glaucoma focusing on the latest advances oriented towards lowering IOP, paying particular attention to the mechanisms used by these drugs and devices.

#### **2. Pharmacological innovation**

Progressive and irreversible loss of vision is the most feared complication for glaucoma. While current treatments focus on lowering IOP, up to date no therapies have been approved to address the critical issue of visual field deterioration. For this reason, high unmet needs remain for the development of innovative approaches to treat such aspect of the disease. Fortunately, beyond classic eye drop medicines, various exciting therapies are emerging in the field. Overall, these techniques aim to provide either a novel alternative to reduce IOP or a protection and regeneration of retinal ganglion cells (RGCs) to ultimately reverse vision loss. In this section, we will describe novel techniques for the treatment of glaucoma. Interestingly, these complex strategies have been combined in order to achieve broader efficacy.

Traditionally, drug discovery has been based on screening large libraries of compounds to select products with specific activities. However, in the last decades, advances in the molecular biology field have allowed the search for active candidates to become more rational. As a result, new pharmaceuticals with innovative mechanisms of action, target specific design and improved pharmaceutical properties have emerged. Some of the advantages of these new compounds include increased specificity, reduced toxicity and the ability to address targets that cannot be engaged by traditional small molecules. Among these new pharmaceuticals are biologicals, stem cells, gene therapy and therapies based on oligonucleotides.

Developing innovative compounds for the treatment of glaucoma entails thus targeting the function of cells and tissues related to IOP control; these tissues include the ciliary muscle, the TM, the SC, collector channels and aqueous veins. The latest discoveries in molecular biology allow the identification of key molecules that control AH dynamics in these tissues, and these molecules can be used as starting point for new therapeutic strategies and discovery of new targets. In order to fully take advantage of these approaches, the characteristics of these specific tissues have to be taken into account when designing the therapeutic strategy. The following characteristics are particularly interesting when developing innovative glaucoma therapies:

#### **2.1. New drug targets**

posterior chambers; thus, reduction in IOP is achieved either by reducing the amount of aqueous humour (AH) secreted into the eye or by increasing its outflow [1]. AH is produced by the epithelial cells of the ciliary processes through a complex mechanism that involves ultrafiltration, active transport and diffusion; AH is thereafter secreted into the posterior chamber. AH circulates from the posterior chamber around the lens and through the pupil into the anterior chamber and exits the eye trough one of two pathways: the conventional pathway or the uveoscleral route. AH exiting the anterior chamber through the conventional route crosses the trabecular meshwork (TM) to reach the Schlemm's canal (SC) located at the limbus. Contraction of the ciliary muscle causes the TM to expand and SC to open resulting in increased outflow through this route. The main source of outflow resistance through this route is the extracellular matrix of the TM and the inner wall endothelium of the SC. Through the unconventional or uveoscleral route, AH flows from the iris angle through the anterior face of ciliary muscle into the connective tissue located between the muscle bundles to finally reach the suprachoroidal space. The fluid is thereafter drained through the sclera or the perivascular spaces into the episcleral tissue where it enters the venous circulation. In contrast to the conventional route, the main source of AH outflow resistance is the ciliary body [3].

The eye is a contained organ that is partially isolated from the rest of the body; this isolation provides a certain immune privilege and limits the amount of compound needed to perform proof-of concept studies. In addition, the eye has a sophisticated structure with many different cell types and specialized barriers making it an ideal organ for studying delivery of larger compounds. As such, the eye has often been used to study new mechanisms of action and to obtain initial data for new compounds in development. Therefore, it is not surprising that most innovative new classes of drugs have, at some point, been tested in the eye, among these new classes of drugs are aptamers, antisense oligonucleotides (ASO), short interfering RNAs (siRNA), antibodies, stem cells, gene therapy and different types of delivery approaches and devices.

Here, we will review innovative programs developing drugs for the treatment of glaucoma focusing on the latest advances oriented towards lowering IOP, paying particular attention to

Progressive and irreversible loss of vision is the most feared complication for glaucoma. While current treatments focus on lowering IOP, up to date no therapies have been approved to address the critical issue of visual field deterioration. For this reason, high unmet needs remain for the development of innovative approaches to treat such aspect of the disease. Fortunately, beyond classic eye drop medicines, various exciting therapies are emerging in the field. Overall, these techniques aim to provide either a novel alternative to reduce IOP or a protection and regeneration of retinal ganglion cells (RGCs) to ultimately reverse vision loss. In this section, we will describe novel techniques for the treatment of glaucoma. Interestingly,

Traditionally, drug discovery has been based on screening large libraries of compounds to select products with specific activities. However, in the last decades, advances in the molecular

these complex strategies have been combined in order to achieve broader efficacy.

the mechanisms used by these drugs and devices.

**2. Pharmacological innovation**

88 Glaucoma - Intraocular Pressure and Aqueous Dynamics

As mentioned above, current approaches to develop new drugs for the treatment of glaucoma are aimed towards decreasing IOP to avoid further damage to the glaucomatous eye. Five drug-types are currently approved to treat glaucoma: alpha agonists, beta-blockers, carbonic anhydrase inhibitors, prostaglandin analogues and cholinergic drugs. Given the information available from the field of genetics, it is surprising that no new targets have reached the clinic since prostaglandin analogues, but this may change in the near future. Here, we review the information and mechanistic data existing for three new targets against which several compounds are currently under clinical development.

#### *2.1.1. Rho-associated protein kinase (ROCK) inhibitors*

#### *<IP_ADDRESS>. Description and biological function*

The Rho family consists of three guanosine triphosphate (GTP)-binding proteins named RhoA, RhoB and RhoC, which belong to the Ras-superfamily of GTPases [4]. Rho proteins bound to guanosine diphosphate (GDP) remain inactive in the cytoplasm of the cell; upon binding of GTP, these proteins become active and translocate to the cellular membrane where they exert their function [5]. The Rho activation and inactivation cycle is regulated by GTP-ase activating proteins (GAP) and guanine nucleotide-exchange factors (GEFs) that catalyse GTP and GDP exchange [6]. Rho proteins are ubiquitously expressed and participate in the regulation of cytoskeletal dynamics, thus playing a central role in cell morphology, adhesion and migration, as well as in numerous signalling pathways [7].

One of the most comprehensively studied effectors of Rho proteins is the Rho-associated coiled-coil containing protein kinases (ROCK), which are serine-threonine kinases composed of a catalytic domain, a coiled-coin Rho-binding site and an auto-inhibitory domain [9]. In humans, ROCKs exist as two isoforms, ROCK1 and ROCK2, expressed in a wide variety of tissues including the TM and the ciliary muscle cells [10]. Multiple studies indicate that ROCK regulate the contractile properties of the TM, synthesis of extra-cellular matrix (ECM) and outflow of AH through the TM; factors known to be involved in AH dynamics [11]. In addition, ROCK1 and ROCK2 knock-out mice exhibit lower values of IOP when compared to those of their wild-type littermates [12].

#### *<IP_ADDRESS>. Mechanism of action*

Upon activation, ROCK phosphorylates a large number of substrates inducing their activation or inhibition; many of these substrates, such as the myosin light chain (MLC), the myosin phosphatase (MLP) and actin-binding LIM kinase (LIMK), actively participate in cytoskeletal dynamics and cell motility of the TM, SC and ciliary muscle [13, 14]. The contraction/relaxation status of these structures influences the resistance to AH outflow and as a result modulate IOP homeostasis. In consequence, the results of multiple studies have proposed a role of ROCK inhibitors in enhancing AH drainage through the TM by altering the cytoskeleton [15]. NF-κβ is another downstream effector of the ROCK pathway; its activation controls the translation of pro-inflammatory mediators such as interleukins or TNF-α.

The anti-fibrotic activity of ROCK inhibitors seems also to be relevant to the therapeutic role of these agents in glaucoma. Post-operative scaring is one of the main causes of filtration surgery failure; scaring tissue formed at the TM leads to poor IOP control and allows silent disease progression. The differentiation of fibroblasts to myofibroblasts during wound healing and scar formation is mediated by TGF-β that facilitates the contractile response of fibroblasts. ROCK inhibitors such as Y-27632 and AMA0526 have demonstrated to improve surgical outcome in animal models of glaucoma filtration surgery [16, 17]. Additionally, ROCK inhibitors such as Y-39983 and fasudil have been found to improve blood flow to the optic nerve head, seemingly due to their action on MLC that regulates the contraction of smooth muscle cells in the blood vessels irrigating this area [18, 19]. Finally, it has been shown that ROCK may also have an effect on central nervous system (CNS) targets involved in neuronal survival and axonal regeneration thus giving ROCK inhibitors an added-on value to their role on AH dynamics [20].

#### *<IP_ADDRESS>. Drugs in development*

bound to guanosine diphosphate (GDP) remain inactive in the cytoplasm of the cell; upon binding of GTP, these proteins become active and translocate to the cellular membrane where they exert their function [5]. The Rho activation and inactivation cycle is regulated by GTP-ase activating proteins (GAP) and guanine nucleotide-exchange factors (GEFs) that catalyse GTP and GDP exchange [6]. Rho proteins are ubiquitously expressed and participate in the regulation of cytoskeletal dynamics, thus playing a central role in cell morphology, adhesion and migration, as well as in numerous signalling pathways [7]. Increased levels of RhoA expres-

One of the most comprehensively studied effectors of Rho proteins is the Rho-associated coiled-coil containing protein kinases (ROCK), which are serine-threonine kinases composed of a catalytic domain, a coiled-coin Rho-binding site and an auto-inhibitory domain [9]. In humans, ROCKs exist as two isoforms, ROCK1 and ROCK2, expressed in a wide variety of tissues including the TM and the ciliary muscle cells [10]. Multiple studies indicate that ROCK regulate the contractile properties of the TM, synthesis of extra-cellular matrix (ECM) and outflow of AH through the TM; factors known to be involved in AH dynamics [11]. In addition, ROCK1 and ROCK2 knock-out mice exhibit lower values of IOP when compared to

Upon activation, ROCK phosphorylates a large number of substrates inducing their activation or inhibition; many of these substrates, such as the myosin light chain (MLC), the myosin phosphatase (MLP) and actin-binding LIM kinase (LIMK), actively participate in cytoskeletal dynamics and cell motility of the TM, SC and ciliary muscle [13, 14]. The contraction/relaxation status of these structures influences the resistance to AH outflow and as a result modulate IOP homeostasis. In consequence, the results of multiple studies have proposed a role of ROCK inhibitors in enhancing AH drainage through the TM by altering the cytoskeleton [15]. NF-κβ is another downstream effector of the ROCK pathway; its activation controls the translation of pro-inflammatory mediators such as interleukins or

The anti-fibrotic activity of ROCK inhibitors seems also to be relevant to the therapeutic role of these agents in glaucoma. Post-operative scaring is one of the main causes of filtration surgery failure; scaring tissue formed at the TM leads to poor IOP control and allows silent disease progression. The differentiation of fibroblasts to myofibroblasts during wound healing and scar formation is mediated by TGF-β that facilitates the contractile response of fibroblasts. ROCK inhibitors such as Y-27632 and AMA0526 have demonstrated to improve surgical outcome in animal models of glaucoma filtration surgery [16, 17]. Additionally, ROCK inhibitors such as Y-39983 and fasudil have been found to improve blood flow to the optic nerve head, seemingly due to their action on MLC that regulates the contraction of smooth muscle cells in the blood vessels irrigating this area [18, 19]. Finally, it has been shown that ROCK may also have an effect on central nervous system (CNS) targets involved in neuronal survival and axonal regeneration thus giving ROCK inhibitors an added-on value to their role on AH

sion have been detected in optic nerve head of glaucomatous eyes [8].

those of their wild-type littermates [12].

90 Glaucoma - Intraocular Pressure and Aqueous Dynamics

*<IP_ADDRESS>. Mechanism of action*

TNF-α.

dynamics [20].

**Y-27632** was the first identified ROCK specific inhibitor, and **SNJ-1656** (also known as Y-39983/ RKI983; Senju and Novartis Pharmaceuticals) was the first ROCK inhibitor to demonstrate an IOP-lowering effect in human subjects [21]. Despite its effect in humans, the clinical development of this compound was halted in Phase II due to insufficient efficacy and a poor tolerability profile [22]. ROCK inhibitors currently undergoing clinical trials in glaucoma include ripasudil (KowaCompany, Ltd; Japan), netarsudil (Aerie Pharmaceuticals, Inc; USA), PG324 (Aerie Pharmaceuticals, Inc; USA) and AMA0076 (Amakem; Belgium).

**Ripasudil hydrochloride hydrate**, formerly known as K-115 (Glanatec® ophthalmic solution 0.4%), was approved in Japan in September 2014 for the treatment of glaucoma and ocular hypertension when other therapeutic drugs are not effective or cannot be administered, at the dosage of one drop per eye, twice daily (b.i.d) [23, 24]. Compiled data from Phase II and III clinical trials indicated that this drug achieved a 15% IOP reduction (3.5 mmHg), being conjunctival hyperaemia the most frequently reported adverse event with incidence rates ranging from 55 to 74% [25, 26]. Non-clinical studies proved that ripasudil inhibited both ROCK1 and ROCK2 (IC50 0.051 and 0.019 μmol/L, respectively) and mechanistic studies performed in rabbits demonstrated that its ocular hypotensive effect is due to increased drainage of AH through the TM and SC [27]. Ripasudil induces cytoskeletal changes secondary to ROCK inhibition that lead to the retraction and rounding of the TM cells decreasing the compaction of the TM allowing aqueous outflow. Additionally, *in vitro* studies demonstrated that ripasudil reduced outflow resistance and increased SC endothelial cell permeability [28].

**Netarsudil** (RhopressaTM ophthalmic solution, 0.02%), formerly known as AR-13324, is a ROCK and a norepinephrine transporter (NET) inhibitor currently in Phase III clinical trials [29]. In a Phase II study, this product achieved a 22% reduction in mean diurnal IOP after 28 treatment days when administered in eye drops once daily (*q.d)*. However, non-inferiority versus latanoprost was not met [30]. IOP reduction is thought to be achieved by three different mechanisms of action: increasing AH outflow through the TM, reducing the pressure in the epliscleral vein and reducing AH synthesis. Real-time effect of netarsudil on AH dynamics was evaluated *in vivo* both in albino and pigmented mice using a custom-made optical coherence tomography system [31]. This technique confirmed that the IOP-lowering effect of netarsudil is related to its action both in proximal and distal steps of the outflow pathway. It was noted that the observed cytoskeletal changes induced by netarsudil caused the expansion of the conventional outflow tissues such as the TM and the SC avoiding its collapse at elevated IOP. As a NET inhibitor, netarsudil is thought to decrease AH secretion since elevated norepinephrine levels activate α2 adrenergic receptors responsible for AH production at the ciliary processes [32]. Finally, netarsudil is also thought to have vasodilator properties as it reduces episcleral venous pressure in rabbits facilitating AH drainage to the bloodstream [32]. Similarly to ripasudil, ocular hyperaemia was the most frequently reported AE during clinical development with incidence rates of 57% which improved during the course of the study decreasing to 24% after 28 treatment days.

**Roclatan**TM, formerly known as PG323, is a fixed dose combination (FDC) of netarsudil 0.02% and latanoprost 0.005%, which combines the previously described mechanisms of action with the capacity of the prostaglandins of increasing AH outflow through the uveoscleral pathway [33, 34]. This fixed combination currently in Phase III clinical trials achieved clinically and statistically superiority in terms of ocular hypotensive efficacy when compared to its individual active components at the same concentrations. However, incidence rates of ocular hyperaemia were superior in the FDC than those observed in the latanoprost group (40 vs. 60%).

**AMA0076** is a locally acting ROCK inhibitor currently in Phase II clinical trials specifically designed to reduce IOP while minimizing side effects such as hyperemia. *In vivo* studies conducted in an acute hypertensive rabbit model showed that AMA0076 prevented IOP elevation more efficiently than latanoprost and bimatoprost. *In vitro* studies conducted in rabbit ocular tissues demonstrated that AMA0076 was able to induce reversible changes in cell shape and decreased the number of actin filaments and focal adhesions that may facilitate AH outflow [35].

#### *2.1.2. Adenosine receptor ligands*

#### *<IP_ADDRESS>. Description and biological function*

Adenosine is an endogenous nucleoside modulator of both intracellular and extracellular origin. Adenosine half-life is very limited (~1.5 seconds) as it is rapidly metabolized to inosine and hipoxantine; this is why extracellular levels of adenosine, which usually range from 20 to 200 nM, are considered a good indicator of cellular homeostasis. Adenosine levels increase, even up to the micromolar range, in response to cellular stress conditions such as tissue hypoxia or ischemia. In fact, adenosine levels in AH are significantly elevated in ocular hypertensive individuals when compared to normotensive individuals [36, 37].

Adenosine receptors (ARs) belong to the family of G protein-coupled receptors (GPCR) [37]. Four subtypes of AR have been identified (A1, A2A, A2B and A3), and all of them are involved in regulation of cAMP production through different pathways: A1 and A3 down-regulate cAMP levels inhibiting adenylyl cyclase, whereas A2A and A2B receptors activate adenylyl cyclase, increasing cAMP production. ARs are expressed in numerous ocular tissues such as ciliary body, TM, SC and retina. Activation/inactivation of ARs impacts AH formation, outflow and consequently IOP homeostasis. Additionally, ARs are also involved in retinal function, impacting blood flow and neuronal survival [36].

#### *<IP_ADDRESS>. Mechanism of action*

Activation of A3 receptors results in activation of Cl− channels in the non-pigmented ciliary epithelial (NPE) cells of the ciliary epithelium where AH is produced [38]. Studies conducted in A3AR knockout mice and in mice treated with A3AR antagonists showed that absence or inhibition of this receptor significantly decreases IOP when compared to native or untreated animals [39]. On the contrary, mice treated with A3AR agonists show enhanced chloride release resulting in increased AH production and rise in IOP. AR can also affect AH outflow; there are two pathways by which AR agonists increase conventional outflow through the TM and SC: cell volume modification mediated by ion transport and ECM remodelling [36]. A1, A2A and A3 AR agonists have shown to increase Ca2+ in the cells of the SC and to diminish TM cell volume [40]. Outflow resistance in the TM is dependent on the composition of the ECM; activation of A1 ARs triggers signalling cascades that lead to the expression of high levels of metalloproteases such a MMP-2 and MMP-9, enzymes participating in ECM remodelling enhancing AH outflow and decreasing IOP. On the contrary, A2A and A2B AR activation increase ECM deposition and therefore difficult AH outflow increasing IOP [41].

In view of the previously exposed mechanisms, it can be concluded that AR ligands play a key role in IOP control. In general terms, adenosine binding to A1AR in the TM reduces outflow resistance. However, A2A AR stimulation may result in IOP increase or decrease depending on the alterations to the resistance in the SC. Finally, activation of A3 AR mediates activation of Cl− channels in the NPE cells inducing AH production, while A3 AR antagonists prevent adenosine-induced activation of Cl− channels decreasing IOP.

#### *<IP_ADDRESS>. Drugs in development*

the capacity of the prostaglandins of increasing AH outflow through the uveoscleral pathway [33, 34]. This fixed combination currently in Phase III clinical trials achieved clinically and statistically superiority in terms of ocular hypotensive efficacy when compared to its individual active components at the same concentrations. However, incidence rates of ocular hyperaemia were superior in the FDC than those observed in the latanoprost group (40 vs. 60%).

**AMA0076** is a locally acting ROCK inhibitor currently in Phase II clinical trials specifically designed to reduce IOP while minimizing side effects such as hyperemia. *In vivo* studies conducted in an acute hypertensive rabbit model showed that AMA0076 prevented IOP elevation more efficiently than latanoprost and bimatoprost. *In vitro* studies conducted in rabbit ocular tissues demonstrated that AMA0076 was able to induce reversible changes in cell shape and decreased the number of actin filaments and focal adhesions that may facilitate

Adenosine is an endogenous nucleoside modulator of both intracellular and extracellular origin. Adenosine half-life is very limited (~1.5 seconds) as it is rapidly metabolized to inosine and hipoxantine; this is why extracellular levels of adenosine, which usually range from 20 to 200 nM, are considered a good indicator of cellular homeostasis. Adenosine levels increase, even up to the micromolar range, in response to cellular stress conditions such as tissue hypoxia or ischemia. In fact, adenosine levels in AH are significantly elevated in ocular

Adenosine receptors (ARs) belong to the family of G protein-coupled receptors (GPCR) [37]. Four subtypes of AR have been identified (A1, A2A, A2B and A3), and all of them are involved in regulation of cAMP production through different pathways: A1 and A3 down-regulate cAMP levels inhibiting adenylyl cyclase, whereas A2A and A2B receptors activate adenylyl cyclase, increasing cAMP production. ARs are expressed in numerous ocular tissues such as ciliary body, TM, SC and retina. Activation/inactivation of ARs impacts AH formation, outflow and consequently IOP homeostasis. Additionally, ARs are also involved in retinal

epithelial (NPE) cells of the ciliary epithelium where AH is produced [38]. Studies conducted in A3AR knockout mice and in mice treated with A3AR antagonists showed that absence or inhibition of this receptor significantly decreases IOP when compared to native or untreated animals [39]. On the contrary, mice treated with A3AR agonists show enhanced chloride release resulting in increased AH production and rise in IOP. AR can also affect AH outflow; there are two pathways by which AR agonists increase conventional outflow through the TM and SC: cell volume modification mediated by ion transport and ECM remodelling [36]. A1, A2A and A3 AR agonists have shown to increase Ca2+ in the cells of the SC and to diminish

channels in the non-pigmented ciliary

hypertensive individuals when compared to normotensive individuals [36, 37].

function, impacting blood flow and neuronal survival [36].

Activation of A3 receptors results in activation of Cl−

AH outflow [35].

*2.1.2. Adenosine receptor ligands*

*<IP_ADDRESS>. Mechanism of action*

*<IP_ADDRESS>. Description and biological function*

92 Glaucoma - Intraocular Pressure and Aqueous Dynamics

Numerous AR ligands (both agonists and antagonists) are being developed with the purpose of exploiting their potential for modulation of IOP. The adenosine analogues that are currently in glaucoma clinical trials include trabodenoson (Inotek Pharmaceuticals, US), OPA-6566 (Acucela, US and Otsuka Pharmaceutical, Japan), ATL-313 (Santen Pharmaceutical) and CF-101 (Can-Fite Bio Pharma, US).

**Trabodenoson**, formerly known as INO-8875/PJ-875, is a highly selective A1 AR agonist administered in eye drops currently in Phase III clinical trials. The IOP-lowering effect of four different doses of trabodenoson ranging from 50 to 500 μg was evaluated when administered b.i.d during 28 consecutive days in a Phase II clinical trial [42]. Administration of trabodenoson resulted in a dose-related IOP reduction; the highest dose tested achieved a statistically significant IOP reduction in 25% (6.5 mmHg) when compared to placebo. All the doses tested showed a good tolerability profile. The proposed mechanism of action for trabodenoson involves activation of the A1 adenosine receptor that promotes phosphorylation of the extracellular signal-regulated kinases ERK1 and ERK2, resulting in increased secretion of MMP-2 and changes in the ECM that decrease TM resistance to the AH outflow.

**OPA-6566** and **ATL-313** are two A2A agonists currently in Phase I clinical trials; at this point of development, little information on their clinical development is available. Both drugs are anticipated to increase AH outflow via the conventional pathway of the TM and SC rather than the uveoscleral pathway.

**CF-101** is an A3 AR agonist orally administered presently in Phase II clinical trials. Recently, CF-1001 failed to meet its primary endpoint as no statistically significant differences in IOP were found between the CF101 group and the placebo group after 16 treatment weeks.

#### *2.1.3. Nitric oxide donors*

#### *<IP_ADDRESS>. Description and biological function*

Nitric oxide (NO) is a gaseous endogenous signalling molecule synthesized by nitric oxide synthases (NOS) that catalyse the oxidation of the amino acid L-arginine to form NO and L-citrulline. There are three NOS isoforms: the neuronal NOS (nNOS or NOS-1), the endothelial NOS (eNOS or NOS-3) and the inducible NOS (iNOS or NOS-2). NOS-1 and NOS-3 are activated by the calcium/calmodulin complex in response to an increase in calcium and produce NO in the pico- or nanomolar scale. On the contrary, NOS-2 activation is calcium independent and produces NO in a micro- to millimolar scale [43].

NOS-3 is expressed in TM, SC, ciliary body and uveal vascular endothelium. NOS-1 is located in nerve fibres in the cornea and lens epithelium and iNOS is detected after directed stimulation in the TM and in the ciliary body and vessels. This expression pattern of NOS enzymes in the anterior segment of the eye suggests that NO plays a key role in the regulation of AH dynamics. In fact, levels of NO and NOS expression are diminished in glaucomatous human eyes. In addition, NOS-3 knock-out mice exhibit elevated IOP levels due to a reduction in the conventional outflow, whereas the opposite effect is observed in NOS-3 over expressing transgenic mice [43, 44].

#### *<IP_ADDRESS>. Mechanism of action*

NO stimulates soluble guanylatecyclase (sGC) leading to the elevation of intracellular cyclic guanosine monophosphate (cGMP levels), a secondary messenger that interacts with protein kinases and phosphodiesterases. Multiple studies indicate that the NO-cGMP pathway regulates IOP levels increasing AH outflow through the conventional route and by reducing AH secretion [43].

Cytoskeletal changes at the TM seem to be the underlying mechanism mediating the increase in AH outflow induced by NO. NO induces vascular smooth muscle cells (VSMC) relaxation by stimulation of cGMP synthesis that subsequently activates protein kinase G (PKG). This results in inhibition of the Rho-kinase cascade and leads to inhibition of the MLC-2 phosphorylation. Analogously to VSMCs, TM cells also exhibit contractile properties and modulation of these properties by NO is thought to mediate changes that result in a reduction in outflow resistance. Additionally, it has been demonstrated that inhibition of multi-drug resistanceassociated protein-4 also induces TM cellular relaxation mediated by cGMP-PKG pathway [45]. NO could also mediate its IOP-lowering effects by targeting cells of the SC. In fact, Rhokinase inhibition mediated by NO-cGMP regulates actin dynamics and cell contractility in cultured SC cells [46].

#### *<IP_ADDRESS>. Drugs in development*

**Latanoprostene bunod** (LBN; BOL-303259-X; Bausch & Lomb) is a novel nitric oxide-donating prostaglandin F2a analogue currently in Phase III clinical trials [47]. In the eye, LBN is metabolized to two moieties. The first, latanoprost acid, is an F2alpha prostaglandin analogue, while the second, butanediol mononitrate, releases nitric oxide, which activates the soluble cGMP signalling pathway. LBN achieves IOP control simultaneously enhancing AH outflow through the conventional and the uveoscleral routes. Doses ranging from 0.006 to 0.040% of LBN solution were administered once a day to patients with OAG or OHT for 28 consecutive days. LBN at 0.024 and 0.040% achieved statistically significant reductions in mean IOP when compared to latanoprost. During the Phase III clinical trial LBN 0.024% (QD) was not only non-inferior to timolol maleate 0.5% dosed twice daily (b.i.d) after 3 months of treatment but also provided significantly greater IOP reduction. LBN exhibited a similar safety profile than prostaglandins being conjunctival hyperaemia the most frequently reported AE.

#### **2.2. New mechanisms of action**

L-citrulline. There are three NOS isoforms: the neuronal NOS (nNOS or NOS-1), the endothelial NOS (eNOS or NOS-3) and the inducible NOS (iNOS or NOS-2). NOS-1 and NOS-3 are activated by the calcium/calmodulin complex in response to an increase in calcium and produce NO in the pico- or nanomolar scale. On the contrary, NOS-2 activation is calcium

NOS-3 is expressed in TM, SC, ciliary body and uveal vascular endothelium. NOS-1 is located in nerve fibres in the cornea and lens epithelium and iNOS is detected after directed stimulation in the TM and in the ciliary body and vessels. This expression pattern of NOS enzymes in the anterior segment of the eye suggests that NO plays a key role in the regulation of AH dynamics. In fact, levels of NO and NOS expression are diminished in glaucomatous human eyes. In addition, NOS-3 knock-out mice exhibit elevated IOP levels due to a reduction in the conventional outflow, whereas the opposite effect is observed in NOS-3 over expressing

NO stimulates soluble guanylatecyclase (sGC) leading to the elevation of intracellular cyclic guanosine monophosphate (cGMP levels), a secondary messenger that interacts with protein kinases and phosphodiesterases. Multiple studies indicate that the NO-cGMP pathway regulates IOP levels increasing AH outflow through the conventional route and by reducing AH

Cytoskeletal changes at the TM seem to be the underlying mechanism mediating the increase in AH outflow induced by NO. NO induces vascular smooth muscle cells (VSMC) relaxation by stimulation of cGMP synthesis that subsequently activates protein kinase G (PKG). This results in inhibition of the Rho-kinase cascade and leads to inhibition of the MLC-2 phosphorylation. Analogously to VSMCs, TM cells also exhibit contractile properties and modulation of these properties by NO is thought to mediate changes that result in a reduction in outflow resistance. Additionally, it has been demonstrated that inhibition of multi-drug resistanceassociated protein-4 also induces TM cellular relaxation mediated by cGMP-PKG pathway [45]. NO could also mediate its IOP-lowering effects by targeting cells of the SC. In fact, Rhokinase inhibition mediated by NO-cGMP regulates actin dynamics and cell contractility in

**Latanoprostene bunod** (LBN; BOL-303259-X; Bausch & Lomb) is a novel nitric oxide-donating prostaglandin F2a analogue currently in Phase III clinical trials [47]. In the eye, LBN is metabolized to two moieties. The first, latanoprost acid, is an F2alpha prostaglandin analogue, while the second, butanediol mononitrate, releases nitric oxide, which activates the soluble cGMP signalling pathway. LBN achieves IOP control simultaneously enhancing AH outflow through the conventional and the uveoscleral routes. Doses ranging from 0.006 to 0.040% of LBN solution were administered once a day to patients with OAG or OHT for 28 consecutive days. LBN at 0.024 and 0.040% achieved statistically significant reductions in mean IOP when compared to latanoprost. During the Phase III clinical trial LBN 0.024% (QD) was not only

independent and produces NO in a micro- to millimolar scale [43].

transgenic mice [43, 44].

94 Glaucoma - Intraocular Pressure and Aqueous Dynamics

*<IP_ADDRESS>. Mechanism of action*

secretion [43].

cultured SC cells [46].

*<IP_ADDRESS>. Drugs in development*

New drug targets are certainly playing an important part in modernising the way glaucoma will be treated in the future. But innovation in glaucoma is not only focused in the discovery of new drug targets, but it is also taking advantage of new and exciting mechanisms of action that seek to solve unmet needs that current treatments cannot solve. In the following section, we will give an overview of drugs using innovative mechanisms of action, focusing on those that have in the past few years entered in clinical development for glaucoma. It should be noted that although antibodies fall into this category of drugs using new mechanisms of action, they have not been included in this section since, up to the authors' knowledge, there are no clinical programmes currently developing antibodies for the treatment of glaucoma. A section on neuroprotective drugs has been included at the end of this section to highlight the importance these drugs are acquiring in the glaucoma pipeline.

#### *2.2.1. Oligonucleotide-based compounds*

Oligonucleotides have in the past decades turned out to be an interesting therapeutic approach, particularly due to their ability to address intracellular targets. Oligonucleotides can be designed to target specific genes or RNAs with the aim of altering gene expression or even exert a direct interaction by binding to molecules. The main classes of oligonucleotides that are currently being developed as therapeutic tools are aptamers, ASOs, siRNAs and microRNAs (miRNAs) [48].

#### *<IP_ADDRESS>. Aptamers*

Aptamers are RNA or DNA oligonucleotides that form a 3D structure designed to interact with large or small molecules. Aptamers typically bind to proteins but can be designed to act upon other types of molecules [49]. Aptamers, contrary to antibodies, are chemically synthetized products that do not require biological steps in their production processes; this results in products that are very well controlled without significant variability among batches. Aptamers have not been widely tested in the context of glaucoma. Pegaptanib, a first-in-class FDA-approved aptamer for the treatment of age-associated macular degeneration (AMD), has been briefly studied for the treatment of neovascular glaucoma [50]. This compound binds to a subtype vascular endothelial growth factor (VEGF), VEGF165, hampering its ability to bind to its cell surface receptor thus impairing neovascularization [51]. In addition, ARC81, an anti-transforming growth factor-β (TGF-β) aptamer, has been studied for the reduction in corneal scaring, a common complication of glaucoma filtration surgery [52].

#### *<IP_ADDRESS>. Antisense oligonucleotides (ASOs)*

ASOs are single-strand RNA or DNA oligonucleotides, of approximately 15–25 bp, that mediate mRNA degradation by an RNaseH-mediated mechanism [53]. These compounds have been widely used to study cell function and lately applied to therapeutics. There are two FDA approved ASOs; vitravene, an intravireally administered ASO for the treatment of cytomegalovirus retinitis in AIDS patients and mipomersen, an intravenously administered ASO for the treatment of familiar hypercholesterolemia [54, 55]. The main advantage of this class of compounds is that they modulate gene expression without altering the genetic code, and that their action upon their target gene can be rapidly modified. In addition, as other oligonucleotides, ASOs are chemically synthetized, with all the advantages this entails. On the other hand, ASOs are labile products that require chemical modifications to increase their stability; these modifications can potentially increase their toxicity [48, 56].

There are two ASOs under development for treatment of different types of glaucoma; Aganirsen (GS-101) and ISTH0036. Aganirsen is a 25-bp ASO targeting insulin receptor substrate-1 (IRS-1) administered in eye drops; this compound is being developed for the treatment of corneal neovascularization and has also been tested in neovascular glaucoma [57]. ISTH0036 is a fully modified phosphorothioate 14-bp oligodeoxynucleotide with a 3 + 3 LNAgapmer pattern targeting TGF-β2. TGF-β2 is an anti-proliferative and anti-inflammatory factor that is upregulated in the AH of POAG patients. Increases in TGF-β2 correlate with deposition of fibrillar extracellular matrix in the TM, one of the hallmarks of POAG [58]. These extracellular depositions in the TM hamper AH outflow and consequently result in IOP increase. ISTH0036 is currently being tested in a Phase I dose-finding clinical study in patients with advanced glaucoma undergoing filtration surgery due to uncontrollable elevated IOP. The compound is administered intravitreally (IVT) at the end of trabeculectomy; the outcomes of the study are safety, tolerability and effect on IOP.

#### *<IP_ADDRESS>. Short interfering RNAs (siRNAs)*

siRNAs are double-stranded RNA molecules of approximately 19–25 bp that mediate gene silencing by blocking translation of specific mRNAs into their corresponding protein [59]. These molecules, although usually larger than ASOs, are in general more potent and stable than ASOs. Depending on the target tissue and the level of expression of the target gene, these compounds can be administered in eye drops avoiding invasive administration methods, if deeper regions need to be accessed IVT administration may be required [60]. In addition, once the siRNA has entered the RISC complex its action lasts for quite some time, this means that a single molecule would be able to mediate the degradation of several mRNAs thus amplifying the effect of the compound. This prolonged action is particularly interesting in the case of glaucoma as it could avoid sudden increases in IOP due to skipped doses.

Bamosiran (SYL040012) is a 21-bp unmodified siRNA targeting adrenergic receptor β2 (ADRB2) for the treatment of glaucoma. This compound is administered in eye drops and penetrates the eye to reduce synthesis of AH by blocking the ADRB2 at the ciliary body and possibly also at the TM [61, 62]. In contrast to traditional beta-blockers, bamosiran acts only locally in the eye; this is because the molecule is rapidly degraded when it reaches systemic circulation, thus reducing the likelihood of systemic side effects. This characteristic makes bamosiran a safe compound for the treatment of individuals with risk of heart disease or other alterations in which beta-blockers are contraindicated.

QPI-1007 is a 19-bp siRNA targeting Caspase-2. This product, administered by IVT injection, is under development for the treatment of several optic neuropathies, including glaucoma [63]. Caspase-2 is specifically activated during ganglion cell death leading to irreversible loss of vision, thus reducing its expression could potentially protect retinal ganglion cells from apoptosis.

#### *2.2.2. Gene therapy*

been widely used to study cell function and lately applied to therapeutics. There are two FDA approved ASOs; vitravene, an intravireally administered ASO for the treatment of cytomegalovirus retinitis in AIDS patients and mipomersen, an intravenously administered ASO for the treatment of familiar hypercholesterolemia [54, 55]. The main advantage of this class of compounds is that they modulate gene expression without altering the genetic code, and that their action upon their target gene can be rapidly modified. In addition, as other oligonucleotides, ASOs are chemically synthetized, with all the advantages this entails. On the other hand, ASOs are labile products that require chemical modifications to increase their stability;

There are two ASOs under development for treatment of different types of glaucoma; Aganirsen (GS-101) and ISTH0036. Aganirsen is a 25-bp ASO targeting insulin receptor substrate-1 (IRS-1) administered in eye drops; this compound is being developed for the treatment of corneal neovascularization and has also been tested in neovascular glaucoma [57]. ISTH0036 is a fully modified phosphorothioate 14-bp oligodeoxynucleotide with a 3 + 3 LNAgapmer pattern targeting TGF-β2. TGF-β2 is an anti-proliferative and anti-inflammatory factor that is upregulated in the AH of POAG patients. Increases in TGF-β2 correlate with deposition of fibrillar extracellular matrix in the TM, one of the hallmarks of POAG [58]. These extracellular depositions in the TM hamper AH outflow and consequently result in IOP increase. ISTH0036 is currently being tested in a Phase I dose-finding clinical study in patients with advanced glaucoma undergoing filtration surgery due to uncontrollable elevated IOP. The compound is administered intravitreally (IVT) at the end of trabeculectomy; the out-

siRNAs are double-stranded RNA molecules of approximately 19–25 bp that mediate gene silencing by blocking translation of specific mRNAs into their corresponding protein [59]. These molecules, although usually larger than ASOs, are in general more potent and stable than ASOs. Depending on the target tissue and the level of expression of the target gene, these compounds can be administered in eye drops avoiding invasive administration methods, if deeper regions need to be accessed IVT administration may be required [60]. In addition, once the siRNA has entered the RISC complex its action lasts for quite some time, this means that a single molecule would be able to mediate the degradation of several mRNAs thus amplifying the effect of the compound. This prolonged action is particularly interesting in the case of

Bamosiran (SYL040012) is a 21-bp unmodified siRNA targeting adrenergic receptor β2 (ADRB2) for the treatment of glaucoma. This compound is administered in eye drops and penetrates the eye to reduce synthesis of AH by blocking the ADRB2 at the ciliary body and possibly also at the TM [61, 62]. In contrast to traditional beta-blockers, bamosiran acts only locally in the eye; this is because the molecule is rapidly degraded when it reaches systemic circulation, thus reducing the likelihood of systemic side effects. This characteristic makes bamosiran a safe compound for the treatment of individuals with risk of heart disease or

these modifications can potentially increase their toxicity [48, 56].

comes of the study are safety, tolerability and effect on IOP.

glaucoma as it could avoid sudden increases in IOP due to skipped doses.

other alterations in which beta-blockers are contraindicated.

*<IP_ADDRESS>. Short interfering RNAs (siRNAs)*

96 Glaucoma - Intraocular Pressure and Aqueous Dynamics

Gene therapy is a technique that uses genetic material to modify the disease state, usually using a vector to transfer the genetic material. For ophthalmic affectations, distinct viral vectors can be used for the delivery of such genetic material. Most-studied vectors include adenovirus (AdV), adeno-associated virus (AAV), herpes simplex virus (HSV) and lentivirus; all of them offering distinct pros and cons [64]. While large advancement has been achieved in different eye diseases, gene therapy for glaucoma has faced substantial limitation due to the lack of obvious genetic alterations. Indeed, causative and risk factor genes such as myocilin (MYOC), optineurin (OPTN), Cytochrome P450 1B1 (CYP1B1), caveolin (CAV1/CAV2) and TANK-binding kinase 1 (TBK1), among others, represent less than 10% of glaucoma cases worldwide [65]. Up to date, only one gene therapy trial for glaucoma has been listed (Trial ID: US-0589). This Phase I study proposes to evaluate the safety of SCH-412499 (rAd-p21) after a single injection into the sub-conjunctival space of the eye in glaucoma subjects prior to trabeculectomy. The treatment uses an AdV vector for the expression of p21 WAF-1/Cip1, a potent cyclin-dependent kinase inhibitor to reduce wound healing process and fibroproliferation after filtering surgery [66]. No results have been disclosed yet; however, pre-clinical studies demonstrated a safe profile with strong anti-proliferative effect after filtration surgery in animal models [67, 68].

In collaboration with Mayo Clinic (Rochester, USA), Oxford BioMedica plc (Oxford, UK) is developing a novel gene therapy approach for the treatment of chronic glaucoma. Pre-clinical studies have been undertaken to determine the feasibility of the LentiVector® platform for the delivery and expression of cyclooxygenase (COX-2) and prostaglandin F2α (PGF-2α) genes to reduce IOP in glaucomatous patients. According to the company's announcement, pre-clinical studies demonstrated a good tolerance for the LentiVector® when used at high doses, with the ability to transduce proper target cells following transcorneal injection into the anterior chamber. Moreover, data showed a long-term gene expression for up to 5 months. Although preclinical efficacy studies on IOP have been planned, no additional results have been published.

Besides these two emerging gene therapy treatments for glaucoma, most investigational developments are still in pre-clinical phases, showing relative effect on IOP. For example, the COX-2 and PGF-2α gene therapy models previously described demonstrated a prolonged decrease in IOP in large animal models [69]. Similarly, using lentiviral-based dual expression vector to deliver prostaglandin F synthase, significant reduction in IOP has been achieved [70]. However, the overall reduction in IOP produced by these vectors was not as extensive as that observed for topical PG eye drops. Likewise, different gene therapy strategies to modulate the RhoA or Rho-kinase pathways showed an efficient but moderate IOP-lowering effect [71, 72]. In an elegant way, gene therapy has also been adapted to steroid-induced glaucoma. In these patients, topical instillation of glucocorticoids is known to produce ocular hypertension by producing a downregulation of the matrix metalloproteinase 1 (MMP1) gene in the TM. Because the patients are under an on and off treatment schedule with glucocorticoids, a new self-complementary AAV has been generated for the expression of MMP1 under the control of a glucocorticoid response element. This allows for the induction of MMP1 expression only after the administration of glucocorticoids, a strategy that presents great advantages. Using this novel system, a reduction in IOP was detected in large animal model and would be highly beneficial in clinic [73].

In addition to cell transduction and adequate gene expression modulation, the route of administration is a critical factor for effective gene therapy. Interestingly, optimum delivery and transduction of AdV have been found through the TM and the SC, structures from where the AH flows [74]. However, the complexity of the whole outflow tract, which includes various cell-types harbouring different transduction properties, may impair the overall efficacy of gene therapy. Moreover, the possible systemic exposure with unwanted side effects is an important downside of direct release of viral vectors that can drain into the retinal venous circulation. Although we are still a long way from the instauration of gene therapy in glaucoma treatment, the advancement achieved in the field is encouraging and sustains the feasibility of such technique in human.

#### *2.2.3. Stem cell therapy*