Tesamorelin - an overview

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12.2 Growth hormone-releasing hormone (GHRH) analog

Tesamorelin [88–90], is a long-lasting GHRH analogue, that was shown effective in attenuating visceral fat and lipodistrophy in HIV-infected individuals taking highly active anti-retroviral therapy (HAART). As May 2018, Theratechnologies (www.theratech.com) has announced that tesamorelin will enter a Phase 2b trial in patients with in the current year.

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12 Agents that target classical hormone receptors

12.1 Mineral corticoids

Aparenone (MT3995) is small molecule antagonist of aldosterone and is currently advanced to Phase 2 trial in patients with diabetic nephropaty and NASH [86,87]. Results are pending.

12.2 Growth hormone-releasing hormone (GHRH) analog

Tesamorelin [88–90], is a long-lasting GHRH analogue, that was shown effective in attenuating visceral fat and lipodistrophy in HIV-infected individuals taking highly active anti-retroviral therapy (HAART). As May 2018, Theratechnologies (www.theratech.com) has announced that tesamorelin will enter a Phase 2b trial in patients with in the current year.

12.3 β-Selective thyroid hormone receptor (THR) agonist (MGL-3196)

Since the beneficial effects of thyroid hormone on lipid levels are primarily linked to action on the thyroid hormone receptor β (THR-β) in the liver (Fig. 2), while adverse effects, including cardiac effects, are mediated by THR-α, selective THRβ agonist have been developed to target metabolic disorders. MGL-3196 [91] is a orally active THR-β agonist, that diminish hepatic lipid accumulation in rodents fed a high fat diet and is currently developed for treating NASH. In a Phase 2 trial in patients with biopsy-proven NASH [91], MGL-3196 has meet all the primary endpoints reducing the hepatic fat content and liver fibrosis as measured by MRI-PDFF.

12.4 Leptin analog

Metreleptin, a leptin analogue approved by FDA for the treatment of generalized lipodystrophy, a rare condition characterized by leptin deficiency. Metreleptin has also been tested for its effects on lipid accumulation in lipodistrophic patients [92]. A small pilot trial (ClinicalTrials.gov Identifier: NCT01679197) carried out on 23 patients, of whom 18 completed one year follow up, has shown some signals of steatosis improvement. It is unclear whether development will be continued.

12.5 Adenosine (A)3 receptor antagonist

CF102, also known as namodenoson, is a small orally bioactive, highly selective agonist for the A3 adenosine receptor (A3AR). This agent [93] exert anti-fibrotic and anti-inflammatory activity in rodent models of NASH and liver fibrosis, partially by targeting NKT cells (Fig. 3 and 4). CF102 is currently investigated in a Phase 2 trial in patients NASH. The trial is ongoing and enrollment is expected to finish in 2018.

12.6 Gut microbiota

Intestinal dysbiosis is considered a causative mechanisms of NASH, at least in animal models [26]. A recent meta-analysis has demonstrated a putative relation between intestinal dysbiosis and NAFLD/ NASH [94], and patients are known to develop a signature microbiota that might predict the severity of liver fibrosis [95]. Probiotic and fecal microbiota transplantation have shown beneficial effects in rodent models of NASH [96,97], suggesting that modulation of gut microbiota through probiotics and/or symbiontics (that target protozoa) could provide a beneficial effect in NASH patients. The macrolide antibiotic, solithromycin [98], has been tested with some results in a pilot study and is currently in a phase II trial (NCT02510599). Similarly,

IMM-124 E, a hyperimmune bovine colostrum [99], that modulates the gut-liver axis, is currently evaluated in Phase 2 trial in patients with NASH. Finally, several ongoing trials are currently running to test efficacy of probiotic and fecal microbiota transplantation in NASH patients (NCT02469272, NCT02868164, NCT02721264, NCT01680640, NCT02568605, NCT02642172).

12.7 Miscelaneous agents

The semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1) is an homodimeric glycoprotein adhesion molecule that is widely expressed in the vascular system. During inflammation (Fig. 4) this complex facilitates leukocyte recruitment through its SSAO component and its activation promotes liver inflammation and fibrosis [100]. BI 1,467,335 (formerly known as PXS-4728 A) is a SSAO/VAP-1 inhibitor that was shown effective in reducing liver injury in rodents [100]. A Phase 2 clinical trials in patients with NASH was started in 2017 [101].

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Lipodystrophy

In a separate category, on November 10, 2010, the FDA approved Egrifta (tesamorelin) to treat HIV patients with lipodystrophy (associated with many ART medications), a condition in which excess fat develops in different areas of the body, most notably around the liver, stomach, and other abdominal organs. Although it may decrease visceral abdominal fat in PLWHA, it has not been shown to provide any clinical benefit (e.g., decrease in cardiovascular risk or improved quality of life); it is nonformulary, and the estimated 30-day price is $2042.18 Long-term safety is yet another concern, and a 10-year observational study is currently underway.19

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However, new options are emerging that could help alleviate some of the adverse effects reducing compliance. For example, the growth hormone-releasing factor analog tesamorelin has been shown to reduce visceral adipose tissue in HIV patients on HAART (Falutz et al., 2007). Clinical trials assessing the effect of uridine, pravastatine, and their combination on the recovery from HAART-associated lipoatrophy are being conducted. Although a small study has shown minimal benefit of this approach (Calmy et al., 2009a), the final answer should come from a large ongoing ACTG study (http://www.clinical trials.gov/; study # NCT00307164). It is likely that the exploration of various pharmacological approaches to reduce HAART-associated side effects will continue in future.

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5 Current therapeutic approaches in AD

As of fall of 2016, the TR database contains four agents defined as anti-MCI drugs and being in the stage of preclinical trials. Potential protective effects of these compounds are discussed below.

DP-NDD is an oligopeptide product developed by D-Pharm Ltd. for the treatment of AD, Parkinson’s disease (PD, and MCI. The lead-compound is aimed at correcting pathological protein aggregation inherent in neurodegenerative disorders. In particular, the agent is under preclinical trials for preventing amyloidosis in AD and MCI [18].

Another compound is BCA909, which is under development by BioCrea GmbH, for the treatment of MCI, AD, anxiety, schizophrenia, and depression. BCA909 is a lead drug candidate of original class of selective phosphodiesterase 2 (PDE2) inhibitors, penetrating the CNS through the blood–brain barrier. The PDE2 inhibition enhances the action antagonists of dopamine-2 receptors, useful for treatment of psychosis, and the action of agonists of dopamine 1 receptors, which decrease the side effect liability and contribute to the pro-cognitive profile. In 2011, preclinical trials of the agent were started, which demonstrated that BCA909 has a significant pro-cognitive activity in models of cognitive impairment caused by disruption of cholinergic as well as glutamatergic neurotransmission [19]. More recent data on the development of this drug are not available.

NNZ-2591 is being developed by Neuren Pharmaceuticals. This is an original pyrrolopyrazine derivative, 8a(R)-allylperhydropyrrolo[1,2-a]pyrazine-1,4,-dione. The molecular mechanism of action of this agent has not been clarified; however, it was shown to stimulate cognition and improve motor function in animal models of neurodegenerative disorders [20]. A similar situation occurs for GRE-213 developed by Grespo [21]. GRE-213 is a medical food product that is an oral combination of well-known active ingredients and is under development for the potential treatment of mild cognitive impairment. The first component is generally recognized as safe and the second component is similar to established pharmaceutical products. In preclinical studies, GRE-213 showed an improvement in cognitive behavior, and reinforced neurogenesis and synaptic plasticity in animal models of age-related CNS degeneration. A good safety profile was also demonstrated. No data regarding moving to clinical trials of these agents are currently available.

The DF-302 agent is under preclinical trials for treating MCI. The investigations are underway out in Russia in accordance with State Contract 14.N08.12.1027 with the Ministry of Science and Education. The compound was developed at the Institute of Physiologically Active Compounds of the Russian Academy of Sciences [22] and represents a fluorinated analogue of the Dimebon drug, which demonstrated very promising results in phase II clinical trials for Alzheimer’s patients [23], but on phase III trials did not show improvement in comparison with placebo (it has to be mentioned that patients in this trial did not deteriorate significantly in either the drug-treated group or the placebo group, which makes interpretation of the study more difficult [24]). A study of the mechanism of action of DF-302 demonstrated that it reduces uncontrolled protein aggregation in the animal models of proteinopathies. The Thy1mgSN and P301S transgenic mouse was used as experimental models. The Thy1mgSN transgenic mouse on the C57Bl/6J genetic background are characterized by high level of accumulation of the aggregation-prone gamma-synuclein protein and formation of intracellular amyloid type pathological deposits, which in turn promotes the key steps of proteinopathy pathogenesis. The other P301S line of transgenic mice is characterized by overproduction of the aberrant human tau protein, which is a characteristic histopathological structure inherent in taupathy that seen and/or accompanying AD. According to the trial results, systematic administration of DF-302 considerably reduced the amount of Aβ deposits in the spinal cord tissues of the Thy1mγSN transgenic animals. Indeed, prolonged administration of DF-302 led to a statistically significant decrease in the amount of tau positive neurons in the spinal cord of P301S transgenic mice [25]. Recently, it was shown that DF-302 unlike dimebon exerted pronounced pro-neurogenic activity under normal and stressful conditions [26]. Thus, this compound can be regarded as a disease-modifying drug candidate able to affect pathogenetic mechanisms of the disease.

During 2016, several anti-amyloid drugs underwent phase I trials in recent years. For example, the Sanofi Pharmaceuticals Company is developing SAR-228810–an anti-protofibrillar Aβ monoclonal antibody, for the treatment of MCI due to AD. Protofibrils are metastable intermediates in the formation of amyloid fibrils, which can alter the electric activity of neurons and cause neurodegeneration in the central nervous system. Thus, the SAR228810 drug candidate affecting the Aβ protofibrils can be efficient for treating early AD and MCI [27]. In 2013, the drug candidate was under Phase I parallel-group, single and multiple dose escalation study to assess the safety and tolerability as well as pharmacokinetic parameters of SAR228810 administered as intravenous infusions or subcutaneous injections in patients with MCI and mild to moderate AD. The trial results have not been disclosed up to now and, according to the TR database, the agent is still under active development.

Eli Lilly is developing the LY-3002813 agent for the treatment of MCI due to AD. The medication is an N3pG monoclonal antibody targeting Aβ(p3-42), resulting from cleavage of the amyloid precursor protein (АРР). The monoclonal antibody is able to cross the blood–brain barrier and is active against both soluble and insoluble Aβ peptide, without causing microhemorrhage. Preclinical trials using PDAPP transgenic mice as AD models demonstrated that a modified antibody was able to clear pre-existing amyloid-beta deposits without toxic side effects. The agent is under Phase I trials covering about 100 participants [28]. One more drug developed by the same company, LY-2599666, is meant for subcutaneous administration for the treatment of MCI due to AD and mild or moderate AD. In 2015, the agent was under Phase I clinical trials [29]. The data on the structure or mechanism of action are not disclosed.

In 2017, Pfizer announced the beginning of Phase I clinical trial of the agent Bosutinib. In 2012, the drug was launched in U.S. for the treatment of adult patients with chronic, accelerated, or blast phase Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemia (CML) with resistance, or intolerance to prior therapy. The agent is a multiple kinase inhibitor (in particular, inhibitor of kinases of Src, Bcr and Abl − family, and also signal tranducer and activator of transcription 5) [30]. In in vitro experiments, significant reduction in active Src, MAPK and phosphorylated EGFR and ERK1/2 compared to untreated cells was observed. It is supposed that Bosutinib also prevents formation of abnormal tau-aggregates.

The SAGE-547 agent is being developed by Sage Therapeutics. SAGE-547 is a formulation of allopregnanolone (progesterone metabolite), a positive allosteric modulator of gamma-aminobutyric acid type A (GABA-А) receptor. The potentiating of GABA-mediated chloride current influx is known to induce hyperpolarization of neurons, resulting in a down-regulation of excitation; hence, this can be considered as a possible neuroprotective mechanism in some neural disorders [31]. A preclinical study for evaluation of allopregnanolone activity in blocking of Aβ-induced neurodegeneration on animal models of AD showed that allopregnanolone promotes survival of newly generated neural cells, reduces accumulation of the Aβ oligomer. Furthermore, allopregnanolone modulates phosphorylated-tau protein expression in the 3xTgAD mice and regulates liver X receptor (LXR), pregnane X receptor (PXR), and 3-hydroxy-3-methylglutaryl-СоА-reductase (HMG-CoA-R) expression in 3xTgAD mice. The allopregnanolone treatment inhibits microglial activation and increases a marker of myelination in the brains of the 3xTgAD mice. In July 2014, the University of Southern California in collaboration with the National Institute on Aging (NIA) registered a Phase I and in 2017 it was moved on Phase 1b/2a trials to evaluate the safety and tolerability of allopregnanolone in MCI and early AD [32]. The drug is also under Phase III trials for the treatment of super-refractory status epilepticus and under Phase II phase trials for the treatment of traumatic brain injury, tremor, and for adjuvant therapy of severe postpartum depression.

The AbbVie company had performed Phase I clinical trials of the ABT-957- novel ceplain inhibitor developed for the treatment of MCI [33] and in parallel, the company started clinical investigations of this agent for AD patients [33]. Both trials have been terminated recently.

For a number of years, Suven Life Sciences Ltd. has pursued the search for and investigation of drug candidates for treating AD and MCI based on selective serotonin receptor (5-НТ) antagonists. There are data on several drug candidates (SUVN-501, SUVN-502, SUVN-507, SUVN-512, and SUVN-976), which underwent preclinical studies for the treatment of AD and MCI as selective serotonin type 6 (5-HT6) receptor antagonists. The blockage of this serotonin receptor type is known to enhance neurotransmission in cholinergic and glutamatergic neurons, which is usually disrupted in neurodegenerative diseases. The company does not disclose the chemical structure of the compounds; however, judging by the existing patents, these are arylsulfonyl-indoles and–carbazoles, and their derivatives. Preclinical studies showed that these compounds are 5-HT6 receptor antagonists increasing the extracellular level of acetylcholine and, hence, improving memory acquisition in experimental animals. According to TP database, SUVN-502 is now under intense development. This selective 5-НТ6 receptor antagonist, which readily penetrates the brain upon oral administration [34], is also under Phase II clinical trials for treating early AD when used in combination with Donepezil and Memantine [35,36]. No data about active development of other compounds of this group are available. Another selective 5-НТ6 receptor antagonist − quinoline derivative –Intepirdine was in phase III clinical trials at Axovant Sciences for the treatment of patients with mild-to-moderate Alzheimer's disease and in phase II clinical trials for the treatment of patients with dementia with Lewy bodies. The product had previously been undergoing clinical evaluation at GlaxoSmithKline for the treatment of Alzheimer's-type dementia. However, recently Axovant Sciences has announced that the co-primary efficacy endpoints were not met in the phase III MINDSET trial of intepirdine in patients with mild to moderate Alzheimer's disease who were receiving background donepezil therapy (ClinicalTrials.gov Identifier NCT02585934).

Yet, another class of medications being developed by Suven Life Sciences Ltd. are carboxamide quinoline derivatives, which are selective agonists of serotonin type 4 (5-НТ4) receptors. These receptors are known to be expressed in acetylcholine-innervated CNS regions and to be involved in cognitive function. Being 5-HT4 receptor partial agonists, these agents stimulate the activation of cAMP-response element-binding (CREB) protein and thus provide improved signal transmission and induce neurogenesis. The lead compound in this group is SUVN-D4010 (SUVN-D1108121) which was under Phase I clinical trials for the treatment of moderate cognitive impairment due to AD [37,38]. Recently clinical trials have been completed.

The Takeda company is developing a new muscarinic М1 receptor positive allosteric modulator, TAK-71, for oral administration to treat MCI and early AD [39]. Currently, the drug is in Phase I trials both as a monotherapy and in combination with Donepezil [ClinicalTrials.gov Identifier NCT02769065]. In parallel, the company carries out phase I studies for the treatment of Levy body dementia.

At least four other drug candidates being in Phase 2 trials are positioned as anti-amyloid agents capable of reducing the level of neurotoxic Aβ forms in the brain. For example, BAN2401 is a humanized mAb158 antibody derived from mice immunized with protofibrils, isolated upon the arctic mutation of Аβ42. The mechanism of action includes selective binding, neutralization, and elimination of soluble protofibrils, which are toxic Aβaggregates involved in the development of neurodegenerative processes in AD [40]. In September 2010, Eisai Co., Ltd. initiated the first clinical study of BAN2401, a new monoclonal antibody developed as a new-generation drug for treating AD, with patient enrollment. BAN2401 is the first example of a monoclonal antibody that can selectively bind, neutralize, and eliminate soluble protofibrils–toxic amyloid-beta aggregates. In February 2013, Eisai Inc. completed a Phase I, randomized, double-blind, placebo-controlled, combined single and multiple dose escalation study to assess safety, tolerability, immunogenicity, pharmacodynamic response, and pharmacokinetics of intravenous infusions of BAN2401 in subjects with mild to moderate AD. Since January 2013, Eisai Inc. has started a Phase II study of BAN2401 to evaluate safety, tolerability, and efficacy of BAN2401 in patients with early AD. The study was registered with the US National Institutes of Health. Currently, this drug candidate is in Phase II of development in the USA and EU for treating AD and in Phase I in Japan for treating MCI due to AD and mild AD.

CSP-1103 (CHF-5074) is under development by Cere Spir Inc. as a preventive agent for MCI due to AD and the Batten disease. CSP-1103 is a modulator of gamma-secretase, an enzyme involved in the formation of Aβ, which stimulates neurotrophin expression in primary neurons. The drug candidate has passed preclinical trials, in which it decreased the Aβdeposition in brain tissue and attenuated memory deficit in transgenic mouse models of AD [41]. Phase I trial was performed for three ascending doses of the drug (200, 400, and 600 mg per day for 14 days) in a double-blind, placebo-controlled, parallel-group study involving 48 healthy subjects. No serious or severe adverse effects were reported for any of the subjects. The maximum tolerated dose was about 600 mg per day. Currently, the drug is in Phase II clinical trials for the prevention of MCI due to AD and, according to a parallel drug development program, as an orphan drug for treating the Batten disease.

E-2609 is an inhibitor of beta-secretase (BACE1), an enzyme involved in the formation of Aβpeptide from the АРР protein [42]. In 2014, Eisai Co., Ltd. and Biogen Idec signed an agreement on joint development of this drug. In 2016, this agent successfully passed Phase II and is currently renamed as Elenbecestat in Phase III clinical trials for the treatment of patients with MCIt and mild dementia due to Alzheimer's disease.

Phenserine, a known AChE inhibitor, underwent Phase III clinical trials involving AD patients for the ability to reduce the АРР and Aβlevels in the plasma and CSF of mild to moderate AD patients. The statistical analysis of the results did not show significant improve relative to the placebo group; however, more detailed analysis for separate groups showed a beneficial effect for the highest-dose group of patients. Currently, QR Pharma is performing Phase II clinical trials of (+)-phenserine called Posiphen involving patients with MCI, early Alzheimer’s dementia and PD. The drug is declared as an anti-amyloid agent, which has been shown to reduce the АРР, and Aβlevels in preclinical studies on rodents and to cause no significant side effects [43].

Recently, compounds modulating the activity of nicotinic receptors in the brain have also been classified as agents involved in the neurotoxic action mechanism of Aβpeptides. For example, Tropisetron, which is a well-known in clinical practice as an antemetic. In recent years, this compound, which is a α7 nicotinic receptor partial agonist and serotonin receptor type 3 (5-НТ3) antagonists, was shown to affect cleavage of the АРР, one of the key players in the pathological processes involved in AD. In particular, tropisetron was shown to considerably increase the sAPPα/Aβ1-42 ratio of the normal and pathological peptide formed upon APP cleavage in neuronal cultures of the J20 (PDAPP, huAPP (Swe/lnd)) mice. In in vivo studies involving the J20 mice, tropisetron improved thesAPPα/Aβ ratio and also improved the spatial and working memory in mice, being effective both during the symptomatic and pre-plaque phases [44]. On these grounds, the Buck Institute for Research on Aging initiated, in 2014, Phase I/II randomized, double-blind, placebo-controlled, sequential cohort, multicenter study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics and to estimate the preliminary efficacy of tropisetron in patients with MCI due to AD.

A number of drug candidates in Phase II clinical trials refer to the signal modulating agents, which act at the synaptic transmission level. These are mainly drugs acting on enzymes and receptors and on the second messenger system. Drugs of this type include UE-2343 (Xanamem), which is a small-molecule amidoisothiazole derivative formulated as capsules. This drug candidate targets 11-beta-hydroxysteroid dehydrogenase type 1 (11-beta-HSD1) that regulates the conversion of glucocorticoids from inactive to active form. Furthermore, the expression of this enzyme results in endothelial-dependent vasodilation. Increased cortisol levels lead to irreversible memory loss and formation of amyloid plaques and neurofibrillary tangles (NFT) in the brain, which aggravates the disease. It is expected that by inhibiting this enzyme, the drug could retard the development of Alzheimer’s dementia. In preclinical 11-beta-HSD1 knockout model studies, the 11-beta-HSD1 level in brain was found to be inversely correlated with cognitive decline, and protection against age-related cognitive impairment was detected. The 11-beta-HSD1 inhibition by UE-2343 reduced the formation of Aβplaques and decreased the plasma amyloid-beta level in animal models [45]. After successful completion of Phase I clinical trials, Actinogen Ltd., which received a license for the development of this drug, started Phase II clinical trials involving patients with MCI due to AD in 2016 [46].

Another drug is AN2/AVex-73 being developed by Anavex Life Science. The compound is a multi-target drug, being muscarinic М1 receptor agonist, sigma-1 opioid receptor agonist, NMDA-receptor antagonist, voltage gated sodium channel and tau hyperphosphorylation blocker, and lipid peroxidation inhibitor [47]. Recently, new positive results of Phase II clinical trials of the agent ANAVEX2-73 (AN2/AVex-73) in AD patients have been presented at 10th Clinical Trials on Alzheimer's Disease (CTAD) [48]. Simultaneously, the company started Phase I clinical trials in order to treat Rett’s syndrome and performs preclinical trials for evaluation of applicability of this drug for the treatment of ADС and epilepsy.

DAOI-B acts via indirect activation of NMDA receptors − a subgroup of ionotropic glutamate receptors of the CNS, which regulate the influx of calcium ions into nerve cells. These receptors are known to play an important role in memory consolidation and cognitive function. Meanwhile, hyperactivation of these receptors contributes to the development of neurodegenerative processes in brain, and NMDA receptors may regulate nerve growth factor expression, thus providing neurotrophic regulation in brain. Hence, moderate activation of NMDA receptors may form a promising strategy for improving cognitive function in patients with early dementia. Although direct data on the DAOI-B structure and molecular mechanism are lacking, presumably, this drug is a D-amino acid oxidase inhibitor, causing accumulation of d-serine − NMDA-receptor glycine binding site co-agonist − and thus enhancing the NMDA receptor. DAOI-B for the treatment of MCI and mild Alzheimer’s disease is under study at the Chang Gung Memorial Hospital (Taiwan). In January 2012, a Phase II trial was initiated to evaluate the applicability of the NMDA-activating agent for treatment of MCI and mild AD. The purpose of the study was to investigate whether DAOI-B would be more efficacious than a placebo for cognitive function in patients with MCI or mild AD. The study was completed in May 2013 [49]. In February 2014, another Phase II trial was initiated in order to investigate the effect of DAOI-B for the treatment of cognitive function and behavioral and psychological symptoms of dementia (BPSD). It was assumed that DAOI-B might show better efficacy than placebo for cognitive function and clinical symptoms in BPSD patients. This study was expected to be completed in the late 2015; however, the results have not been published as yet. Recently, researchers from Takeda Company published new date on DAOI-B structural analogue–the agent PGM030756–on increasing d-serine concentration in experiments in vivo, thus giving promise for further development of this compound as a potential therapeutics for AD and MCI treatment [50].

Ladostigil developed by Avraham Pharmaceuticals Ltd. seems to advance the furthest towards clinical use. Ladostigil is a deliberately designed binary drug containing a carbamate moiety, known as acetylcholinesterase and butyrylcholinesterase inhibitor, and a propargyl moiety, which is a selective monoamine oxidase B (МАО-B) inhibitor [51]. Since inhibition of cholinesterase group enzymes is considered as an approach to AD therapy and MAO-B inhibition is a known approach to the development of antiparkinsonian drugs, ladostigil is expected to be efficient for treating both AD and PD. The mechanism of action includes also neuroprotection and immune modulation associated with the ability to reduce oxidative stress. Initially, the drug candidate was tested on AD patients. However, in October 2012, Avraham Pharmaceuticals Ltd. terminated the AD treatment program in Phase II as the drug candidate failed to meet the endpoint. Earlier, Teva (the primary licensee) also stopped Phase II investigations of this drug candidate for treating dementia and AD. Currently, Avraham Pharm. Inc. performs one more multi-center, double-blind, placebo-controlled, randomized study to evaluate the safety and efficacy of low doses of ladostigil in patients with MCI. The trial was designed as an interventional, randomized, parallel-group, double-blind study of the treatment safety and efficacy. The primary outcome measures included the conversion from MCI to AD compared to placebo and the total number of conversions from MCI to AD across the entire 3-year study period. This conversion is defined by a clinical dementia rating (CDR) score of greater than or equal to one.The secondary outcome measures included a change in geriatric depression scale for ladostigil versus placebo administration, a change in neuropsychiatric test scores for ladostigil versus placebo administration, a change in disability assessment in dementia for ladostigil versus placebo administration, and safety of 10-mg ladostigil versus placebo treatment. These parameters, as well as clinical laboratory safety data, are collected within 3, 6 12, 18, 24, 20, and 36 months. The data such as the time to development of probable or possible AD, the mean time to development of probable or possible AD, and the time to conversion from MCI to AD are analyzed by means of Kaplan-Meier’s survival method.

Levetiracetam (AGB101) is under development to slow the progression of AD and had been shown to improve the memory of patients with amnestic mild cognitive impairment (aMCI). The compound is an N-type calcium channel blocker (Ca(v) 2.2) and a synaptic vesicle protein 2А (SV2A) ligand [52]. Recently, levetiracetam was found to inhibit the Aβpeptide-induced glutamate release from human astrocytes [53]. Initially, it was brought to the market by the UCB Company for adjuvant therapy of convulsions of various etiologies. The anti-epileptic action is based on targeting the SV2A protein, which is involved in the regulation of secretion in neural and endocrine cells, by selectively enhancing low-frequency neurotransmission. In December 2013, AgeneBio Inc. patented the use of low doses of levetiracetam (AGB101) to treat cognitive impairment and cognitive decline [54]. In March 2014, AgeneBio Inc. and US Food and Drug Administration (FDA) had Pre-IND meeting concerning Phase III clinical trial of AGB101 for the prevention of AD. In July 2014, AgeneBio Inc. announced that they are preparing to initiate a large-scale Phase II/III clinical trial of AGB101 in 2015–2016.

Yet, another target that attracts close attention of companies in the search for drugs for the treatment of MCI and early AD is represented by insulin receptors. The hypothesized neuroprotective mechanism is related to activation of insulin receptors, which are expressed in the brain of AD patients mainly in the desensitized state. Impel NeuroPharma is developing insulin-based INP-102 agent for the treatment of AD and MCI using precision olfactory delivery (POD) technique [55] according to which aerozoled drug is delivered to the upper nasal cavity for direct transport into the brain. The drug candidate is in Phase II development, and the company has performed three Phase II studies and is planning a Phase III program and subsequent commercialization [56].

One more insulin receptor-acting agent that was recently proposed for MCI treatment is a peptide compound–Exenatide [57]. Exenatide (glucagon-like peptide analogue), the first in a new class of drugs known as incretin mimetics, was launched in 2005 as adjunctive therapy to improve blood sugar control in patients with type 2 diabetes who have not achieved adequate control with metformin and/or sulfonylurea therapy. The National Institute on Aging (NIA) is conducting phase II clinical trials with exenatide for the treatment of early-stage Alzheimer's disease or mild cognitive impairment.

For a number of years, attempts have been made to create efficient vaccines for the treatment of AD and MCI on the basis of various forms of immunoglobulins. Human normal immunoglobulin contains mainly immunoglobulin G (IgG) with a broad spectrum of antibodies against infectious agents. Immunoglobulin G competitively blocks gamma-Fc receptors, preventing binding and ingestion of phagocytes and suppressing platelet depletion. It is actively used to treat primary immunodeficiency syndromes such as congenital agammaglobulinemia and hypogammaglobulimenia, severe combined immunodeficiencies, chronic lymphatic leukemia with severe secondary hypogammaglobulinemia, and some other diseases. Octapharma AG is developing Octagam, a drug based on human immunoglobulin for intravenous administration. The drug has been in Phase III clinical trials for the treatment of relapsing remitting multiple sclerosis and in Phase II studies for MCI in the European Union. The grounds were provided by the clinical studies of Octagam in AD patients carried out earlier [58]. Although the study was too small-scale and the duration was too short to draw final conclusions, a 18F FDG PET examination demonstrated a significant dose-dependent attenuation of the regional reduced glucose metabolism, which is inherent in AD, for all patients who received Octagam 10% compared with the placebo group, low doses being more efficient than high doses. Recent date on results of Phase III clinical trials showed good tolerability of treatment with low-dose human IVIg for 18 months but did not show beneficial effects on cognition or function relative to participants who received placebo [59].

Verubecestat (MK-8931) developed by Merck & Co is in Phase III clinical trials for the treatment of MCI. The drug is classified as an anti-amyloid agent, as it has been shown to inhibit beta-secretase 1 (BACE1), which ensures cleavage of АРР to give the Aβpeptide. The compound is also in Phase II/III clinical trials for the treatment of Alzheimer type dementia [60]. Already in February 2017 Merck announced that they will stop EPOCH trial of verubecestat in mild to moderate AD. A pre-specified interim analysis by an external data-monitoring committee reportedly judged the chance of a clinical benefit to be near zero, according to Merck press release. Another Merck’s trial (APECS) in people with prodromal AD will continue; results are expected by 2019.

One more strategy in the search for new drugs is investigation of novel applications for the drugs that have already been introduced in the medical practice for some other indication. This approach, which is called drug repositioning, is actively used in the search for drugs for the treatment of various forms of dementia, in particular, AD and MCI [61]. Below we present data from the Thomson Reuters Integrity database on known drugs that have undergone clinical trials for the possible use in MCI therapy.

Pioglitazone (Actos, Glustin, Zactos) was brought to the market in the 1990s by Lilly, Novo Nordisk, and Takeda for treating type 2 diabetes. Currently, Takeda together with Zinfandel Pharmaceuticals performs Phase III clinical trials of this drug in combination with the ТОММ40 biomarker to treat MCI. The hypothesized mechanism of neuroprotective action is related to the ability of this compound, as a peroxisome proliferator-activated receptor γ (PPAR γ) agonist, to affect the CRMP2 phosphorylation level disrupted at early stages of Alzheimer’s dementia preceding the formation of Aβstructures [62].

Three other drugs that are currently in use for the treatment of AD were investigated as preventive agents for MCI patients. Eisai performed Phase III clinical trials of Donepezil but no evident beneficial effects were noted [63]. No data on further development of these studies are available. Clinical investigations of Galantamine for the treatment of MCI patients did not show any promising good results either [64]. The Schwabe company, which has developed a Ginkgo Biloba extract-based agent, carried out Phase III trials involving patients with MCI; however, later, the investigations were terminated. Nevertheless, recently it was reported that Ginkgo Leaves Tablet (GLT) improves some memory characteristics of MCI patients, especially in recognition, regeneration, understanding, and recitation tests [65].

Tesamorelin, a growth hormone releasing factor (GRF) developed by Serono, which was approved in the USA in 2010 for treating HIV-associated lipodystrophy syndrome, is now in Phase II studies. The clinical trials involving MCI patients are currently performed by the National Institute of Aging [66].

There are data about initiation of clinical trials of Sagramostim (Leukine) [67], which is a glycoprotein consisting of 127 amino acids and resembling the granulocyte-macrophage colony-stimulating factor (GM-CSF), with the only difference being arginine instead of leucine in position 23. The Genzyme Company performs Phase II clinical trials of this agent for the therapy of MCI.

In recent years, Melatonin and its analogues have attracted considerable attention as safe drug candidates for the treatment of MCI. This is an endogenous compound produced in the body by the secretory cells of epiphysis and normalizing the circadian rhythms. Previously, it was found in a number of studies that melatonin and its biological precursor, N-acetylserotonine, exhibit clear-cut neuroprotective properties [68,69]. Therefore, the study of this compound for the therapy of MCI initiated by Neurium Pharm. was reasonable. No data about results of this study are available in the TR database; however, the results of another study were published [70], indicating that melatonin is promising as an open label add-on drug for the therapy of MCI.

Two other drugs that have been used previously for a different indication are in Phase I clinical trials. The first one is Guanfacine, which was first proposed by Novartis for the treatment of hypertension and later approved by the FDA for the therapy of attention deficit hyperactivity disorder in children was under investigation initiated by the US National Institute of Health involving MCI patients [71]. Recently, the clinical trials have been completed. The participants in both the investigational agent group and the placebo group showed statistically significant improvement in their symptoms and functioning over the course of the trial. The mechanism of action of this drug is attributable to neurotransmission modulation, as this compound is an alpha-2-adrenergic receptor agonist and hyperpolarization- and cyclic nucleotide-gated (HCN) channel blocker [72].

Another drug, Montelukast, was developed by Merck in the late 1990s to treat asthma and allergic rhinitis. The mechanism of anti-allergic action is related to its action as an antagonist of leukotriene receptors, in particular, LTD4. According to TR data, IntelGenx is developing an oral film formulation of the compound, which is in phase I a clinical trial for the treatment of mild cognitive impairment and Alzheimer’s disease. Presumably, the cognitive stimulation action of this drug is associated with the possible positive modulation of leukotriene receptor inhibition of cyclooxygenase- (COX) and lipoxygenase-mediated (LOX) inflammatory processes [73]. The key data on the compounds that have undergone preclinical and clinical trials as drug candidates for the treatment of MCI are summarized in Table 1. Known structures of agents for MCI treatment currently in preclinical and Phase I, II and III clinical trials are displayed in Figs. 1 and 2.

Table 1. Drug- candidates for MCI treatment in clinical and preclinical trials.

Agent Company Main type of action Trials phase SAR-228810 Sanofi Anti-amyloid I LY-3002813 Eli Lilly Anti-amyloid I LY-2599666 Eli Lilly Anti-amyloid I SAGE-547 Sage Therapeutics Anti-amyloid I/IIa ABT-957 AbbVie Signal modulation I SUVN-502 Suven Life Sci.Ltd. Signal modulation I SUVN-D4010 Suven Life Sci.Ltd. Signal modulation I TAK-71 Takeda Signal modulation I Guanfacinea NIA (NIH) Signal modulation I Montelukasta IntelGenx Anti-inflammatory I Bositunib Pfizer (Anti-tau?) Decreases MAP phosphorylation I Exenatide NIH Insulin secretion modulator II BAN-2401 Eisai Anti-amyloid II CSP-1103 (Itanapraced) Cere Spir Inc. Anti-amyloid II Posipen QR Pharma Anti-amyloid II Tropisetron Back Inst. Res. Aging Signal modulation II UE-2343 Actinogen Ltd. Signal modulation II AN2/AVex-73 Anavex Life Sci. Signal modulation II DAOI-B Kaohiung Chang Gung Memorial Hospital, Taiwan Signal modulation II Ladostigil Avraham Pharmaceuticals Ltd. Signal modulation II Levitiracetam Agene Bio Inc. Signal modulation II INV-102 Impel NeuroPharma Insulin receptor modulation II Octagam Octapharma AG Immunocorrector II Tesamorelina NIA (NIH) Neurotrophic II Sagramostima Genzyme Neurotrophic II Melatonina 1. NeuriumPharm. Neuroprotective II 2. Facultad de Medicina, Universidad de Buenos Aires E-2609 Eisai и Biogen Anti-amyloid III Verubecestat Merck&Co. Anti-amyloid III terminated Pioglitazonea Takeda и Zinfandel Pharm. Neuroprotective III Ginkgo Leaves Tablet (GLT)a 1. Schwabe 2. HuadongHospital, FudanUniversity, Shanghai. China. Neuroprotective III DP-NDD D-Pharm Ltd. Anti-amyloid, аnti-таu preclinic BCA909 BioСrea Signal modulation preclinic NNZ-2591 Neurwn Pharmaceuticals Not disclosed preclinic GRE-213 Grespo Not disclosed preclinic DF-302 IPAC Rus.Acad.Sci. Anti-aggregative preclinic

a agents that were launched earlier for another application.

Fig. 1. Known structures of agents for MCI treatment currently in preclinical and Phase I clinical trials.

Fig. 2. Known structures of agents for MCI treatment currently in Phase II and Phase III clinical trials.

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18 Peptide drugs targeting growth hormone releasing hormone receptor

Growth hormone releasing hormone (GHRH) receptor is a class II G protein–coupled receptor required for normal growth hormone (GH) production and release from the pituitary, and for the normal growth and proliferation of somatotrophs within the pituitary [105]. GHRH stimulates the release and secretion of GH by activating GHRH receptor. Tesamorelin (sequence: N-[(3E)-1-oxo-3-hexen-1-yl]-YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL) is a stabilized synthetic peptide analog of GHRH, used to reduce excessive abdominal fat in HIV-infected patients with lipodystrophy [106]. Lipodystrophy is a metabolic condition characterized by insulin resistance, fat redistribution, and hyperlipidemia associated with antiretroviral therapy for HIV infection. By acting on pituitary cells in the brain, tesamorelin stimulates the production and release of the endogenous hormone.

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Treatment may involve substituting TDF or ABC for thymidine analogues, polylactic acid injections for facial lipoatrophy, weight loss and exercise for fat accumulation, liposuction for dorsocervical fat accumulation and breast reduction surgery. Tesamorelin (growth hormone-releasing factor analog), completed phase 3 clinical trials for visceral fat reduction. Results showed that patients treated for 26 weeks achieved an average 11% decrease in visceral adipose tissue (measured by Dexa scan) from baseline without significant changes in subcutaneous adipose tissue. In addition, a trend for improvement in triglyceride levels was recorded for the treated group versus placebo and was significantly different versus baseline (Falutz et al., 2009).

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12.21.4.2.3 Sustaining ADAs

Some binding ADAs prolong or prevent the clearance of protein therapeutics and are termed sustaining ADAs. The mechanism for sustaining the exposure is likely binding by ADAs with moderate or low affinity. These ADAs do not bind tightly enough to the drug to rapidly facilitate clearance of the therapeutic through formation of immune complexes and degradation in phagolysosomes, but are bound long enough to prevent normal antibody clearance through the FcRn pathway. Sustaining ADAs can increase circulating drug concentrations, potentially leading to longer exposure to the protein therapeutic and exaggerated pharmacology. Sustaining ADAs have been demonstrated in nonclinical and clinical settings. For example, dogs administered tesamorelin had a ∼ 100-fold increase in drug exposures over six months of dosing with highly exaggerated pharmacodynamic effects (Mihalcik, 2010). Some human subjects administered aldesleukin (rhuIL-2) who developed ADAs had increased exposure (up to 68% higher) compared to ADA-negative subjects (Chiron, 2015).

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2 Peptide injectables

As demonstrated in Fig. 2, parenteral routes are the most frequently used routes for the administration of peptide medications. The term parenteral refers exclusively to subcutaneous (SC), intravenous (IV), and intramuscular (IM) injections. These injections do not pass through the gastrointestinal tract, which protects peptides from the degradation induced by dramatic changes in pH and from numerous enzymatic digestion processes. As a result, peptides maintain their ideal bioavailability and efficacy. Additionally, injections avoid the limitations of peptides in regard to permeating biological membranes.

Peptide formulation scientists not only must meet the challenges and manage the risks of injectables in general, they must also solve the particular problems that occur in peptide injections. Generally, injection is not viewed as the best choice of delivery in most cases due to the inconvenience it poses for healthcare providers and poor patient compliance due to its invasive nature and the pain associated with injections. The need for special operations also increases the expense for both patients and health insurance providers. As mentioned in the introduction, the half-life of peptides is very short, so repeated doses are required within a short time interval, and this exacerbates the disadvantages. As a result, there is a strong demand for the development of modified-release formulations to resolve these problems. Moreover, injections bypass the body’s natural defenses and thus pose a greater safety risk. Consequently, injection formulations must be rigorously sterilized and must remain free of pyrogens and particulates during their shelf life (typically two years). The pH, injection volume, and tonicity must be appropriate for the injection route to prevent local irritation, discomfort, and hemolysis (Bak et al., 2015; Maggie Bryans, 2016; Mathaes et al., 2016).

2.1 Physicochemical properties and stability of peptides

The physicochemical properties of therapeutic peptides are a barrier to formulating stable and effective dosage forms. The solubility of peptides depends on the pH of the solution, which results in minimum solubility at the peptides’ isoelectric point (PI). As such, pH should be adjusted to a value at which the peptide is completely soluble and in the tolerable range of injection (e.g., pH 3–10 for subcutaneous, intravenous, and intramuscular injectables) (Gervasi et al., 2018; Röthlisberger et al., 2016; Thomas et al., 2000).

For injection routes other than intravenous, a higher concentration is usually required to deliver a certain amount of the peptide given the limited injection volume (typically considered 1.5 ml for SC) (Mathaes et al., 2016; Chadwick and Withnell, 1987). This may introduce another peptide-specific concern: peptide self-association. Amphiphilic peptides tend to associate through interactions between the hydrophobic regions, which is enhanced as peptide concentration increases. As a result, the peptides in higher-concentration formulations may present in a highly associated state instead of as independent monomers. Theoretically, the peptide micelles formed from this behavior can enhance the peptide’s chemical stability due to diminished solvent accessibility. These micelles can also influence the peptide’s pharmacokinetics and bioavailability as well as its physical stability (Payne and Manning, 2009; Clodfelter et al., 1998; Silvestri et al., 1993; Fung et al., 2003).

However, these molecular interactions can result in peptide aggregation, most commonly perceived as gelation and sometimes as precipitation (Payne and Manning, 2009; Velichko et al., 2008). Other than self-association at high concentrations, peptides (unlike proteins) may exist in multiple conformations in solution rather than as a single stable secondary structure, and peptide conformations may change as the environment changes. Certain conformations are less stable and may lead to undesirable outcomes such as loss of function, increased adsorption, self-association, fibril formation, gelation (i.e., poor colloidal stability), aggregation, or precipitation (Bak et al., 2015; Palladino et al., 2012; Balaram et al., 1983; Zhang et al., 2018). Therefore, the conditions of the formulation (e.g., pH, temperature, ionic environment) are critical to the physical stability of peptides.

In addition to physical stability risks, chemical stability should also be carefully considered when formulating peptides. Proteins usually have folded structures and thus prevent side chains from interacting with potential hazards. However, the typical peptide is fully solvent exposed, causing complete contact with reactive factors. Peptides containing oxidation-sensitive amino acids are particularly more susceptible. These amino acids include cysteine, methionine, histidine, tyrosine, and tryptophan residues (Davies, 2016; Elizabeth et al., 2010). Light exposure can also accelerate oxidation, which is why the warning “protect from light” appears on most peptide product inserts. In addition to oxidation, peptides are susceptible to hydrolysis and deamidation due to the high degree of flexibility in the peptide chain and the lack of steric bulk in the amino acid adjacent to the carboxyl end of Asn (Hovgaard et al., 2012). Deamidation is more likely to occur when the pH is above 6, and it can occur in both solid and liquid peptide formulations. Typically, Asn–Gly, Asn–Ala, Asn–Ser, and Asn–Asp sequences have captured the attention of pharmaceutical researchers (Payne and Manning, 2009).

2.2 Formulations and excipients in approved non-modified-release peptidic injection products

The FDA, EMA, and PMDA have collectively approved more than 70 therapeutic peptide products since the 1950 s, with more than 75% administered by injection (see Fig. 2). Subcutaneous, intravenous, and intramuscular delivery are the most frequently developed routes. In contrast, only one intrathecal product and one intracavernous product have been developed. These products are manufactured and stored in the form of a lyophilized powder or in a frozen or liquid state (i.e., solution or suspension). Among the products approved in the last 20 years that have not been discontinued, over 55% are stored and injected in solution form (see Fig. 3), while much of the remaining products are in a solid state as a lyophilized powder. Almost all these suspensions are modified-release formulations, and the majority of these are polymer-based formulations. Table 1 lists examples of non-modified-released injectable peptide formulations. The modified-released formulations will be discussed in the next section. It is important to note that long-acting peptides or peptide analogs (i.e., those that have a long half-life) are defined differently than modified-release products, whose prolonged effect depends on formulation technologies rather than modifications of the peptide molecule.

Fig. 3. The number of forms of injectable peptide products that have been approved in the last 20 years and are not in discontinued status. The black and grey bars represent products in the solid state that are reconstituted either as a solution (black bar) or suspension (gray bar) before use.

Table 1. Examples of approved non-modified-release peptide injectable formulations.

Molecule Brand name Excipients pH Form* Adm. route Remark Reference Glucagon Glucagon Lactose and hydrochloric acid. Diluent: glycerin and water for injection. N/A Solid → solution SQ, IV, IM (Glucagon [package insert], 2018) Bivalirudin Bivalirudin in 0.9% sodium chloride Sodium chloride, sodium hydroxide and/or hydrochloric acid, and water for injection. 5.2–6 Frozen → solution IV (Bivalirudin in 0.9% Sodium Chloride Injection [package insert], 2000) Aviptadil Invicorp Sodium chloride, disodium edetate, phosphoric acid, hydrochloric acid, sodium hydroxide, and water for injection. N/A Solution Intracavernous Combined with 2 mg phentolamine mesilate (Invicorp, 2015) Ziconotide Prialt L-methionine and sodium chloride. Diluent: 0.9% sodium chloride injection. 4–5 Solution Intrathecal Diluent must not contain preservatives. (Prialt [package insert], 2004) Pramlintide Symlin Metacresol, D-mannitol, acetic acid, sodium acetate, and water for injection. 4 Solution SQ (Symlin [package insert] 2014) Degarelix Firmagon Mannitol. Diluent: water for injection. N/A Solid → solution SQ (Firmagon [package insert], 2015) Liraglutide Victoza, Saxenda Disodium phosphate dihydrate, propylene glycol, phenol, and water for injection. N/A Solution SQ (Victoza [package insert], 2019; Saxenda [package insert], 2018) Xultophy Glycerol, phenol, zinc, and water for injection; hydrochloric acid and sodium hydroxide. 8.15 Solution SQ Combined with insulin degludec. (Xultophy [package insert], 2019) Teduglutide Gattex L-histidine, mannitol, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate heptahydrate. Diluent: water for injection. N/A Solid → suspension SQ (Gattex [package insert], 2019) Carfilzomib Kyprolis Sulfobutylether beta-cyclodextrin, anhydrous citric acid, and sodium hydroxide. Diluent: 5% dextrose injection. 3.5 Solid → solution IV (Kyprolis [package insert], 2019) Lutetium Lu 177 dotatate Lutathera Acetic acid, sodium acetate, gentisic acid, sodium hydroxide, ascorbic acid, diethylenetriaminepentaacetic acid, sodium chloride, and water for injection. 4.5–6 Solution IV Peptide is radiolabled (Lutathera [package insert], 2018) Desmopressin Ddavp Sodium chloride, hydrochloric acid, and chlorobutanol. 4 Solution IV, SQ Chlorobutanol is added only in high-concentration formulations. (Ddavp (Injection), 2019) Enalapril Enalaprilat sodium chloride, sodium hydroxide, water for injection, and benzyl alcohol. N/A Solution IV Stored at 20–25 °C. (Enalaprilat [package insert], 2019) Abarelix Fuzeon mannitol, sodium carbonate (anhydrous), sodium hydroxide, and hydrochloric acid. Possible diluents: 5% dextrose injection, 0.9% sodium chloride, 5% dextrose injection in 0.9% sodium chloride, or 5% dextrose in Lactated Ringer's Injection. 9 Solid → solution SQ (Fuzeon [package insert], 2019)

*Form refers to the packaged form (before reconstitution), solid means lyophilized powder, and frozen means frozen liquid. The symbol “→” represents reconstitution.

Generally, the pH of protein formulations are buffered in the range of 4.6–8.2 (Gervasi et al., 2018). In addition, peptide formulations are composed of fewer categories of excipients than protein formulations. Protein formulations typically contain buffer agents, amino acids, stabilizers or bulking agents, surfactants, and tonicity agents. In contrast, typical peptide formulations exclude surfactants and amino acids, and they have a broader pH range. For instance, the pH of a reconstituted glucagon solution is as low as 2.5, while the pH of Fuzeon® rehydrated solution is as high as 9 (Steiner et al., 2010; Fuzeon [package insert], 2019; Glucagon [package insert], 2016).

In this review, we classify the excipients added to approved peptide formulations into seven categories, which are summarized in Table 2 below. The categories are buffering agents, pH-adjusting agents, tonicifying agents, bulking agents or stabilizers, preservatives, antioxidants, and other excipients.

Table 2. Summary of excipients used in currently active non-modified-release peptide injection products.

Excipient class Agent Buffering and pH-adjusting agents Disodium phosphate, monobasic sodium phosphate, and/or phosphoric acid Trisodium citrate and citrate acid Sodium carbonate Acetic acid and trihydrate sodium acetate Succinic acid Tartaric acid Hydrochloric acid Sodium hydroxide Histidine Isotonicity agent Mannitol Sodium chloride 5% dextrose Glycerin Propylene glycol Bulking agent/stabilizer Mannitol Lactose Histidine Antioxidant Methionine Ascorbic acid Gentisic acid Chelator Disodium edetate DTPA* Preservative Chlorobutanol Phenol Benzyl alcohol M-cresol Other Sulfobutylether beta-cyclodextrin Zinc

Note: Information is for current products in United States, Europe, and Japan that have not been discontinued. This information may not be comprehensive due to dynamic changes in the database and the authors’ accessibility to information. *DTPA: Diethylenetriaminepentaacetic acid.

2.2.1 Buffering and pH-adjusting agents

The pH level of the solution plays a significant role in aqueous formulations of therapeutic peptides. The ability to maintain the formulation pH at a value that differs from the peptides’ pI enhances its solubility. Many physical and chemical degradation pathways are also pH dependent. In addition, a change in pH can cause the release of extractables and leachables from the container or closure of the products, leading to safety and stability risks. Furthermore, the pH of therapeutic peptide formulations must be adjusted to an acceptable range to prevent irritation, pain, or extravasation at the site of injection. As a result, in therapeutic peptide formulations, buffers and pH-adjusting agents are critical components that maintain a consistent pH over the product’s shelf life.

Buffer systems are typically composed of two chemical species that are related to a change in protonation state. When choosing buffer systems, the primary considerations include compatibility with other components of the formulation and the buffer capacity of certain salt concentrations. It is known that higher concentrations of buffer salts typically help improve buffer capacity. However, undesirable outcomes should be considered when increasing the buffer concentration. Some of these undesirable outcomes include the salting-out effect and crystallization-induced pH shifting upon freezing, as well as reactions at injection sites upon dosing. For example, it is reported that lower pH and higher buffer capacity are associated with increased myotoxicity of acetate buffers (Napaporn et al., 2000).

Table 3 lists the buffer systems deployed in approved peptide parenteral products, of which acetate is the most popular (greater than50%), followed by citrate and phosphate. Acetate buffers has been so prevalent maybe because peptide purification processes commonly use acetate counter-ions as a substitute for trifluoroacetate (TFA) due to safety concerns. In addition to an acetate buffer, approved products such as Integrilin®, Natrecor®, and Kyprolis® contain citrate buffer species, while Invicorp, Victoza®, Ozempic®, and Gattex® are buffered by a phosphate system(Invicorp, 2015; Victoza [package insert], 2019; Gattex [package insert], 2019; Kyprolis [package insert], 2019; Integrilin [package insert], 2019; Natrecor [package insert], 2019; Ozempic [package insert], 2019).

Table 3. Examples of buffer systems used in therapeutic peptide injection products.

Buffer system Controlled pH range (25 °C) Acid Base Example product Product pH Reference Phosphate 5.8–7.8 Monosodium phosphate Disodium phosphate Ozempic 7.4 (Ozempic [package insert]; Revised:, 2019) Acetate 3.8–5.8 Acetic acid Sodium acetate Ganirelix Acetate 5 (Acetate, 2019) Citrate 3.0–7.4 Citric acid Sodium citrate Integrilin 5.35 (Integrilin [package insert], 2019) Carbonate 5.4–7.4, 9.3–11.3 Sodium bicarbonate Sodium carbonate Sandostatin 4.2 ± 0.3 (Sandostatin [package insert]:, 2019) Fuzeon 9 (Fuzeon [package insert], 2019) Succinate 3.3–6.6 Succinic acid Sodium succinate Parsabiv 3.3 (Parsabiv [package insert], 2019) Tartrate 2.5–6.2 Tartaric acid Sodium tartrate Signifor N/A (Signifor [package insert], 2019)

Salting-out salts, sodium phosphate, and sodium citrate were reported to be extremely effective in raising melting temperatures, which increases the shelf life of liquid protein formulations (Ohtake et al., 2011). This may be an important consideration when choosing buffer systems for peptides that have higher-order structures. Moreover, citrate salts can remain in an amorphous state and minimize pH shifts before and after lyophilization, unlike sodium phosphate, which tends to crystallize and cause a pH shift of up to 4 units, possibly causing peptide degradation (Gomez et al., 2001; Pikal-Cleland and Carpenter, 2001).

Histidine is found in the phosphate-buffered lyophilized formulation Gattex®, which is most likely employed in order to minimize the pH shift provoked by freezing. Agents with a wide buffering range have been selected for use in liquid products such as Tymlos®, which uses succinic acid, and Signifor®, which uses tartaric acid(Tymlos [package insert], 2018; Signifor [package insert], 2019). It has been reported that succinate and tartrate crystalize during lyophilization, which causes a pH shift. Tartaric acid can be amorphous in its buffering range (other than pH 3–4), while succinate crystallizes throughout its buffering pH range. Tartaric acid can also serve as an antioxidant in parenteral formulations (Pramanick et al., 2013). In addition, some popular buffering agents in protein formulations (e.g., Tris buffer and glycine) are not yet found in peptide injection products. It has been noted, and widely cited, that Tris buffer degrades peptides by reacting with tyrosine residues and by liberating formaldehyde at a storage temperature of 70 °C (Song et al., 2001).

Buffering species must be selected carefully in solid formulations (Gervasi et al., 2018). Despite the above examples, the addition of buffer agents is not always beneficial or necessary for peptide formulations, especially for some peptides that can maintain a critical pH on their own. Products such as Cetrotide® (solid), GlucaGen® (solid), Copaxon® (solution), and Prialt® (solution) do not contain any buffer or pH-adjusting agents(Prialt [package insert], 2004; Cetrotide [package insert], 2018; GlucaGen [package insert], 2018; Copaxon [package insert], 2019). Finally, hydrochloride and sodium hydroxide are typically applied in pH adjustments, even though acetic acid and sodium acetate are identified as pH-adjusting agents in some formulations, including Pitocin® and Symlin® (Symlin [package insert], 2014; Pitocin [package insert], 2018).

2.2.2 Tonicifying agents

When developing injectable formulations, tonicity must be considered in order to create isotonic solutions for parenteral administration. Tonicifying agents are included in peptide injection formulations to ensure proper tonicity. This is because a non-isotonic solution can lead to tissue damage and increased pain at the injection site. Isotonicity in human plasma is equivalent to 290 mOsm/L. Therefore, this should be a target value for intravenous solutions or suspensions (Stranz and Kastango, 2013). However, hypertonicity is actually required in some formulations. Wang, et al. reviewed the tolerability of hypertonic products, and they propose 600 mOsm/kg as the upper osmolality limit for intramuscular or subcutaneous injections and 500 or 1000 mOsm/kg for intravenous and intravascular injections, depending on the injection volume (Wang, 2015).

Sodium chloride, mannitol, dextrose, and glycerin are specifically reported in product inserts as tonicifying excipients, as listed in Table 2. Sodium chloride is the most common tonicifying agent in peptide injection products. In several products, sodium chloride (NaCl) in various concentrations (e.g., 0.75% in Miacalcin®, 0.85% in Parsabiv®, 0.69% in Lutathera®) is added directly to liquid formulations(Revised:, 2018; Parsabiv [package insert], 2019; Miacalcin [package insert], 2018). Normal saline (0.9% NaCl) often serves as diluent and is absent in the freeze-dried powder of solid products. This is likely due to the fact that NaCl could greatly reduce the collapse temperature of the formulation, resulting in a low drying temperature and thus an increase in drying time and cost (Carpenter et al., 2002). In addition, NaCl can not only crystallize on its own but also induce the crystallization of other excipients upon storage, which would impact the stability of the formulation (Hawe and Frieß, 2006). Interestingly, the only frozen product, Bivalirudin in 0.9% Sodium Chloride® is frozen with 0.9% NaCl, which generates an osmolality of 300 mOsmol/kg for intravenous injection (Bivalirudin in 0.9% Sodium Chloride Injection [package insert], 2000). This could indicate that the impact of NaCl on freeze-dried product is during or after the drying stage. Freezing with NaCl does not necessarily harm the peptide.

Mannitol is demonstrated to be a suitable alternative to NaCl as a tonicifier. Mannitol often appears in subcutaneous solutions (e.g., Forteo®, Copaxone®, Signifor®), although only two pramlintide products, Symlin® and Byetta®, distinctly claim in their inserts that the purpose of adding mannitol is to adjust their tonicity (Symlin [package insert], 2014; Signifor [package insert], 2019; Copaxon [package insert], 2019; Forteo [package insert], 2019; Byetta [package insert], 2018). Mannitol is frequently found in solid products as a bulking agent, some of which contain mannitol as their only bulking agent. The diluent for these products can be water for injection alone instead of isotonic solutions, which indicates that mannitol is not only a bulking agent in lyophilization but also a tonicifier for injectables. Examples of these products include Cetrotide®, Angiomax®, Firmagon®, and Egrifta® (Firmagon [package insert], 2015; Cetrotide [package insert], 2018; Angiomax [package insert], 2019; Egrifta [package insert], 2019). Similarly, lactose, which is the only inactive ingredient in Glucagon®, may have the same use (Glucagon [package insert], 2016).

For injection, the selected diluents are normally water, normal saline, or 5% dextrose. It is worth mentioning that 0.9% NaCl is the diluent for Mepact, the only product that used a liposome delivery system (Mepact [package insert], 2019). Normal saline and 5% dextrose are used interchangeably for some lyophilized products, which gives both health providers and patients more practicality and flexibility. For example, Natrecor® lists 5% dextrose with 0.45% sodium chloride, USP, and 5% dextrose with 0.2% sodium chloride as alternatives other than 5% dextrose and 0.9% NaCl (Natrecor [package insert], 2019).

A pH-adjusted glycerin solution is the diluent of another glucagon peptide injectable formulation, Glucagon® (Glucagon [package insert], 2018). In the reconstituted solution, glycerin (12 mg/ml) serves multiple purposes: It is a tonicifying agent and a cosolvent or solubilizing agent. It not only adjusts the osmolality of the solution but also helps the peptide dissolve in an environment with a pH between 3 and 9.5 (Steiner et al., 2010). In addition, solution injectable formulations, the liraglutide product Xultophy®, the bremelanotide product Vyleesi®, and both lixisenatide products (i.e., Soliqua® and Adlyxin®) used glycerin for tonicifying the formulation (Xultophy [package insert], 2019; Knudsen et al., 2006; Vyleesi [package insert], 2019; Soliqua [package insert], 2019; Adlyxin [package insert], 2019). It is worth mentioning here that glycerin is also a useful wetting agent for suspension formulations (Pramanick et al., 2013).

Propylene glycol is another common cosolvent or solubilizing agent. Propylene glycol is incorporated in the Ozempic® and Victoza® formulations, in which both semaglutide (the API of Ozempic®) and liraglutide (the API of Victoza®) are reported to be “good [sic] soluble in an aqueous solution” at their final pH (Victoza [package insert], 2019; Ozempic [package insert], 2019). Thus, propylene glycol most likely serves as a tonicifying agent in these two formulations. Attention should be paid when using propylene glycol because it generates relatively high osmolality even in low working concentrations, despite being reported to inhibit gel formation of peptides and to has a wetting effect in suspension formulations (Payne and Manning, 2009; Doenicke et al., 1992; Powell et al., 1991). The diluent for modified-release formulations will be discussed in the next section.

Achieving isotonicity can be challenging, especially for high-concentration formulations. Even though the acceptable range of osmolality is relatively broad, it is still worth noting that developers should alert healthcare providers and patients about the risk of injection-related pain under the “Adverse Reaction” or “Warnings and Precautions” section of the package inserts for formulations with high solute concentrations.

2.2.3 Bulking agents or stabilizers

Sugars, sugar alcohols, and amino acids appear in both solid and solution peptide products as bulking agents or stabilizers (Table 4). They protect peptides and proteins from aggregating in liquid forms via interrelated mechanisms, including the excluded volume effect, increasing water surface tension, preferential hydration at high concentrations, or increased melting temperature (Tm), which usually applies to larger peptides that have a complexed structure. In dry-state formulations, peptide structure can be perturbed by crystallization during freezing or by the removal of water from the vicinity during drying. This results in physical and chemical degradation. Sugars, sugar alcohols, and some amino acids can help peptides withstand these stresses and preserve their activity (Ohtake et al., 2011; Kamerzell et al., 2011; Li et al., 2005). In addition to working as a stabilizer, some sugars (or sugar alcohol) and amino acids can act as bulking agents in a freeze-dried formulation matrix to ensure a desirable structure and appearance of the finished product. This is particularly important for formulations with low solid concentrations of substances (<2%) (Jennings, 1997).

Table 4. Critical temperatures of bulking or stabilizing agents used in approved peptide non-extended-release injection products.

Agent Function Teu (°C)* Tg’ (°C)* Tc (°C)* Ref. Mannitol Bulking −1.5 – – (Kim et al., 1998) Lactose Bulking/stabilizer – −28 −32 (Cook and Why, 2009; Eva Meister, 2008) Histidine Stabilizer −33 – (Baheti et al., 2010)

* Teu refers to eutectic temperature (with water). Tg’ refers to glass transition temperature. Tc refers to collapse temperature.

Mannitol is the most extensively selected sugar alcohol excipient of approved peptide parenteral products in both liquid and dry states. As discussed in the previous subsection, mannitol also plays a role in balancing the tonicity of formulations. The strength of mannitol ranges from 4 mg/ml to 54.8 mg/ml in approved peptide products. In dry-state formulations, mannitol serves as a bulking agent because of its crystallinity, high eutectic temperature, and matrix properties (Akers, 2002). These traits enable mannitol to maintain the structure of the lyophilized cake while simultaneously allowing drying under relatively aggressive conditions, which results in a shortened drying time and a lower cost (Patel et al., 2010; Johnson et al., 2002).

One should be careful when adding mannitol to a formulation, for a variety of reasons. First, although not been found in peptide formulations yet, it has been widely reported that the crystallization of mannitol during freeze-drying damages protein stability in both the presence and absence of amorphous excipients such as sucrose. Second, mannitol tends to form various crystalline modifications during storage, and this negatively impacts its storage stability (Ohtake et al., 2011; Al-Hussein and Gieseler, 2012; Izutsu et al., 1994). Finally, impurities from mannitol supplies can induce oxidative degradation of a cyclic heptapeptide from a lyophilized formulations (Wu et al., 2011).

As discussed above, mannitol is found in almost all non-extended-released injectable formulations that contain sugars or sugar alcohols as their excipient, with the single exception of Glucagon®, which uses lactose(Glucagon [package insert], 2016). Special attention should be paid when selecting lactose, glucose, or maltose, because their reductivity can lead to the Maillard reaction (Kato et al., 1989).

Histidine is a multifunctional additive in lyophilized bioproducts. Gattex® is the only peptide product that contains histidine (Gattex [package insert], 2019). Despite employing a phosphate buffer system in its formulation, histidine still likely acts as a buffer agent and a stabilizer rather than a bulking agent to mitigate the pH shift during lyophilization. This is because 7.76 mg/ml is not a typical working concentration for histidine as a bulking agent (Gervasi et al., 2018).

Sugars, polyols, and some amino acids are effective in stabilizing a variety of therapeutic proteins by protecting them from aggregation, denaturation, and other degradation pathways in both dried and solution states. As such, they are incorporated in almost every protein formulation (Gervasi et al., 2018). Nevertheless, approximately half of peptide formulations are sugar free, particularly those formulations invented in earlier decades, such as Oxytocin®, Enalaprilat®, and Eptifibatide® (Enalaprilat [package insert], 2019; Oxytocin [package insert], 2019; Eptifibatide [package insert], 2016). This could be a result of the conformational simplicity of peptides compared to proteins, which require a higher-order structure (i.e., secondary, tertiary, and quaternary) to maintain activity. Sugars can preserve their structural integrity, and they ensure the protein remains in its native state through various mechanisms. The experience gained from protein formulation is valuable for stabilizing peptides, notably peptides with more complex structures. For example, other bulking agents and stabilizers (e.g., glycine, arginine, sucrose, sorbitol) can be considered as alternatives to mannitol.

2.2.4 Preservatives

Antimicrobials are added to therapeutic peptide formulations, and they are required in multidose vials as preservatives to inhibit or reduce microbial growth. An antimicrobial is expected to satisfy several requirements. First, the antimicrobial must be effective against a broad spectrum of microbes, including bacteria, yeasts, fungi, and molds at low inclusion levels. Next, the antimicrobial must maintain its activity throughout the product’s manufacture, shelf life, and usage. In addition, the antimicrobial must have no influence on the quality or performance of both the active ingredient and other inactive additives. Last, the antimicrobial must have no adverse safety concerns (David and Elder, 2012). Only a few antimicrobials have been approved for inclusion in pharmaceutical products, and even fewer are allowed to be used in parenteral products (Meyer et al., 2007). Only four preservatives have been identified in peptide injectable products: phenol, chlorobutanol, m-cresol (i.e., metacresol), and benzyl alcohol. Preservatives are usually added in small amounts (<1%). Table 5 lists the working strengths of several preservatives documented in the literature or applied in approved peptide injectables.

Table 5. Preservatives in approved peptide injection products.

Preservative Reported Range in parenteral products Range in approved peptide formulations Example Product Reference Chlorobutanol 0.25–0.5% 0.5–0.525% Oxytocin® (Pramanick et al., 2013; Oxytocin [package insert], 2019; Meyer et al., 2007) Phenol 0.0715–0.5% 0.5–0.57% Victoza®, Ozempic® (Victoza [package insert], 2019; Enalaprilat [package insert], 2019; Pramanick et al., 2013; Meyer et al., 2007) Benzyl alcohol 0.75–5% 0.9% Enalaprilat® (Pramanick et al., 2013; Meyer et al., 2007) M−cresol 0.1–0.315% 0.22–0.3% Byetta®, Soliqua® (Pramanick et al., 2013; Byetta [package insert], 2018; Soliqua [package insert], 2019; Meyer et al., 2007)

Phenol and m-cresol are the most frequently used preservatives in liquid peptide formulations. Phenol is categorized as a phenolic compound and has a broad inhibiting effect on microorganisms, including mycobacteria, fungi, and viruses. The activity of phenol increases in acidic and concentrated solutions as well as at higher temperatures (Gervasi et al., 2018). Aqueous solutions of phenol can be sterilized by dry heat or by autoclaving, but they should be protected from light exposure (Meyer et al., 2007). Another commonly used phenolic preservative, m-cresol, is found in later-stage developed peptide injectable formulations such as Byetta® and Soliqua®(Byetta [package insert], 2018; Soliqua [package insert], 2019). M-cresol has been found to induce fewer adverse events. It is active against Gram-positive bacteria, but less active against Gram-negative bacteria, yeasts, and mold, and it is entirely inactive against spores.

Aside from phenol and m-cresol, chlorobutanol is an additive in most early-developed peptides such as vasopressin, oxytocin, and desmopressin. Chlorobutanol has a relatively low effective pH, as it is only stable at pH 3 in aqueous solutions. Increasing the pH results in a decrease in its antibacterial activity (Elder and Crowley, 2012). The pH of Vasostrict® (vasopressin), Oxytocin® (oxytocin), and Ddavp® (desmopressin) are 3.8, 4, and 4, respectively (Ddavp (Injection), 2019; Oxytocin [package insert], 2019; Vasostrict [package insert], 2016). Chlorobutanol has bacteriostatic effects on both Gram‐positive and Gram-negative organisms, and it exhibits some activity against yeasts and fungi (Rowe et al., 2006). Finally, benzyl alcohol is one of the most routinely added preservatives in parenteral protein formulations, and it is known for its less deleterious effect on protein stability. However, among all peptide injectables, benzyl alcohol is applied only in Enalaprilat®, in a concentration of 0.9% (Enalaprilat [package insert], 2019; Heljo et al., 2015).

Compatibility studies should be conducted when considering whether to add preservatives. This is a consequence of their reactivity with other ingredients, which can sabotage the activity of the active pharmaceutical ingredients and excipients. In addition, preservatives can also react with packaging and other materials during the manufacturing process (Akers, 2002; Meyer et al., 2007). For example, Hejo et al. proposed that preservatives can adsorb oligomeric state peptides, and this interaction leads to both perturbed peptide self-interaction and impairment of antimicrobial efficiency (Heljo et al., 2015).

2.2.5 Antioxidants and chelators

During manufacture and storage, three main sources give rise to oxidants in formulations: trace metal ions from containers or handling tools, hydrogen peroxide from sanitizing agents, and oxidant impurities from other excipients. Many amino acid residues in peptides and proteins are susceptible to oxidants, and they undergo chemical degradation when encountering them. Sulfur-containing amino acids (e.g., methionine, cysteine) are particularly sensitive to oxidation (Stadtman and Levine, 2003). Metal ions (e.g., Fe2+ and Cu2+) can catalyze oxidative reactions in proteins or peptides through generating a redox oxygen species (ROS) either by binding directly to side-chain groups on the amino acids or by reacting indirectly with O2. The formed ROS can react with amino acid residues, which leads to peptide degradation (Bridgewater et al., 2006; Stadtman, 1990; Li et al., 1995).

Chelators and antioxidants are typically used to prevent the oxidation of active ingredients and excipients. Chelators stabilize proteins by forming multiple coordinate bonds with metal ions to reduce available ions for metal-catalyzed oxidative peptide degradation. Only two products, Invicorp (an intracavernous injectable) and Lutathera® (used in peptide receptor radionuclide therapy (PRRT)), incorporate the chelators disodium edetate (e.g., EDTA disodium salt) and diethylenetriaminepentaacetic acid (DTPA), respectively, in their formulations (Invicorp, 2015, 2018). EDTA is the most commonly used chelator in both chemical and biological drug formulations. It can chelate with metal ions, removing them from the solution in a process called sequestering. The working concentration of EDTA in parenteral formulations is reported to be between 0.01% and 0.2% (w/v) (Pramanick et al., 2013). In addition to complexing with metal ions, EDTA can also be used synergistically as an antioxidative and as an antimicrobial with other antioxidants and preservatives (Rowe et al., 2006). The use of DTPA in Lutathera® is unique because the chelation of DTPA with metal ions not only stabilizes the formulation but also facilitates the clearance of free lutetium from blood via renal excretion, which subsequently reduces the risk of severe radiotoxic effects and other adverse effects (Lutathera [package insert], 2018; Liu and Edwards, 2001).

The addition of antioxidants can minimize oxidations caused by metal ions and other oxidation sources, such as trace impurities from other additives and hydrogen peroxide from sterilizing agents. Antioxidants can serve as electron donors to inhibit the oxidation of molecules in the formulation (Chi, 2016). Methionine, ascorbic acid, and gentisic acid are included in approved non-modified-released peptide injection formulations. Prialt® and Soliqua® have incorporated methionine in their formulations, and Lutathera® contains ascorbic acid and gentisic acid (Prialt [package insert], 2004; Lutathera [package insert], 2018; Soliqua [package insert], 2019). Radiolytic degradation occurs frequently due to the high radioactive concentration of the therapeutic radiopharmaceuticals. The combination of ascorbic acid and gentisic acid act as free radical scavengers to provide better stability for the radiopharmaceutical products. Gentisic acid can be added to a formulation either before or after radiolabeling as long as the thermal decomposition caused by heating during labeling is within an acceptable range. Otherwise, post-labeling addition is more favorable (Liu and Edwards, 2001a, 2001b).

In addition to the molecules mentioned above, glutathione and several other amino acids (e.g., histidine, cysteine) also contain antioxidant properties. They are found in commercialized protein formulations, and they can serve as alternatives to methionine (Gervasi et al., 2018). Acids such as tartaric acid also have antioxidative activity, but they simultaneously influence pH. Ascorbic acid can also serve as an antioxidant for non-radio-labeled molecules (Pramanick et al., 2013; Choksi, 2015).

2.2.6 Other excipients

Two other excipients are identified in approved, non-modified-release peptide injectable formulations: sulfobutylether beta-cyclodextrin and zinc.

Sulfobutylether beta-cyclodextrin (SBECD), one of the polysubstituted derivatives of cyclodextrin, is employed in the peptidic product Kyprolis®, whose active peptide, carfilzomib, is a crystalline substance that is practically insoluble in water and only slightly soluble in acidic conditions (Kyprolis [package insert], 2019). Decreasing pH cannot achieve the desired concentration of carfilzomib, and researchers have found that this peptide is more prone to degrade in low-pH conditions. Therefore, the major responsibility of SBECD in a formulation is to solubilize the peptide to a necessary aqueous concentration without precipitation upon dilution. In addition to enhancing carfilzomib’s solubility, SBECD has been shown to provide isotonicity, and it acts as a bulking agent to support the integrity of the lyophilized cake (Shimpi et al., 2005).

Zinc is an additive in products that combine an insulin analog with a GLP-1 analog. The zinc ions in specific concentrations (55 μg/ml in Xultophy® and 30 μg/ml in Soliqua®, respectively) promote the association of insulin molecules in hexamers to increase the activity of proteins and improve both PK and physical stability (Xultophy [package insert], 2019; Soliqu, 2019; Brange and Langkjaer, 1992; R.D.D. Michael Rosario DeFelippis, 2002). In addition, the formed complex results in delayed action of insulin (Hagendorf et al., 2012; Plum et al., 2015). However, it has been demonstrated that the GLP-1 analog in Xultophy® (liraglutide) can form a zinc-binding liraglutide diheptamer in the presence of equimolar concentrations of zinc ions and diheptamers in stored insulin–liraglutide formulations(Xultophy [package insert], 2019). It has been proposed that zinc-chelating compounds, such as histidine or imidazole, can be added to reduce the formation of liraglutide diheptamers (Plum et al., 2015).

2.3 Approved modified-release therapeutic peptide products

Long-acting formulations are frequently pursued for treating chronic diseases because they simplify the dose regimen and improve patient compliance by reducing administration frequency over the long-term health management period (Ingersoll and Cohen, 2008). Even though proteins and peptides are highly potent molecules, they are constrained by their relatively short in vivo half-life and their poor bioavailability. These limitations result in the need for frequent and multiple doses, which leads to a poor quality of life for the patient, reduced compliance, and a loss of patient adherence to the treatment regimen. Aside from prolonging the half-life of a peptide by chemical modification, extended release can be achieved using formulation technologies such as nanoparticles, microparticles, peptide self-assembly, nanotubes, in situ gels, or a combination of different strategies (Patel et al., 2014). These approaches enable peptides to be released in a specific pattern to improve the bioavailability and dosing regimen. The most prevalent delivery strategies for sustained-release peptides are implants and parenteral injections, primarily intramuscular and subcutaneous injections. These strategies enable the drug to avoid first-pass metabolism. In this section, we will focus on peptide injections; implants will be discussed in a later section.

2.3.1 Polymer-based formulations

Approved PLGA–based peptidic products are usually provided in the form of lyophilized powder, and they are reconstituted to form a suspension before subcutaneous or intramuscular injection. The majority of approved modified-release peptidic products (e.g., Bydureon®, Triptodur®, Signifor Lar®) employ a polymer-based system, preferably a system based on a combination of polylactic acid and polyglycolic acid (a combination referred to as PLGA) (Bydureon [package insert], 2019; Triptodur [package insert], 2018; Lar, 2019).

PLGA is a general name for the family of biocompatible and biodegradable polymers composed of polylactic acid (PLA) and polyglycolic acid (PGA). PLGA usually refers to poly D,L-lactic-co-glycolic acid, where the D- and L- lactic acid forms are in a 1:1 ratio, unless specifically stated otherwise (Makadia and Siegel, 2011). This polymer system has been extensively studied for delivering a variety of drug modalities, including small molecules, proteins, genetic therapeutics, and peptides, due to its favorable degradation properties, its sustained-release characteristics, and its versatility in achieving a desired dosage and release interval (Makadia and Siegel, 2011; Kapoor et al., 2015). PLGA degrades in aqueous media via hydrolysis of its ester linkage, which makes it biodegrade in an in vivo physiological environment. PLGA’s biodegradation rate is determined by several factors, including polymer composition, molecular weight, end groups, and the nature of the surrounding media. Given the same composition, higher molecular weights degrade faster than lower molecular weights. The monomer ratio of lactic acid to glycolic acid is critical in determining the degradation rate. The presence of the methyl group makes PLA more hydrophobic than PGA, thus systems with higher PLA ratio degrades more slowly because less water is available to trigger hydrolysis. However, the 50:50 ratio of PLA: PGA is an exception because it exhibits the fastest degradation rate (Vyleesi [package insert], 2019; Doenicke et al., 1992).

In the case of peptides, the PLGA drug delivery system dramatically extends the effective duration of the peptide formulations and consequently reduces the dosing frequency to a time scale of one month. Furthermore, a variety of timeframes is achieved by adjusting the PLA:PGA ratio or by adjusting the loading amount in the system. This allows more flexibility in treatment regimen design. As discussed above, the degradation time of the polymers greatly depends on their composition, such as the ratio of PLA to PGA. This is well reflected in the correlation between PLGA composition and dosing intervals in the approved products (see Table 6). Generally, a PLA:PGA ratio of 50:50 is chosen for a release term of one month. Higher PLA content gives a longer extension of up to six months, regardless the half-lives of the API peptides.

Table 6. Examples of PLGA–based sustained-released peptide injectable products.

Product API Half-Life of API PLA: PGA Dosing interval Reference Bydureon® exenatide 2.4 h 50:50 1 week (Bydureon [package insert], 2019; Bond, 2006) Lupron Depot® leuprolide 3 h 75:25 100:0 1 month 3,4,6 months (Depot, 2019; Zhou et al., 2018) Trelstar® triptorelin 6 min, 45 min, 3 h 50:50 1 month (Trelstar [package insert], 2018; Garner et al., 2015) Sandostatin® Lar Depot octreotide 1.7 h 55:45 1 month (Petersen et al., 2011; Lar, 2019) Signifor® Lar pasireotide 12 h 50–60:40–50, 50:50 1 month (Lar, 2019; DrugBank, 2008)

Lupron Depot® is supplied for one, three, four, and six months dosing schedules, although the half-life of its active ingredient peptide (leuprolide) is only 16 min (Okada, 1997; Depot, 2019). The application of the PLGA system prolongs the release of the peptide. The length of the period of continuous sustained release depends heavily on the adjustment of the molecular weight of the polymer and the composition ratio of PLG to PLA. The microspheres made with PLGA (75/25)-14,000 and PLA-15,000 achieved one month and three months of zero-order release, respectively (Okada, 1997; Okada et al., 1996). In addition, multiple dose options for the same dosing frequency are available by means of altering the amount of peptides loaded into the PLGA system. This allows products to be developed for different indications, and it facilitates the dose adjustment based on responses and tolerability during the treatment. Signifor® LAR® is a good example, since it is provided in 10 mg, 20 mg, 30 mg, 40 mg, and 60 mg doses for a 1-month supply, and the recommended doses for its two major indications, acromegaly and Cushing’s disease, are 40 mg and 10 mg, respectively (Lar, 2019).

Other excipients commonly found in PLGA–based peptidic products include mannitol, carboxymethylcellulose, polysorbate, buffering agents, and pH-adjusting agents. As in non-modified-release formulations, mannitol is a popular excipient that is widely employed in products. Mannitol is either added in lyophilized formulations as a bulking agent to prevent the aggregation of microspheres during the freeze-drying process, or it is added to diluent solutions to help the resuspension of the microspheres before administration (Zhou et al., 2018; Ravi et al., 2008). One exception is the group of exenatide products, which uses sucrose instead of mannitol, most likely as a protective agent (Zhengxing Su et al., 2016). Carboxymethylcellulose (CMC) sodium salt is added to the diluents of several products, including Lupron Depot®, Trelstar®, and Signifor® LAR® (Lar, 2019; Depot, 2019; Trelstar [package insert], 2018). It provides viscosity to the system and stabilizes the suspension. Surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188) are also routinely incorporated in diluents for wetting purposes. Moreover, the diluent of Bydureon® is buffered by a phosphate system, while other diluents adjust pH using acetic acid (Bydureon [package insert], 2019). Some other excipients are selected for specific products. In another exenatide product, Bydureon Bcise® (a medium-chain triglyceride (MCT) suitable for use in an autoinjector) functions as a non-aqueous vehicle, which consequently generates an oily suspension (Bydureon [package insert], 2019; Non-clinical Review(s), 2017). Gelatin is added in leuprolide formulations to increase encapsulation efficiency because of the high viscosity it generates in the primary emulsion (Depot, 2019; Zhou et al., 2018).

PLGA is a well-developed system for prolonged-release formulations. It is also the most widely used platform for modifying the release of peptidic drug molecules. As such, it is a good reference for formulation scientists. However, the PLGA system has some inherent disadvantages that must be managed. One problem is that microsphere-based formulations usually cause pain in intramuscular injections, and they require large injection volumes. This can limit the dose administrated at one time and can consequently shorten the interval of injections. Second, toxic organic solvents are involved in the manufacturing process, which could cause safety problems. The generation of acidic metabolites induces the risk of peptide degradation and increased local acidity, as well leading to irritation at the depot site. Third, as with all modified-release formulations, burst release and incomplete release should be major concerns. A high initial burst is a problem unique to the PLGA system when used for peptide delivery. In addition, the compatibility between the polymers and the peptide must be examined in pre-formulation (Ansary et al., 2014; Uhrich et al., 1999).

2.3.2 Other sustained-released formulations

Somatuline Depot® (lanreotide) is the first marked sustained-release formulation produced via peptide self-assembly. This formulation is provided in a single-dose, prefilled syringe attached to an automatic needle guard system for deep subcutaneous injections (Depot, 2019). Upon injection, lanreotide forms a depot at the injection site, and the precipitated drug is continuously discharged into surrounding tissues from the depot, likely through passive diffusion, followed by absorption into the blood. After a single administration, lanreotide is continuously released from the depot with a half-life of 23–30 days, and the achieved bioavailability in healthy subjects is up to 78.4% (for a 120 mg dose) (Depot, 2019).

Wolin et al. have thoroughly discussed this product in their review paper, and the sustained release mechanism of the lanreotide depot has been sufficiently addressed (Wolin et al., 2016). Briefly, as illustrated in Fig. 4, the cyclic octapeptide noncovalently dimerizes (mainly through hydrogen bonding), forming building blocks for β-sheet-rich filaments that constitute hollow nanotubes with a highly uniform diameter and wall thickness. A very dense packing of these constructed nanotubes creates a semi-solid gel structure. These nanotubes primarily form an organized hexagonal lattice with lanreotide concentrations of up to 10–15% (w/w). However, at higher peptide concentrations, such as 24.6% in the product, a densely packed embedded tube structure begins to form. These nanotubes are also constructed by noncovalent forces such as hydrophobic interactions and π–π stacking of the aromatic side chains within the filaments. Consequently, in vitro experiments have revealed that the nanotube assembly is reversible, which enables the release of the drug (Wolin et al., 2016; Pouget et al., 2010; Valery et al., 2004; Pellet and Bismuth, 2003).

Fig. 4. Lanreotide is a cyclic octapeptide that can form noncovalently bonded dimers. The dimers constitute β-sheet-rich filaments that are stabilized by hydrogen bonding. The hollow nanotubes are composed of 26 filaments, which have a highly uniform diameter of 24 nm. Depending on the lanreotide concentration, nanotubes in the semi-solid gel can be organized in a hexagonal lattice or they can form densely packed tube-within-a-tube structures (i.e., embedded nanotubes). Reprinted from Wolin et al. (2016) with permission. Link to the Creative Commons license: http://creativecommons.org/licenses/by/4.0/.

It has also been noted that pH is critical for the formation of the depot. Acetic acid is the only excipient in the formulation, and its purpose is to maintain the pH at a level that ensures the injectability of the formulation and the solubility of the lanreotide (Montes et al., 2016).

Acthar® Gel is one of the first approved therapeutic peptide products (approved in 1952) that is still available. The active ingredient is adrenocorticotropic hormone (ACTH), which is eliminated rapidly with a half-life of around only 15 min (Acthar Gel [package insert]). It is administered via intramuscular or subcutaneous injection twice daily or once daily depending on the indication and the medical condition. The prolonged release is achieved by formulating ACTH with 16% gelatin. It is reported that the hydrolyzed gelatin counteracts the tissue inactivation phenomenon and enhances the stability and activity of the ACTH (Thompson, 1962). In addition, 0.5% phenol and no more than 0.1% cysteine are added as a preservative and an antioxidant, respectively. The pH is adjusted using sodium hydroxide and/or acetic acid (Acthar Gel [package insert]).

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2.2.2 Tonicifying agents

When developing injectable formulations, tonicity must be considered in order to create isotonic solutions for parenteral administration. Tonicifying agents are included in peptide injection formulations to ensure proper tonicity. This is because a non-isotonic solution can lead to tissue damage and increased pain at the injection site. Isotonicity in human plasma is equivalent to 290 mOsm/L. Therefore, this should be a target value for intravenous solutions or suspensions (Stranz and Kastango, 2013). However, hypertonicity is actually required in some formulations. Wang, et al. reviewed the tolerability of hypertonic products, and they propose 600 mOsm/kg as the upper osmolality limit for intramuscular or subcutaneous injections and 500 or 1000 mOsm/kg for intravenous and intravascular injections, depending on the injection volume (Wang, 2015).

Sodium chloride, mannitol, dextrose, and glycerin are specifically reported in product inserts as tonicifying excipients, as listed in Table 2. Sodium chloride is the most common tonicifying agent in peptide injection products. In several products, sodium chloride (NaCl) in various concentrations (e.g., 0.75% in Miacalcin®, 0.85% in Parsabiv®, 0.69% in Lutathera®) is added directly to liquid formulations(Revised:, 2018; Parsabiv [package insert], 2019; Miacalcin [package insert], 2018). Normal saline (0.9% NaCl) often serves as diluent and is absent in the freeze-dried powder of solid products. This is likely due to the fact that NaCl could greatly reduce the collapse temperature of the formulation, resulting in a low drying temperature and thus an increase in drying time and cost (Carpenter et al., 2002). In addition, NaCl can not only crystallize on its own but also induce the crystallization of other excipients upon storage, which would impact the stability of the formulation (Hawe and Frieß, 2006). Interestingly, the only frozen product, Bivalirudin in 0.9% Sodium Chloride® is frozen with 0.9% NaCl, which generates an osmolality of 300 mOsmol/kg for intravenous injection (Bivalirudin in 0.9% Sodium Chloride Injection [package insert], 2000). This could indicate that the impact of NaCl on freeze-dried product is during or after the drying stage. Freezing with NaCl does not necessarily harm the peptide.

Mannitol is demonstrated to be a suitable alternative to NaCl as a tonicifier. Mannitol often appears in subcutaneous solutions (e.g., Forteo®, Copaxone®, Signifor®), although only two pramlintide products, Symlin® and Byetta®, distinctly claim in their inserts that the purpose of adding mannitol is to adjust their tonicity (Symlin [package insert], 2014; Signifor [package insert], 2019; Copaxon [package insert], 2019; Forteo [package insert], 2019; Byetta [package insert], 2018). Mannitol is frequently found in solid products as a bulking agent, some of which contain mannitol as their only bulking agent. The diluent for these products can be water for injection alone instead of isotonic solutions, which indicates that mannitol is not only a bulking agent in lyophilization but also a tonicifier for injectables. Examples of these products include Cetrotide®, Angiomax®, Firmagon®, and Egrifta® (Firmagon [package insert], 2015; Cetrotide [package insert], 2018; Angiomax [package insert], 2019; Egrifta [package insert], 2019). Similarly, lactose, which is the only inactive ingredient in Glucagon®, may have the same use (Glucagon [package insert], 2016).

For injection, the selected diluents are normally water, normal saline, or 5% dextrose. It is worth mentioning that 0.9% NaCl is the diluent for Mepact, the only product that used a liposome delivery system (Mepact [package insert], 2019). Normal saline and 5% dextrose are used interchangeably for some lyophilized products, which gives both health providers and patients more practicality and flexibility. For example, Natrecor® lists 5% dextrose with 0.45% sodium chloride, USP, and 5% dextrose with 0.2% sodium chloride as alternatives other than 5% dextrose and 0.9% NaCl (Natrecor [package insert], 2019).

A pH-adjusted glycerin solution is the diluent of another glucagon peptide injectable formulation, Glucagon® (Glucagon [package insert], 2018). In the reconstituted solution, glycerin (12 mg/ml) serves multiple purposes: It is a tonicifying agent and a cosolvent or solubilizing agent. It not only adjusts the osmolality of the solution but also helps the peptide dissolve in an environment with a pH between 3 and 9.5 (Steiner et al., 2010). In addition, solution injectable formulations, the liraglutide product Xultophy®, the bremelanotide product Vyleesi®, and both lixisenatide products (i.e., Soliqua® and Adlyxin®) used glycerin for tonicifying the formulation (Xultophy [package insert], 2019; Knudsen et al., 2006; Vyleesi [package insert], 2019; Soliqua [package insert], 2019; Adlyxin [package insert], 2019). It is worth mentioning here that glycerin is also a useful wetting agent for suspension formulations (Pramanick et al., 2013).

Propylene glycol is another common cosolvent or solubilizing agent. Propylene glycol is incorporated in the Ozempic® and Victoza® formulations, in which both semaglutide (the API of Ozempic®) and liraglutide (the API of Victoza®) are reported to be “good [sic] soluble in an aqueous solution” at their final pH (Victoza [package insert], 2019; Ozempic [package insert], 2019). Thus, propylene glycol most likely serves as a tonicifying agent in these two formulations. Attention should be paid when using propylene glycol because it generates relatively high osmolality even in low working concentrations, despite being reported to inhibit gel formation of peptides and to has a wetting effect in suspension formulations (Payne and Manning, 2009; Doenicke et al., 1992; Powell et al., 1991). The diluent for modified-release formulations will be discussed in the next section.

Achieving isotonicity can be challenging, especially for high-concentration formulations. Even though the acceptable range of osmolality is relatively broad, it is still worth noting that developers should alert healthcare providers and patients about the risk of injection-related pain under the “Adverse Reaction” or “Warnings and Precautions” section of the package inserts for formulations with high solute concentrations.

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