Topical Medication Therapy for Glaucoma and Ocular Hypertension

Glaucoma is one of the most common causes of blindness, thus seriously affecting people’s health and quality of life. The topical medical therapy is as the first line treatment in the management of glaucoma since it is inexpensive, convenient, effective, and safe. This review summarizes and compares extensive clinical trials on the topical medications for the treatment of glaucoma, including topical monotherapy agents, topical fixed-combination agents, topical non-fixed combination agents, and their composition, mechanism of action, efficacy, and adverse effects, which will provide reference for optimal choice of clinical medication. Fixed-combination therapeutics offer greater efficacy, reliable security, clinical compliance, and tolerance than non-fixed combination agents and monotherapy agents, which will become a prefer option for the treatment of glaucoma. Meanwhile, we also discuss new trends in the field of new fixed combinations of medications, which may better control IOP and treat glaucoma.


INTRODUCTION
Glaucoma is a neurodegenerative ophthalmologic disease characterized by the progressive degeneration of the retinal ganglion cells and axonal death, resulting in an irreversible blindness (Marshall et al., 2018;Garcia-Medina et al., 2020). Glaucoma is commonly related to high intraocular pressure (IOP) with a subsequent injury of the optic nerve, and eventually blindness (Goel et al., 2010). The normal physiological IOP is 5-20 mmHg, which in turn depends on the adjustment of production and absorption of the aqueous humor (AH) (Goel et al., 2010;Skrzypecki et al., 2018). The ciliary muscle produces the AH that leaves the eye by passive flows through two pathways: the conventional (trabecular meshwork, TM) and unconventional (uveoscleral) pathway (Huang et al., 2018). Normal tension glaucoma (NTG) is characterized by a IOP usually below 21 mmHg, while the primary open-angle glaucoma (POAG) has a IOP of over 22 mmHg (Razeghinejad and Lee, 2019), the latter being the main and modifiable risk factor for glaucoma and its progression (Fogagnolo and Rossetti, 2011;Jayaram, 2020). Ocular hypertension (OHT) is defined as a IOP over 21 mmHg without the damage of the optic disk and view field (Chamard et al., 2020), but it is most likely to develop into open-angle glaucoma (OAG) (Kass et al., 2021).
Currently, the treatments to cure glaucoma mainly consist of surgical intervention and topical medication therapy (Guglielmi et al., 2019;Webb, 2021). Surgical intervention is required when a patient's visual independence is at risk (Lusthaus and Goldberg, 2019). The risks associated with the surgical procedure and the postoperative recovery are complicated with each patient and the cost is relatively expensive (Razeghinejad and Lee, 2019). Therefore, topical medication therapy with reliable effectiveness and safety is often as the preferred approach in the management of glaucoma (Conlon et al., 2017;Guglielmi et al., 2019;Webb, 2021). The classes of topical monotherapy medications to cure glaucoma include prostaglandin analogs, rho-kinase inhibitors, β-adrenergic blocking agents, α-2 adrenergic agonists, and carbonic anhydrase inhibitors, but they are often inadequate to keep IOP under control (Schehlein et al., 2017). Therefore, the combination of multiple medications is significantly necessary for an adequate control of IOP.
The lowering effect of IOP by the fixed combination and non-fixed combination medications are similar and both the combinations are superior to their constituent agents. Importantly, the fixed combination and non-fixed combination medications have reliable security with no additional new side effects comparing to monotherapy agents. Fixed combination medications have a greater  convenience, compliance, and tolerance than non-fixed combination medications, since they can decrease dosing frequency and eliminate the washout effect by decreasing the number of instilled drops, the total number of bottles of medication in use, and the exposure to preservatives (Babic, 2015).
This review collects and compares extensive clinical trials on the topical anti-glaucoma medications, and summarized their   mechanism of action, efficacy, adverse effects (AEs), and safety on Tables 1-6. And the structure of this review is shown on Figure 1.

METHODS
This review summarizes the effectiveness and safety of topical anti-glaucoma agents based on an overall review of related literatures published. Extensive references were obtained by searching keywords: prostaglandin analogs, rho-kinase inhibitors, β-adrenergic blocking agents, α-2 adrenergic agonists, carbonic anhydrase inhibitors, fixed-combination agents, glaucoma and so on. Systematic and large-scale clinical trials, meta-analysis and review articles were the main priority; because of the lacking of related articles, small-scale experiments and related cases were referenced. The main clinical trials more elaborated were summarize on Table 6.

Prostaglandin Analogs (PGAs)
Prostaglandins (PGs) are potent biologically active metabolites of the arachidonic acid that modulate many biological responses in various tissues including the eye (Matsou and Anastasopoulos, 2018). PGAs represent the first-line medical therapy to cure POAG and OHT, since they target different PG receptors to reduce IOP (Diaconita et al., 2018;Angeli and Supuran, 2019) ( Table 1).
PGF2 analogs exert different IOP lowering effects, as demonstrated by clinical trials and meta-analysis: bimatoprost exerts the greatest IOP lowering effect and the highest incidence of hyperemia than other PGF2 analogs (Aptel and Denis, 2011;Yu and Welge-Lussen, 2013;Takagi et al., 2018). In addition, they are well tolerated and induce mild AEs including conjunctival hyperemia, increase in the eyelash growth, as well as changes in periocular pigmentation and iris color (Aptel and Denis, 2011;Yu and Welge-Lussen, 2013).
Aganepag Isopropyl (AGN-210961). AGN-210961 has been formulated as a proprietary ophthalmic solution (Impagnatiello et al., 2019). Both AGN-210961 and bimatoprost 0.03% exert a similar IOP lowering effect at day 7 and week 4, as demonstrated by a clinical trial (NIH, 2014a). AGN-210961 do not exert any serious AEs, and the incidence of AEs in AGN-210961 group is also lower than bimatoprost group (NIH, 2014a).
AGN-210669. AGN-210669 is a sustained-release formulation of AGN-210961 (Impagnatiello et al., 2019) used in clinical trials for the treatment of POAG and OHT. But the safety and effectiveness of AGN-210669 is not superior to bimatoprost (NIH, 2013b; a). The combination of AGN-210669 (0.05%) and bimatoprost does not induce a significant change in the IOP lowering effect, and the AEs of the combination therapy also increased (NIH, 2013b; a). Therefore, AGN-210669 may not be a better medicine to control IOP.
A phase I clinical trial in OHT and POAG patients demonstrated that ONO-9054 reduces IOP in a dosedependent manner and the greatest reduction is 29.6% at 9 h after the dose of ONO-9054 of 30 μg/ml on day 1 (Harris et al., 2016). In healthy volunteers, the mean reduction is 8.29 mmHg (28.2%) at 9 h after a dose of ONO-9054 of 30.0 μg/ml (Suto et al., 2015). The morning (AM) and evening (PM) dosage of 30 μg/ml ONO-9054 have similar sustained reduction in IOP, whereas the incidence of hyperemia and dryness is slightly increased in the patients subjected to the PM dose of ONO-0954 (Berlin et al., 2016). Miller Ellis, et al. observed that ONO-9054 (30 μg/ml) has a better IOP lowering effect than Xalatan once daily (Miller Ellis et al., 2017). The rate of the mean IOP reduction of ≤ −25%, ≤ −30%, and ≤ −35% in the ONO-9054-treated patients is 2.39, 2.37, and 4.85 times higher than the rate in the Xalatan-treated  (Harris et al., 2016;Miller Ellis et al., 2017).

Nitric Oxide-donating Prostaglandin F2-Alpha Analog
Nitric oxide (NO)-donating prostaglandin is composed of a prostaglandin F2 analogue moiety and NO-donating moiety, thus providing an IOP-lowering effect by two independent mechanisms (Impagnatiello et al., 2019). NO is an endogenous gas working as a signaling molecule inducing TM and Schlemm's canal cell relaxation, decreasing cell volume, enlarging cell gap, and decreasing the outflow resistance through the activation of the soluble guanylyl cyclase/cyclic guanosine monophosphate (sGC-cGMP) signaling pathway (Krauss et al., 2011;Kaufman, 2017;Cavet and DeCory, 2018;Mao et al., 2020). In addition, it decreases AH production by inhibiting the Na + , K + -ATPase pump eventually lowering IOP (Wareham et al., 2018). NO also regulates the eye blood flow and protects the optic nerve by the sGC-cGMP signaling pathway (Wareham et al., 2018;Mao et al., 2020).
Safety and AEs. LBN is well-tolerated with no serious AEs and system AEs. Conjunctival hyperemia, eye irritation, and eye pain are the most reported ocular AEs (Medeiros et al., 2016). Blood pressure or heart rate does not change in correlation with LBN treatment (Kawase et al., 2016;Liu et al., 2016).
Safety and AEs. Little evidence was found of treatment-related systemic effects or drug-related serious AEs associated to NCX-470. Conjunctival hyperemia was the most frequent AE in 16.8% of patients treated with NCX-470 (0.065%) versus 6.5% of patients treated with latanoprost (NIH, 2019a).

Rho-Kinase Inhibitors
The rho-kinases are serine/threonine kinase isoforms and effectors (Schehlein and Robin, 2019). Topical rho-kinase inhibitors inhibit rho-kinase isoforms in TM, thus directly increasing AH outflow in the TM pathway and consequently reducing IOP in a significant manner (Berrino and Supuran, 2019;Moura-Coelho et al., 2019). Rho-kinase inhibitors also exert a neuroprotective effect that in turn can exert a greater positive impact on the ocular blood flow, and even an antifibrotic effect that may be helpful if conventional glaucoma surgery is needed (Tanna and Johnson, 2018) ( Table 2).

Launched Rho-Kinase Inhibitors
Ripasudil (Glanatec ® ) Efficacy. Ripasudil is the first rho-kinase inhibitor approved for the treatment of glaucoma (Berrino and Supuran, 2019). It promotes AH outflow by changing the TM cytoskeleton to reduce the outflow resistance and increasing the endothelial permeability of the Schlemm's canal (Schehlein and Robin, 2019). Multiple clinical trials reported that ripasudil provides a great IOP lowering effect and exerts an additional reduction of IOP when used an add-on medication to PGAs and β-blockers (BBs) (Tanihara et al., 2013;Tanihara et al., 2016;Inoue and Tanihara, 2017). A phase 2 clinical study demonstrated that IOP-lowering effect of ripasudil is dosedependent. When patients are treated with 0.1, 0.2, and 0.4% ripasudil for 8 weeks, the mean IOP reductions are 3.4 mmHg, 3.2 mmHg, and 3.5 mmHg, respectively (Tanihara et al., 2013).
Safety and AEs. Conjunctival hyperemia is the most frequent AE caused by ripasudil (Tanihara et al., 2013;Tanihara et al., 2016;Sakamoto et al., 2019) due to the relaxation of the smooth muscle in the blood vessels because of the inhibition of rho-kinase. However, this AE is mild, transient, and can resolve spontaneously in glaucoma patients (Sakamoto et al., 2019;Kusuhara and Nakamura, 2020). The other frequently observed AEs are blepharitis and allergic conjunctivitis (Tanihara et al., 2016).

Clinical Rho-Kinase Inhibitors
RKI-983/SNJ-1656/Y-39983 SNJ-1656, also called Y-39983 or RKI-983, promotes the regeneration of the axon in damaged retinal ganglion cells (Yang et al., 2013). However, the IOP lowering effect is not very remarkable. In a phase I clinical trial, the mean IOP reductions are 2.2, 3.8, 4.3, and 4.0 mmHg before the instillation of the eyedrops in the morning and 1.5, 5.0, 4.4 and 4.5 mmHg at 2 h after the instillation in the morning in placebo and SNJ-1656 (0.03, 0.05 or 0.1%) group . AEs are mild to moderate, and conjunctival hyperemia is the most frequently reported .

Y-27632
Y-27632 is used in the treatment of OHT since it modulates the cytoskeletal alterations in TM cells to increase the conventional outflow, and relax the ciliary muscle contraction Luo et al., 2021). At present, there is no related clinical study, but glaucomatous transgenic mice experiment demonstrated that Y-27632 can significantly decreased IOP and increased outflow facility, which greatly influenced the long-term IOP-lowering effect .

β-adrenergic Blocking Agents
BBs actively block the β-adrenergic receptors and their pharmacological properties depend on their various effects on the adrenergic signaling pathway (Bejan-Angoulvant and Angoulvant, 2020). The ocular AEs include conjunctival hyperemia, ocular surface discomfort, reduction in tear flow, and worsening of the dryness in the eye, while the systemic AEs include bradycardia, heart block and arrhythmia, bronchospasm, and worsening of underlying asthma or chronic obstructive pulmonary disease (Dikopf et al., 2017). These AEs are caused by the direct systemic absorption of BBs after ocular administration through the nasolacrimal system or the conjunctiva, without being subjected to first pass metabolism (Morales et al., 2016;Shukla et al., 2020). Although topical BBs is not associated with excess cardiovascular mortality as revealed in a meta-analysis with large population-based studies (Pinnock et al., 2016), ophthalmic BBs are not recommended in patients with asthma, chronic obstructive pulmonary disease, bradycardia, heart block, or uncontrolled heart failure (Marshall et al., 2018) ( Table 3).

Selective β-2 Adrenergic Blocking Agents
Bamosiran (SYL040012) Efficacy. SYL040012 targets the human β2-adrenergic receptor to inhibit its expression and consequently reduces the production of AH (

Carbonic Anhydrase Inhibitors
Human carbonic anhydrase (hCA) is present in all living organism and it catalyzes carbon dioxide hydration that is regulating many physiological processes Supuran, 2020). hCA inhibitors exert IOP lowering effect by decreasing bicarbonate and AH secretion through the inhibition of hCA II, IV, and XII isoforms. Ghorai et al., 2020). The first generation CAs inhibitors are sulfonamide hCA-II inhibitors including acetazolamide, methazolamide, and dichlorophenamide, which decrease IOP by 25-30% (Scozzafava and Supuran, 2014;Ghorai et al., 2020). They have been used as systemic antiglaucoma drugs for more than 50 years, but certain undesirable AEs are reported due to the inhibition of the enzymes present in the tissues than the ones in the eye (Carradori et al., 2015). The second generation of CAs inhibitors topically administered are sulfonamides, such as dorzolamide hydrochloride (Trusopt ® ) and brinzolamide (Azopt ® ), whose IOP lowering effect is comparable with the one of BBs, but less effective than PGAs (Eichhorn, 2013).
Trusopt ® and Azopt ® are characterized by a better tolerance and less AEs compared to the first generation inhibitors, and the common reported AEs include stinging, burning or reddening of the eye, blurred vision, pruritus and bitter taste (Scozzafava and Supuran, 2014) (Table 4).

Adenosine Receptor Agonists
Adenosine is an essential component of the energy production and utilization systems of the body by providing the energetics necessary for the muscle movements, heartbeat, nerve signals, and chemical reactions (Jamwal et al., 2019). It exerts its functions by activating four adenosine receptor (AR) subtypes (A1, A2A, A2B, and A3) (Ciancetta and Jacobson, 2017). Adenosine is neuroprotective against excitotoxicity and metabolic dysfunctions that may be present in neurological and ocular diseases (Jamwal et al., 2019) (Table 4).

Trabodenoson (INO-8875) Efficacy
Trabodenoson is an adenosine mimetic that selectively acts on the A1 receptor subtype (Qiu, 2021). Trabodenoson can involve in matrix metalloproteinase signaling pathway associated with glaucoma (Qiu, 2021), consequently increasing TM outflow and exerting IOP lowering effect (Li et al., 2018). Trabodenoson exerts a dose-dependent and time-dependent IOP lowering effect . When patients receive of trabodenoson topical administration at the dose of 50, 100, 200, or 500 μg twice daily exerts a mean IOP reduction at day 14 to 1, 1.67, 2.5, and 3.2 mmHg, respectively, and a reduction to 4.1 mmHg with the dose of 500 μg at day 28 .

Safety and AEs
Trabodenoson has a good safety profile (Laties et al., 2016;Myers et al., 2016). The common systematic AEs such as headache, eye pain, back pain, dermatitis, and excoriation, were more frequently observed in the placebo-treated patients than in the trabodenosontreated patients (Laties et al., 2016). Ocular AEs are uncommon and not serious, present for not longer than 24 h, and they are selflimited, and are usually mild in intensity (Laties et al., 2016).

TOPICAL FIXED COMBINATION OF TWO AGENTS 4.1 Fixed Combination of Prostaglandin F2a
Analogs and β-blockers

Fixed Combination Latanoprost and Timolol (Xalacom ® ) Efficacy
LT-FC is currently one of the most widely used topical antiglaucoma fixed-combination eye drops, which is usually chosen to cure patients with POAG and OHT who are insufficiently responsive to BBs, PGs, or other IOP lowering agents (Konstas et al., 2013c). LT-FC exerts a superior IOP lowering effect than the monotherapy using latanoprost or timolol. Mean IOP at 12 weeks is reduced to 10.2 mmHg in the LT-FC-treated patients, 8.9 mmHg in the latanoprost-treated patients and 7.2 mmHg in the timolol-treated patients and 73.5, 57.5, and 32.8% patients achieve a diurnal IOP reduction over 30% (Higginbotham et al., 2010). Zhao, et al. reported the IOP lowering effect of LT-FC versus non-fixed combination of latanoprost and timolol (LT-nFC), and their results revealed that LT-FC is noninferiority to LT-nFC regarding the mean diurnal IOP reduction after 8 weeks (8.6 versus 8.9 mmHg) (Zhao et al., 2011). Importantly, LT-FC shows a better compliance than LT-nFC (Zhao et al., 2011).

Safety and AEs
Various studies show similar AEs in patients treated with LT-FC, LT-nFC, latanoprost, and timolol monotherapies, and the most commonly reported one is conjunctival hyperemia (Higginbotham et al., 2010).

Fixed Combination Bimatoprost and Timolol (Ganfort ® ) Efficacy
BiT-FC has present in the market of more than 30 countries and regions worldwide OHT used for the reduction of IOP in patients with POAG (Fang et al., 2015). BiT-FC significantly decreases IOP in a greater extent than PGA, or timolol monotherapy (Lewis et al., 2010;Lequeu et al., 2013). BiT-FC also has better 24 h-IOP controlling effect than latanoprost monotherapy (Konstas et al., 2013b). The rates of the mean diurnal IOP reduction over 20% are 68.1, 58.1, and 38.0% in BiT-FC, bimatoprost, and timolol-treated patients, respectively, and the rates of IOP less than 18 mmHg are 23.3, 18.1, and 8.0%, respectively (Lewis et al., 2010). The mean 24-h IOP reduction Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 749858 was 12.2 mmHg (39.2%) in BiT-FC group, which was better than that in latanoprost group (9.9 mmHg, 31.9%) (Konstas et al., 2013b).

Safety and AEs
No addition AEs and serious AEs are reported in BiT-FC treatment group. Although, the incidence of AEs is more in the bimatoprost-treated patients (60.0%) than in the BiT-FC-(48.0%) and timolol-treated patients (31.6%), it is acceptable. Conjunctival hyperemia is the most frequently observed AE, with an incidence of 43.4% by bimatoprost, followed by 25.7% of BiT-FC and 8.7% of timolol (Fang et al., 2015).

Safety and AEs
The most frequently observed AEs after TrT-FC treatment are ocular hyperemia, ocular discomfort, pruritus, and dryness (Hoy et al., 2006). Although, timolol has systemic side effects, no significant change from the baseline value of the mean systolic blood pressure is observed when patients switch to TrT-FC from PGA monotherapy (Nakano et al., 2015).

Fixed Combination of Tafluprost and Timolol (Tapcom ® ) Efficacy
PF-TfT-FC was used for the reduction of IOP in adults with OAG or OHT who require a combination therapy because they are insufficiently responsive to topical monotherapy with BBs or PGs (Hoy, 2015). PF-TfT-FC has a superior IOP lowering effect compared with agents used as monotherapy (Pfeiffer et al., 2014;Kaarniranta et al., 2016;Konstas et al., 2018), which is more suitable for evening administration (Konstas et al., 2018). In a phase 1 study, the result of the mean IOP was 10.3 mmHg for PF-TfT-FC, 10.9 mmHg for PFtafluprost, and 11.1 mmHg for PF-timolol after 8 days in healthy volunteers (Kaarniranta et al., 2016). In another clinical trial, 189 patients treated with timolol who switched to PF-TfT-FC (n 95) or PF-timolol (n 94), showed a reduction of IOP to 7.1-9.0 mmHg in the PF-TfT-FC-treated patients and 6.5-8.1 mmHg in the timolol-treated patients and 375 patients treated with PGA who switched to PF-TfT-FC (n 188) or PF-tafluprost (n 187) for 3 months showed a reduction of IOP to 8.2-9.0 mmHg in the PF-TfT-FC-treated patients and 6.8-7.4 mmHg in the PF-tafluprosttreated patients (Pfeiffer et al., 2014).

Safety and AEs
The rate of AEs is more in the PF-TfT-FC-treated patients than in those treated with the single agent (Pfeiffer et al., 2014). Patients treated with PF-timolol show the lowest incidence (15.2%) of AEs in contrast to patients treated with PF-tafluprost (36.4%) and PF-TfT-FC (48.5%). The commonly reported ocular AEs related to the treatment with PF-TfT-FC include eye pain, eye pruritus, ocular hyperemia, and photophobia (Kaarniranta et al., 2016), while hyperemia is less common in patients treated with PF-TfT-FC than in those treated with PF-latanoprost (7.1% vs 21.4%) (Konstas et al., 2018).

Fixed Combination of Latanoprost and Carteolol (Mikeluna ® ) Efficacy
LC-FC has the same IOP lowering effect as the non-fixed combination of carteolol and latanoprost (LC-nFC) and LT-FC, and exerts a greater effect than carteolol and latanoprost monotherapy Inoue et al., 2018;Inoue et al., 2020b). In the study 1 of a two phase 3 clinical trial, 220 patients were treated with LC-FC (n 113) or latanoprost (n 116) for 8 weeks, while in the study 2, 175 patients were treated with LC-FC (n 76), carteolol (n 76), or LC-nFC (n 37) for 8 weeks. The adjusted mean IOP reduction in the study 1 was 2.9 and 1.6 mmHg in the LC-FC and latanoprost-treated patients, in the study two was 3.5, 3.5, and 1.6 mmHg in LC-FC, LC-nFC, and carteolol-treated patients . When patients with POAG or OHT were treated with LC-nFC and then switched to LC-FC, they showed an IOP of 15.0 ± 2.6, 15.1 ± 2.4, and 15.0 ± 2.4 mmHg at baseline, month 1 and 3, respectively (Inoue et al., 2018). When patients with POAG, NTG, or OHT were treated with LT-FC and then switched to LC-FC, they showed a not significantly changed mean IOP after 1 month (15.9 ± 3.1 mmHg) and 3 months (16.3 ± 3.8 mmHg) compared to the baseline IOP with LT-FC (16.1 ± 3.1 mmHg) (Inoue et al., 2020b).

Safety and AEs
The AEs related to LC-FC observed in clinical trials are mild , and common ocular AEs include hyperemia, irritation, itching, pain, and blurred vision (Inoue et al., 2018). The incidence of ocular AEs decreases when the therapy switches from LC-nFC to LC-FC (Inoue et al., 2018), so LC-FC may provide better tolerance. The pulse rate and blood pressure do not significantly change in patients treated with LC-FC Inoue et al., 2018).

Comparison of the Fixed Combination of Prostaglandin F2a Analogs and β-blockers
When comparing the efficacy of TrT-FC with that of LT-FC, the former showed a greater IOP lowering effect (Shoji et al., 2013;Konstas et al., 2014). Mean 24-h IOP reduction was 2.6 mmHg in patients treated with PF-TrT-FC and 2.2 mmHg in those treated with LT-FC (Konstas et al., 2014). Mean reduction in IOP from the baseline (14.8 ± 3.3 mmHg) at 12 weeks is significantly greater in patients treated with TrT-FC (2.4 ± 2.3 mmHg) than in those treated with LT-FC (1.1 ± 2.3 mmHg) (Shoji et al., 2013). No significant difference in ocular or systemic AEs is observed between TrT-FC-and LT-FC-treated patients, but stinging is significantly more in patients treated with LT-FC (19%) than in those treated with latanoprost (4.8%) (Konstas et al., 2014). A comparative study between BiT-FC and LT-FC discovered a significant reduction in IOP from the baseline without any change of the anterior ocular parameters. The biggest difference in IOP between treated and untreated eyes was 1.67 mmHg (13.6%) at 8 h after the instillation of BiT-FC and 1.93 mmHg (17.8%) at 10 h after the instillation of LT-FC. Conjunctival hyperemia is the most frequent AE in BiT-FC-and LT-FC-treated patients (33.3% versus 25.0%) (Shim et al., 2014). A comparative study showed that patients treated with BiT-FC have a lower IOP than those treated with TrT-FC and the mean IOP reduction is 11.17 and 7.89 mmHg after 6 months, respectively (Macky, 2014). However, in another clinical trial in which patients whose IOP were uncontrolled on BiT-FC accepted TrT-FC showed a decrease of the mean IOP by additional 16.5% at week 8 and 69.2% patients reached the target IOP (≤18 mmHg) (Scherzer et al., 2011). The common AEs are similar in BiT-FC-and TrT-FC-treated patients and include ocular hyperemia, ocular burning, blurred vision, foreign body sensation, and allergic reaction, while stinging/burning is less severe in patients treated with TrT-FC (Scherzer et al., 2011). PF-TfT-FC is as effective as LT-FC in terms of IOP-reducing effect, and induces fewer AEs such as eye irritation and eye pain, which were significantly reduced in patients using PF-TfT-FC (Suzuki et al., 2018). Patients in a phase IV study who switched from BiT-FC to PF-TfT-FC for 12 weeks showed an IOP after the treatment with PF-TfT-FC that was clinically insignificant and statistically noninferior compared with BiT-FC (0.34 mmHg difference). However, the related ocular AEs including lacrimation, eye pruritus, and pruritus increase (Bourne et al., 2019). In a prospective, observer-masked, randomized study including 54 patients who received BiT-FC, LT-FC, or TrT-FC, a statistically significant reduction in diurnal/nocturnal IOP was observed with all medications, such as 4.6 ± 2.3 mmHg/3.2 ± 2.8 mmHg, 5.8 ± 2.4 mmHg/2.9 ± 1.9 mmHg, and 4.3 ± 1.7 mmHg/3.0 ± 1.6 mmHg, respectively, but no significant difference was observed among the three groups (Guven Yilmaz et al., 2018).
Overall, fixed combination and non-fixed combination of PGAs and β-blockers result in a similar IOP lowering effect, and both are superior to monotherapy, but fixed-combination medicines can significantly improve patient compliance and tolerence. Comparing to eye drops containing preservatives, preserver free medicines can better decrease the incidence of ocular side effects and increase ocular comfortable. So, it is necessary to modify the eye drops to be preservative-free to improve patient compliance and tolerance.

Fixed Combination of Latanoprost and Netarsudil (Rocklatan ® ) Efficacy
NL-FC is a glaucoma eye drop marketed as last and is recently approved by FDA for the treatment of OAG or OHT (Asrani et al., 2020). Multiple comparative studies observed that NL-FC had a superior IOP-lowering effect than netarsudil and latanoprost monotherapy (Asrani et al., 2019;Asrani et al., 2020). The percentage of IOP reduction observed in a phase 3 trial was 30.9-36.7% in patients treated with NL-FC compared to the 21.8-24.9% and 23.3-28.8% in patients treated with latanoprost or netarsudil monotherapy at month 3 (Asrani et al., 2019). Another phase 3 trial showed a mean diurnal IOP reduction to 7.8, 5.2, and 6.2 mmHg, respectively. NL-FC (32.3%) achieved a mean diurnal IOP ≤14 mmHg was 3 times higher than that observed after netarsudil (10.8%) and latanoprost (11.8%) monotherapy (Asrani et al., 2020).

Safety and AEs
NL-FC has a safety profile consistent with that of its individual components (Asrani et al., 2019). Conjunctival hyperemia is the most commonly reported in NL-FC group and netarsudil group (Asrani et al., 2020), which may be blood vessel smooth muscle relaxation and blood vessel dilation due to rho-kinase of calcium sensitization (Mehran et al., 2020).

Fixed Combination of Prostaglandin F2a
Analogs and α-2 Agonists

Fixed Combination of Bimatoprost and Brimonidine Efficacy
BiBr-FC has been evaluated in a clinical trial for the treatment of glaucoma and OHT as an ophthalmic solution in 2013. IOP lowering effect of BiBr-FC was similar to the one of bimatoprost (0.01%) monotherapy and superior to the one of brimonidine (0.2%) monotherapy (NIH, 2015b).

Safety and AEs
The incidence of AEs is more in patients treated with BiBr-FC (71.05%) than in those treated with bimatoprost (69.44%) and brimonidine tartrate (52.63%) (NIH, 2015b), and common eye disorders include conjunctival hyperemia, eye pruritus, eye discharge, blepharospasm, ocular burning, punctate keratitis, eye pain, foreign body sensation, eye irritation, and blurred vision NIH, 2015b). Conjunctival hyperemia and eye pruritus show significantly higher rates in BiBr-FC-and bimatoprost-treated patients than in those treated with brimonidine, while the other AEs are similar in the 3 groups (NIH, 2015b). The similar IOP lowering effect and higher AEs may limit the development of BiBr-FC. 8AM, 10AM, 12PM, 4PM, and 8PM after 6-weeks treatment (NIH, 2015a).

Safety and AEs
TrBz-FC is well-tolerated with no serious AEs and the reported AEs include ocular hyperemia, conjunctival hyperemia, eye irritation, eye pain, and dysgeusia (NIH, 2015a).

Fixed Combination of Latanoprost and Dorzolamide
The fixed combination of latanoprost 0.005% and dorzolamide 2% was used in phase II clinical studies at Alleanza Pharmaceuticals for the treatment of glaucoma in 2013 (NIH, 2014b). The fixed combination of latanoprost (0.0025%) and dorzolamide (2%) will be completed in 2022 (NIH, 2020).
Another fixed combination of the ophthalmic solution composed of 0.1% brimonidine tartrate and 0.5% timolol (Aibeta ® ) was recently approved in Japan (Suzuki et al., 2020). Many clinical studies showed that BrT-FC had superior IOP lowering effect as well as effects in keeping it under control than their constituent agents (Spaeth et al., 2011;Kim et al., 2016), and it was noninferiority to latanoprost in reducing IOP (Katz et al., 2012). Katz, et al. found that 60.3% of patients treated with BrT-FC achieved diurnal IOP <18 mmHg compared to the 52.0% of patients treated with latanoprost after 12 weeks (Katz et al., 2012).

Safety and AEs
Tolerability and side effects were similar with the constituent parts without additional side effects. The most common local AEs of BrT-FC include eye irritation, dry eye, allergic reactions, lid erythema, lid edema, conjunctival follicles, corneal staining/ erosion, and lens opacity (Katz et al., 2012;Kim et al., 2016). Systemic AEs in patients treated with BrT-FC included asthma (Kim et al., 2016).

Fixed Combination of Timolol and Brinzolamide (Azarga ® ) Efficacy
BzT-FC was launched in several European countries in 2009. It has the lowest daily cost and best effectiveness in China compared to other treatments (Xu et al., 2020). A prospective study demonstrated that bimatoprost and BzT-FC exert similar IOP lowering effects in patients with POAG at 8 weeks (Akyol et al., 2017). Importantly, BzT-FC maybe an appropriate choice for patients after uneventful phacoemulsification cataract surgery using Viscoat and Provisc, because BzT-FC can significant prevents IOP increase during the first 24 h after surgery (Georgakopoulos et al., 2013). In a clinical trial, when patients are treated with BzT-FC immediately after surgery or do not receive any ocular hypotensive medication, IOP changes from a preoperative value of +6.7 ± 2.98, +5.3 ± 3.26, and +1.4 ± 2.46 mmHg at 6, 12, and 24 h, to a postoperative value of −0.3 ± 2.95, +0.23 ± 3.49, and −1.76 ± 2.83 mmHg (Georgakopoulos et al., 2013).

Fixed Combination of Timolol and Dorzolamide (Cosopt ® ) Efficacy
DzT-FC (fixed combination of Timolol 0.5% and Dorzolamide 2%) was the first IOP-lowering fixed combination approved by the US Food and Drug Administration (FDA) in the treatment of POAG and NTG (Konstas et al., 2021). PF-DzT-FC 1 (preservative-free fixed combination of Timolol 0.5% and Dorzolamide 2%) and PF-DzT-FC 2 (preservative-free fixed combination of Timolol 0.5% and Dorzolamide 1%) have also been launched in the market in recent year. In an open-label 2center study, patients with NTG received DzT-FC for 12 weeks, and IOP was reduced by 21.7% at the peak drug effect and by 23.9% at 8 h after drug administration . DzT-FC has the same efficacy as latanoprost monotherapy in newly diagnosed NTG patients, and the difference of IOP reduction was 0.39 mmHg (Lee et al., 2016b). Visual-related quality of life and the Glaucoma Symptom Scale were significantly improved at week 8 when patients switched from DzT-FC to PF-DzT-FC 1 (Abegao Pinto et al., 2014), and PF-DzT-FC 1 showed a mean IOP reduction of 6.3% more than DzT-FC after 12 weeks (Renieri et al., 2010). Therefore, preservative-free DzT-FC may be a better choice than preservative DzT-FC.
Generally, both fixed-combination medications represent highly effective without clear superiority of one agent over another; however, patients show more ocular comfortable in BzT-FC group. Comparing to DzT-FC, PF-DzT-FC significantly improved effectiveness and safety, which will be equal to or better than BzT-FC.

Fixed Combination of Brinzolamide and Brimonidine (Simbrinza ® ) Efficacy
BzBr-FC was approved in United States and EU for the reduction of high IOP in patients with OAG and OHT, and it is the fixedcombination ophthalmic suspension to treat glaucoma available without timolol (Greig and Deeks, 2015). BzBr-FC induces a significantly lower mean IOP and exerts a more IOP reduction than either brinzolamide or brimonidine monotherapy Nguyen et al., 2013;Realini et al., 2013;Aung et al., 2014). Importantly, BzBr-FC can significant lower IOP in patients after phacoemulsification cataract surgery. The mean change in postoperative IOP at 6, 12, and 24 h was −0.12, −1.12, and −1.89 mmHg in patients treated with BzBr-FC versus +3.85, +3.46, and +0.85 mmHg in patients of the control group (Georgakopoulos et al., 2020). BzBr-FC 3 times daily exerts a significantly more IOP reduction during the nocturnal period and the entire 24-h period compared to timolol 0.05% twice daily, and IOP lowering effect during the diurnal period is similar in both treatments (Seibold et al., 2017).

Safety and AEs
BzBr-FC is security with no serious AEs and other safety variables appeared , which also has no significant changes in visual acuity, anterior or posterior segment examination, pachymetry, and perimetry are observed . The most common AEs are blurred vision, dysgeusia, ocular hyperemia, dry mouth, and eye allergy Realini et al., 2013;Aung et al., 2014;Weinreb et al., 2019).

Fixed-Combination of α2-Adrenoceptor
Agonists and Rho-Kinase Inhibitors

Fixed-Concomitant Brimonidine/Ripasudil
BrRi-FC (K-232) has been used in phase III clinical trials for the treatment of POAG and OHT since 2020. At present, no new reports on this topic are available.

Comparison of the Fixed Combination of Prostaglandin F2a Analogs and Timolol With Carbonic Anhydrase Inhibitors and Timolol
Several clinical trials demonstrated that the fixed combination of prostaglandin F2a analogs and timolol has a greater IOP lowering effect than the fixed combination of carbonic anhydrase inhibitors and timolol (Eren et al., 2012;Babic et al., 2013). The mean diurnal IOP/peak IOP reduction is greater after the use of LT-FC (8.79 mmHg/7.4 mmHg) than after DzT-FC (7.79 mmHg/ 5.19 mmHg) at week-6 (Eren et al., 2012). The incidence of reported AEs is statistically lower after LT-FC therapy (Eren et al., 2012). A total of 86.7% of patients who received TrT-FC show a IOP reduction>25% compared with the 76.9% of patients treated with DzT-FC after 3 months (Babic et al., 2013). The most frequent ocular AEs in patients treated with TrT-FC are hyperemia (50%), blurred vision, and pruritus (6.7%), but dry eye sensation (30.8%) and foreign body sensation (23.1%) are more reported in patients treated with DzT-FC (Babic et al., 2013).
Overall, the IOP lowering effect of fixed-combination prostaglandin F2a analogs and timolol is significantly superior to fixed-combination carbonic anhydrase inhibitors and timolol, and both of them have no difference in ocular AEs. But the price of fixed-combination carbonic anhydrase inhibitors and timolol is lower, and patient can accept easily.

Comparison of the Fixed Combination of Prostaglandin F2a Analogs and Timolol With Prostaglandin F2a Analogs and Brimonidine
Both brimonidine tartrate (0.1%) and timolol maleate (0.5%) used as adjunctive therapies to PGAs have similar effective, and the difference is only 0.36 mmHg in IOP reduction (Mizoue et al., 2017). No unexpected AEs were reported during the study, and the highest AEs was conjunctival hyperemia (2.8%) in brimonidine-treated patients and pruritus (4%) in timololtreated patients (Mizoue et al., 2017).

Comparison of the Fixed Combination of Prostaglandin F2a Analogs and Timolol With Brimonidine and Timolol
When compared directly of LT-FC, BiT-FC, and BrT-FC, no significant difference among them is observed. The reduction in IOP is 6.8 mmHg (32.1%), 6.7 ± 2.8 mmHg (30.2%), and 10.6 mmHg (42.2%) in LT-FC, BiT-FC, and BrT-FC group, respectively (Yavas et al., 2013). Hommer A, et al. also found that LT-FC and BrT-FC are effective in an equal manner in reducing IOP and they have no effect on the blood flow of the optic nerve head and the velocity of the retrobulbar flow (Hommer et al., 2012).

Comparison of the Fixed Combination of Carbonic Anhydrase Inhibitors and Timolol With Brimonidine and Timolol
When compared directly to BrT-FC, BzT-FC shows more effective and tolerated. Patients who switched from BrT-FC to BzT-FC show a mean IOP reduction from the baseline (on BrT-FC) to 3.6 mmHg Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 749858 (17.1%), and 55.3% of patients achieve an IOP <18 mmHg at week 8 (Alezzandrini et al., 2014). BzT-FC is also more effective than BrT-FC in reducing IOP after phacoemulsification surgery, and IOP value after surgery at day 3 and 5 is −1.1 ± 2.70 and −2.19 ± 2.46 mmHg in the BzT-FC-treated patients, and −1.48 ± 2.53 and −1.72 ± 2.64 mmHg in the BrT-FC-treated patients (Balsak et al., 2018).
Multiple clinical trials reported that BrT-FC is better than DzT-FC in reducing IOP, although no statistically significant difference is found (Gulkilik et al., 2011;Feke et al., 2013;Seymenoglu et al., 2015). The side-effect profile of BrT-FC is similar to that of DzT-FC. But the incidence of the burning feeling and foreign body sensation are significantly higher in patients treated with DzT-FC (43 and 28%, respectively) than in patients treated with BrT-FC (19 and 12%, respectively) (Gulkilik et al., 2011).

Comparison of the Fixed Combination of Carbonic Anhydrase Inhibitors and Timolol With Carbonic Anhydrase Inhibitors and Brimonidine
A clinical trial demonstrated that BzBr-FC is an effective and safe alternative β-blocker free fixed combination. A significant difference in mean morning IOP reduction is observed between DzT-FC-(7.0 ± 2.8 mmHg) and BzBr-FC-treated patients (8.4 ± 1.9 mmHg) for 12 weeks, but mean afternoon IOP reduction had no significant difference (Kozobolis et al., 2017). The common reported AEs are similar after both the two treatments (Kozobolis et al., 2017).

Non-fixed Combination of Latanoprost and Brimonidine Efficacy
An important adjunctive (two-medicine) therapy to reduce IOP is represented by the non-fixed combination therapy of brimonidine tartrate (0.1, 0.15, 0.2%) twice daily and latanoprost (0.005%) once daily (Stewart et al., 2004). A multicenter, open-label, prospective evaluation study demonstrated that the adjunctive therapy with brimonidine purite 0.15% lead to an additional mean IOP reduction from a baseline value of 5.8 mmHg (26%) at peak drug effect after the treatment with 0.005% latanoprost and 3.3 mmHg (15%) at trough drug effect after 1 month (Mundorf et al., 2007). LBr-nFC (brimonidine 0.2% and latanoprost 0.005%) exerted a mean IOP reduction to 9.0 mmHg (33.9%) compared to DzT-FC 6.5 mmHg (25.3%) at 12 weeks in patients with pigmentary and pseudoexfoliative glaucoma (Zabriskie and Netland, 2003). Importantly, brimonidine can act as a supplement to therapy also in prevention as a neuroprotective agent (Conti et al., 2021).

Safety and AEs
No serious AEs are reported, and the most common AEs are ocular allergy (n 2; 4.7%) and foreign body sensation (n 2; 4.7%) in LBr-nFC-treated patients (Mundorf et al., 2007). While patients treated with LBr-nFC have a higher rate of conjunctival hyperemia than LT-FC-treated patients (Stewart et al., 2004).

Non-fixed Combination of Travoprost and Brimonidine Efficacy
A comparative clinical trial showed that timolol 0.5% treatment is associated with a significantly greater reduction in IOP compared with brimonidine 0.2% when added to travoprost 0.004%. The mean IOP reduction is 3.9 mmHg for timolol and 2.3 mmHg for brimonidine and the IOP reduction is 20.2 and 13.4% on day 28, respectively (Reis et al., 2006).

Safety and AEs
No systemic AEs are reported, and slight occasional conjunctival hyperemia is the only AEs in TrBr-nFC-treated patients (Reis et al., 2006).

Non-fixed Combination of Latanoprost and Brinzolamide Efficacy
Several randomized clinical trials reported that the non-fixed combination of latanoprost 0.005% and brinzolamide 1% exerts a better IOP reducing effect than latanoprost monotherapy (Shoji et al., 2005;Nakamoto and Yasuda, 2007;Nakano et al., 2016). A comparative study demonstrated that LBz-nFC provides a more sustained IOP lowering effect than LT-nFC. LBz-nFC reduces both daytime and nighttime IOP, whereas LT-nFC only reduced IOP during daytime, with little effect on the nighttime value (Liu et al., 2009).

Safety and AEs
LBz-nFC represents a safe and effective treatment with no serious AEs reported during the observation period (Shoji et al., 2005). No significant difference in corneal endothelial cell density, systolic, diastolic, mean blood pressures, and pulse rates is observed before and after LBz-nFC administration (Nakamoto and Yasuda, 2007;Miura et al., 2008).

Non-fixed Combination of Bimatoprost and Dorzolamide Efficacy
Non-fixed combination of 0.03% bimatoprost once in the morning and 2% dorzolamide twice daily in patients with POAG exerts an additional hypotensive effect and reduces vascular resistance in the ophthalmic artery compared to bimatoprost monotherapy (Stankiewicz et al., 2010;Stankiewicz et al., 2011). However, BiDz-nFC led to a significant IOP lowering effect only at 4:00 h time point and show a lower IOP fluctuation compared to bimatoprost (4.6 mmHg versus 6.0 mmHg) (Stankiewicz et al., 2010). No serious AEs are reported during study and systemic hypotension is the most common AEs in patients with BiDz-nFC (Stankiewicz et al., 2010).

Non-fixed Combination of Betaxolol and Brimonidine Efficacy
Non-fixed combination of 0.2% brimonidine tartrate and 0.5% betaxolol twice per day exerts statistically greater IOP reduction than their respective monotherapies (Chi et al., 2013). A comparative clinical trial revealed that the decrease IOP rate is 13.8-21.2% in patients treated with betaxolol, 19.8%-25.5% in patients treated with brimonidine and 22.2-33.2% in patients treated with BeBr-nFC after 8-weeks treatment (Chi et al., 2013).

Safety and AEs
No systemic AEs or statistical significant difference are found among patients treated with BeBr-nFC and those treated with monotherapies (Chi et al., 2013). The reported AEs include ocular foreign bodies, irritation, dizziness, headache, fatigue, and dryness of the mouth and nose (Chi et al., 2013).

Non-fixed Combination of Betaxolol and Brinzolamide Efficacy
DzT-FC is more effective after 24 h exposure compared to the effect of the fixed combination of 0.5% betaxolol and 1% brinzolamide, and the rate of IOP reduction is 18-24% and 14-19% in DzT-FC and BeBz-nFC-treated patients, respectively (Brubaker et al., 2000).

Safety and AEs
Anticipated AEs of BeBz-nFC are consistent with the AEs after betaxolol and brinzolamide monetherapy, and the incidence of systemic AEs does not increase (Brubaker et al., 2000).

Safety and AEs
The most common ocular and systemic AEs are mild and include stinging/burning, itching, conjunctival hyperemia, irritation, dry mouth, fatigue, and bitter taste. No serious AEs are reported (Ermis et al., 2002;Ozturk et al., 2005).

Non-fixed Combination of Latanoprost and Ripasudil Efficacy
A clinical trial showed that the lowering effect of IOP exerted by LRi-nFC is superior than that exerted by latanoprost monotherapy, and mean IOP reduction in LRi-nFC and latanoprost-treated patients is 2.2 and 1.8 mmHg respectively, before instillation (9 AM) and 3.2 and 1.8 mmHg respectively, after instillation (11 AM) .

Safety and AEs
No new AEs are observed when ripasudil is added to latanoprost compared to the old AEs exerted by latanoprost, and conjunctival hyperemia is the most frequent AE in patients treated with LRi-nFC and ripasudil (55.9% vs 8.7%) .

Non-fixed Combination of Timolol and Ripasudil Efficacy
A clinical trial demonstrated that TRi-nFC leads to a more IOP reduction than timolol monotherapy at 9 AM (before instillation) (2.4 versus 1.5 mmHg) and 11 AM (after instillation) (2.9 and 1.3 mmHg) .

Safety and AEs
No new AEs are reported when ripasudil is combined with timolol compared to the old AEs exerted by timolol, and the most common AE is conjunctival hyperemia in patients treated with TRi-nFC with 65.4% compared to 5.8% in the timolol-treated patients .

Comparison of Topical Non-fixed Combination of Two Agents
Most clinical trials demonstrated that the non-fixed combination of prostaglandin F2a analogs and carbonic anhydrase inhibitors exerts a greater IOP-lowering effect than the non-fixed combination of prostaglandin F2a analogs and alpha-2 agonists, and both of them have similar systemic and ocular AEs (Konstas et al., 2005;Feldman et al., 2007). A 3-months randomized clinical trial demonstrated that 79 patients who were treated with brinzolamide and 84 patients who were treated with brimonidine twice-daily as adjunctive therapy to travoprost have a diurnal IOP reduction to 2.8 and 2.1 mmHg, and the reduction of diurnal IOP ≥15% is 40 and 27.8% in TrBz-nFC-and TrBr-nFC-treated patients, respectively (Feldman et al., 2007). Konstas, et al. reported that the mean diurnal IOP reduction is 2.2 mmHg in LDz-nFC-treated patients versus 2.1 mmHg in LBr-nFC-treated patients (Konstas et al., 2005). Patients treated with LDz-nFC and LBr-nFC had similar ocular and systemic AEs, while a statistically more incidence of conjunctival hyperemia and bitter taste is observed in patients treated with LDz-nFC (n 9 and 8) than in those treated with LBr-nFC (n 3 and 0) (Konstas et al., 2005).
However, several clinical studies demonstrated that IOP lowering effect of PRO-122 is not inferior to Krytantek Ofteno (NIH, 2019b). And a phase III comparative study demonstrated that the crossover between PRO-122 and TDzBr-FC does not affect IOP, and the mean IOP reduction is 0.11 mmHg difference from the baseline after 60 days (Gomez-Aguayo et al., 2018;NIH, 2019b). Furthermore, an investigator-masked, crossover study demonstrated that BiT-FC once daily exerts a greater IOP lowering effect than TDzBr-FC twice daily (Garcia-Lopez et al., 2014).

Safety and AEs
The incidence of AEs is similar between PF-TDzBr-FC and TDzBr-FC-treated patients, and ocular burning is the one with the highest incidence among the eye disorders in both groups (Gomez-Aguayo et al., 2018;. The cup/disk ratio and visual field are not statistically different in BiT-FC and TDzBr-FCtreated patients (Garcia-Lopez et al., 2014).

Fixed Combination of Bimatoprost, Timolol, and Brimonidine Efficacy
Two comparative clinical studies demonstrated that TBiBr-FC has clinically and statistically significantly superior IOP lowering effects than BrT-FC (Hartleben et al., 2017;Belfort et al., 2020). A phase 3 study conducted in Mexico and Colombia revealed that the mean IOP changes from baseline are 10.03 and 9.18 mmHg, and percentage of IOP ≤13 mmHg is 33.7 and 14.8% for TBiBr-FC and BrT-FC respectively, after 12 weeks (Hartleben et al., 2017). Another phase III study conducted in Brazil demonstrated that the mean IOP reduction at 12 weeks is 10.45 mmHg in patients treated with TBiBr-FC and 8.28 mmHg in those treated with BrT-FC. A total of 28.9, 36.8, and 71.1% of patients treated with TBiBr-FC achieved levels of IOP ≤13, ≤14, and ≤16 mmHg compared to 15.7, 22.9, and 55.4% in patients treated with BrT-FC (Belfort et al., 2020). TBiBr-FC maybe a new promising fixed-combination three agents for patients whose IOP cannot be inadequate by combination two agents, but more clinical trials are needed to verify the effectiveness and safety.

Safety and AEs
The most common AEs are ocular side effects and none are serious in TBiBr-FC and BrT-FC-treated patients (Hartleben et al., 2017;Belfort et al., 2020). The most common AEs include conjunctival hyperemia, eye irritation, allergic blepharitis, allergic conjunctivitis, and dry eye. Conjunctival hyperemia, eye irritation, and eye pruritus are statistically significant in patients treated with TBiBr-FC than those treated with BrT-FC (Hartleben et al., 2017;Belfort et al., 2020).

TOPICAL NON-FIXED COMBINATION OF THREE AGENTS
Topical non-fixed combinations of three agents are usually composed of a fixed combination of two agents and a monotherapy agent, and they are used in patients whose IOP cannot be well controlled by combination two agents. Importantly, there is no a safe and reliable fixed-combination three agents in widespread use today.

Safety and AEs
The ocular and systemic AEs are more frequently reported after the use of the non-fixed combination of three agents compared with travoprost monotherapy (Konstas et al., 2013a). Transient blurred Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 749858 vision occurred significantly more frequently in TBzTr-nFC-treated patients than in those treated with BzT-FC. The incidence of itchiness, hyperemia, and fatigue are more in the TBrTr-nFCtreated patients, and other AEs were similar in the two treatments (Konstas et al., 2013a).

Non-fixed Combination of Brimonidine, Timolol and Latanoprost Efficacy
A 12-weeks, randomized, multicenter study demonstrated that LTBr-nFC exerts a greater IOP lowering effect than LT-nFC in patients whose IOP is not adequately controlled by latanoprost 0.005% alone (Fechtner et al., 2011). The results showed an additional IOP reduction of 8.3 mmHg (35.5%) and 6.2 mmHg (27.0%) at 10 am, the incidence of IOP less than 18 mmHg at both peak and trough measurements is 59.6% versus 42.6%, and IOP reduction over 20% is 72.3 and 57.5% in LTBr-nFC and LT-nFCtreated patients, respectively (Fechtner et al., 2011).

Safety and AEs
No statistically significant difference in overall AEs is found between patients treated with LTBr-nFC and LT-nFC, and the most common AEs are ocular allergy in the patients treated with LTBr-nFC (3.9%) and punctate keratitis in those treated with LT-nFC (2.9%) (Fechtner et al., 2011 Goldberg et al., 2012). Goldberg I, et al. reported that brinzolamide reduces IOP by 1.1-1.5 mmHg (6-9%) when added to TrTFC, and an additional IOP reduction is observed at diurnal IOP, specifically at 08:00 and 16:00 o'clock (Goldberg et al., 2012). Another comparative study demonstrated that the average decrease of diurnal IOP is 6.2 mmHg (21.8%) after the use of travoprost, 9.3 mmHg (32.6%) after the use of TrT-FC, and 11.2 mmHg (39.3%) after the use of TBrTr-nFC (Hollo and Kothy, 2008).

Safety and AEs
No serious AEs are reported during the study (Hollo and Kothy, 2008) and common AEs including conjunctival hyperemia, eyelashes, cataract, blurring, superficial punctate keratitis, conjunctival redness, reduced visual acuity, and itching are no statistically different between patients treated with TBrTr-nFC and those treated with TrT-FC (Hollo and Kothy, 2008).

Safety and AEs
LTDz-nFC provides a safety profile consistent with that of its individual components. The majority of AEs are mild or moderate (Lesk et al., 2008), and included burning/stinging, superficial punctate keratitis, watering, itchiness, conjunctivitis hyperemia, dry eye, ocular irritation, dry mouth, foreign body sensation, and blurred vision (Konstas et al., 2008).

Safety and AEs
The rates of AEs in PF-TDzTf-nFC and latanoprost-treated patients are similar, and they are were more than those in PFtafluprost-treated patients. Burning (6.9%), stinging (20.9%), and bitter taste (11.6%) are significantly more common in patients treated with PF-TDzTf-nFC than in those treated with PFtafluprost monotherapy (Konstas et al., 2017).

Comparison of Non-fixed Combination of Prostaglandin F2a Analogs, β-blockers, and Carbonic Anhydrase Inhibitors
When patients received brinzolamide 1% (2 times a day) or dorzolamide 1% (3 times a day) plus a fixed combination therapy of latanoprost and a BB, the mean IOP reduction is 1.9 and 1.8 mmHg at week 8, respectively (Tsukamoto et al., 2005). The common AEs observed in this study include ocular irritation and blurred vision. Dorzolamide causes more intense ocular irritation than brinzolamide (74% vs 16%), and no significant difference in blurred vision is observed between the two groups (52% vs 37%) (Tsukamoto et al., 2005).
Both brinzolamide and dorzolamide added to a fixedcombination latanoprost-β-blockers show a similar IOP lowering effect, but brinzolamide (2 times a day) presents a better patient compliance and ocular comfortable than dorzolamide (3 times a day), which is more meaningful for glaucoma patients.

Safety and AEs
The incidence of AEs is higher in patients treated with BzBrTr-nFC compared to those treated with travoprost (30.8% 14.7%), and the most common AE in BzBrTr-nFC group is conjunctival hyperemia (Feldman et al., 2016).

Comparison of Non-fixed Combination of Prostaglandin F2a Analogs, α-2 Adrenergic Agonists, and Carbonic Anhydrase Inhibitors
Several clinical trials reported that BzBr-FC as an adjunct to PGA exerts an additional IOP lowering effect (Feldman et al., 2016;Topouzis et al., 2021). The percentage of mean IOP reduction is 24.7% in patients treated with BzBr-FC plus PGA and 8.2% in those treated with vehicle plus PGA at week 6 (Feldman et al., 2016). 60.0% of patients achieving the goal of IOP ≤18 mmHg after BzBr-FC plus PGA treatment versus 20.7% of patients treated with vehicle plus PGA (Topouzis et al., 2021).

DISCUSSION AND OPINION
Nowadays, multiple classes of topical eye drops are available for the treatment of glaucoma, but it is still a challenge to establish the most reasonable and optimal prescription for every patient because systematically extensive clinical studies on different classes of medications are still limited. In this review, the most recent articles on topical anti-hypertensive ophthalmic agents and their characteristics were summarized, which will provide a reference for glaucoma patient care. Monotherapy is usually used for initial treatment, which cannot usually reduce IOP to a physiologically level for long time. Therefore, combination multiple agents play a vital role for long-term control of IOP. Fixed combination and non-fixed combination of two agents results in similar IOP-lowering effect, which are also superior to monotherapy, and no significant difference in local and systematic AEs was observed among them. If combination two agents fail to reduce IOP sufficiently, the second drug can be replaced or a third medication can be added to the fixed combination, or a fixed-combination of three agents can be used. Now, fixed combination three agents, such as TDzBr-FC, cannot be widely available in various countries due to its obvious increase of AEs and no significant improvement in IOP, and TBiBr-FC is still being in clinical trial. Research and development of new, reliable, effective and safe fixed combination three agents is a trend of great importance. Netarsudil is a new launched Rhokinase inhibitor, which provides a better neuroprotection effect superior to other eye drops. Rho-kinase inhibitors might be combined with β-blockers and be suitable for those who are not candidates for PGAs. Furthermore, another potential fixed combination including latanoprost, netarsudil and timolol, may provide better IOP lowering effect and patient compliance, lower preservative exposure and the incidence of side effects by targeting more mechanisms with once per day comparing to traditional marketed medicines.
The current review revealed that the use of a fixed combination of topical anti-hypertensive ophthalmic agents is recommended as early as possible in patients with glaucoma, to better control its development and decrease the damage of the optic nerve. Fixed combination agents not only provide a better effect than monotherapy but also result in a better convenience and tolerance, and less AEs and ocular discomfortable than nonfixed combination. Apart from the effect and safety, local availability and costs are also important factors to consider. Thus, fixed combination agents are a promising choice in the treatment of glaucoma and ocular hypertension.

CONCLUSION
Glaucoma is still one of the most common causes of irreversible vision loss, and the reduction and control of IOP are necessary to Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 749858 decrease the rate of visual field deterioration to treat glaucoma. Despite numerous surgical approaches and classes of IOP-lowering drugs available, glaucoma remains a challenging chronic disease. Many new emerging strategies including medicines and surgery are developed for the treatment of glaucoma by affecting different pathways, providing a new choice to patients and offering additivity and complementary to current therapeutics, although they need long-term rigorous verification of safety and efficacy. The current recent studies revealed that fixed combination therapy with reliable safety and effectiveness is significantly important for patients with glaucoma and effective on glaucoma, and it's of great importance to develop fixed combination agents with a high effect for a better control of glaucoma.

AUTHOR CONTRIBUTIONS
TZ and QJ designed and revised this manuscript. TW wrote the manuscript. LC polished language in English. All of the authors have read and approved the final manuscript.  Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 749858