Use of glu k1 receptor agonists for the preparation of a medicament for the treatment of chronic pain

Abstract

The application belongs to the field of neurobiology and pain treatment, and particularly relates to application of a GluK1 receptor agonist in preparation of a drug for treating chronic pain. The GluK1 receptor agonist is ATPA or a pharmaceutically acceptable salt or solvate thereof. The application finds that, under a chronic pain state, kainate receptor-mediated synaptic plasticity in an anterior cingulate cortex (ACC) is significantly impaired, and is manifested as loss of long-term depression ability, and selective activation of a kainate receptor subtype GluK1 can reverse the abnormality. After chronic pain, GluK1 expression in the ACC is up-regulated, and activation of GluK1 by using a GluK1 receptor agonist ATPA has a significant therapeutic effect on neuropathic pain and inflammatory pain. The results show that, targeted activation of GluK1 can produce an analgesic effect by restoring the homeostasis of synaptic plasticity in the ACC, and the GluK1 receptor agonist can be used for treating chronic pain.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of neurobiology and pain treatment, and particularly relates to the use of GluK1 receptor agonists in the preparation of a medicament for treating chronic pain. BACKGROUND

[0002] Chronic pain is a kind of disease that seriously endangers human health, and about 20% of the world's population suffers from chronic pain. At present, the treatment of chronic pain is always a big problem in clinic, and the main treatment drug is mainly opiate such as morphine. However, long-term use of opiate drugs can lead to tolerance, addiction and serious withdrawal symptoms. The existing treatment methods have limited effect on chronic pain, and there is a lack of long-term effective and small side effect analgesic drugs. The main reason is that we lack sufficient understanding of the causes and regulation mechanisms of chronic pain. In the transmission loop of pain, the anterior cingulate cortex (ACC) of the cerebral cortex is considered to have a very important role in both pain perception and emotional regulation. A large number of clinical brain imaging and animal electrophysiological studies have shown that both peripheral nerve pain and emotional pain stimuli can activate the ACC region. Overactivation of ACC is closely related to many chronic pain conditions, which are often accompanied by fear, anxiety and cognitive dysfunction.

[0003] The mechanism of chronic pain involves peripheral and central sensitization, abnormal synaptic plasticity and neuroinflammation. Central sensitization, such as long-term potentiation (LTP) of synaptic transmission, is a key cellular mechanism for long-term changes in the mammalian central nervous system under physiological and pathological conditions. On the contrary, studies have shown that impaired long-term depression (LTD) function of glutamatergic synapses in the ACC is an important marker of chronic pain sensitization. Activation or amplification of LTD in the ACC contributes to the relief of pain and the production of analgesic effect. However, there is no analgesic drug targeting the LTD signal pathway at present.

[0004] In recent years, studies have shown that the neurotransmitter glutamate plays a core role in pain signal transmission and regulation. Among them, the dysfunction of kainate (KA) type glutamate receptor GluK1 (formerly known as GluR5) subtype is closely related to the maintenance of chronic pain. GluK1 receptor is widely distributed in the central nervous system, especially highly expressed in the anterior cingulate cortex (ACC). This brain area is not only the integration center of the affective component of pain, but also the key regulatory node of pain-emotion comorbidity. GluK1 receptor participates in the dynamic balance of synaptic plasticity in ACC by regulating presynaptic glutamate release and postsynaptic neuronal excitability. For example, in the nerve injury model, down-regulation or functional inhibition of GluK1 receptor expression can lead to over-activation of ACC glutamatergic circuits, thereby exacerbating the affective component of pain and anxiety-like behavior. However, the existing intervention strategies for GluK1 receptor in the prior art are mainly focused on the development of antagonists, which can relieve pain but often have side effects such as cognitive dysfunction or emotional inhibition, limiting their clinical application. Since GluK1 receptor is expressed on both excitatory and inhibitory neurons, the development of target analgesic drugs for GluK1 receptor agonists has very important prospects and significance. SUMMARY

[0005] To solve the above technical problems, the application provides the application of a GluK1 receptor agonist in the preparation of a drug for treating chronic pain.

[0006] The application researches and finds that the synaptic plasticity mediated by the kainate receptor subtype GluK1 in the ACC is significantly impaired under chronic pain conditions, showing loss of KA-induced LTD ability, and selective activation of GluK1 receptor can reverse this abnormality. After chronic pain, GluK1 expression is up-regulated in the ACC, and activation of GluK1 using GluK1 receptor agonist ATPA has a significant therapeutic effect on neuropathic pain and inflammatory pain. The results show that the targeted activation of KA receptor subtype GluK1 can restore the homeostasis of synaptic plasticity in the ACC, produce analgesic effect, and GluK1 receptor agonist can be used for treating chronic pain.

[0007] Further, the GluK1 receptor agonist is ATPA or a pharmaceutically acceptable salt, solvate thereof; the ATPA is (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid, and the structural formula is: .

[0008] Further, the drug contains GluK1 receptor agonist as the only effective ingredient.

[0009] Further, the drug contains active ingredients for enteral or parenteral administration, and suitable pharmaceutically organic or inorganic inert carrier materials.

[0010] Further, the suitable pharmaceutically organic or inorganic inert carrier material is selected from one or more of water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.

[0011] Further, the dosage form of the drug is a solid dosage form or a liquid dosage form.

[0012] Further, the solid dosage form is a tablet, a coated tablet, a suppository or a capsule.

[0013] Further, the liquid dosage form is a solution, a suspension or an emulsion.

[0014] Further, the drug contains a suitable pharmaceutical excipient.

[0015] Further, the excipient is selected from one or more of a preservative, a stabilizer, a wetting agent, an emulsifying agent, an osmotic pressure adjusting agent and a buffer.

[0016] Further, the chronic pain is neuropathic pain or inflammatory pain.

[0017] The present application has the following beneficial effects:

[0018] 1. Targeted regulatory mechanism: The present application first discloses that the activation of GluK1 receptor in the ACC region (through ATPA) has a specific analgesic effect in chronic neuropathic pain, and the effect is achieved by enhancing the long-term depression (LTD) of synaptic transmission in the ACC brain region.

[0019] 2. Dose-dependent analgesia: The present application significantly improves the mechanical paw withdrawal threshold (PWT) and thermal pain response latency of mice in the sciatic nerve branch ligation (SNI) neuropathic pain model and complete Freund's adjuvant (CFA) inflammatory pain model by microinjection of ATPA (10, 50 and 150 ng / kg), and the effect is dose-dependent, which takes effect 5 minutes after single administration and lasts for about 20 minutes.

[0020] 3. Behavior-specific separation: Unlike traditional analgesic drugs (such as opioids), the analgesic effect of ATPA is not accompanied by improvement of anxiety-like behavior, indicating that it regulates pain perception through an independent pathway, and provides direct evidence for the "analgesia-emotion decoupling" treatment strategy.

[0021] 4. Clinical transformation potential: ATPA has no significant effect on normal animals, and does not induce behavioral changes in the sham operation group, suggesting that its effect is pathologically selective, can avoid systemic side effects, and is suitable for precise intervention of chronic pain patients. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1The results of the correlation analysis between GluK1 receptor-mediated LTD dysfunction in the ACC and chronic pain sensitization are shown. A is a schematic diagram of the experiment recording excitatory postsynaptic potentials (fEPSP) of the ACC using the MED64 multichannel system. B is a schematic diagram of SNI surgery: ligation and transection of the common peroneal nerve (CPN) and tibial nerve (TN), while preserving the sural nerve (SN); C is a comparison of the mechanical withdrawal threshold (PWT) between the SNI group and the Sham group mice (0.066 ± 0.02482 g vs. 0.88 ± 0.1744 g in the sham group). p <0.01); D is the LTD of ACC brain slices from Sham group mice after different KA stimulation times; D is the LTD of ACC brain slices from SNI group mice after different KA stimulation times; E is a comparison of the LTD of ACC brain slices from Sham group mice and SNI group mice after KA stimulation for 180 min.

[0023] Figure 2 The expression of GluK1 in the SNI chronic pain model is shown. A is a typical immunoblot band image, and B is a quantitative statistical plot of GluK1 and GluK2 / 3 protein expression levels in the ACC of SNI mice (*p<0.05; n = 3 / group, based on bilateral unpaired student data). t test).

[0024] Figure 3 This diagram illustrates the analgesic effect of microinjection of the GluK1 receptor agonist ATPA into the SNI model. In the diagram, A shows the mechanical withdrawal threshold (PWT) of mice in the Sham and SNI groups after ACC injection of 50 ng / kg ATPA; B shows the hot plate latency of mice in the Sham and SNI groups after ACC injection of 50 ng / kg ATPA; C shows the change in mechanical withdrawal threshold (PWT) over time in mice in the Sham and SNI groups after ACC injection of different concentrations of ATPA; and D shows the change in hot plate latency over time in mice in the Sham and SNI groups after ACC injection of different concentrations of ATPA.

[0025] Figure 4 This diagram illustrates the analgesic effect of microinjection of the GluK1 receptor agonist ATPA into the ACC of CFA mice. A is a schematic diagram of the CFA-induced inflammatory pain mouse model. B shows the analgesic effect of different concentrations of ATPA (10, 50, and 150 ng / kg) injected into the ACC of CFA mice in response to mechanical hyperalgesia. C shows the change in analgesic effect over time after a single ATPA injection. Detailed Implementation

[0026] The present application is described in detail below with reference to the accompanying drawings and specific examples, but should not be construed as limiting the present application. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0027] Example 1

[0028] 1. Methods and Materials

[0029] 1.1 Experimental materials

[0030] (1) Animals

[0031] The experimental animals were adult male C57BL / 6 mice (6-8 weeks old) purchased from the Medical Experimental Animal Center of Xi'an Jiaotong University. They were raised in the animal house of the Frontier Science and Technology Research Institute of Xi'an Jiaotong University, with room temperature maintained at 24 ± 1°C, air humidity maintained at 55%-60%, and a light cycle of 12 h (light from 8:30 am to 8:30 pm). They were allowed free access to water and food. The animal experimental procedures involved in this example were approved by the Experimental Animal and Management Committee of Xi'an Jiaotong University, and the animal experimental and animal ethics standards were strictly followed during the experiment.

[0032] (2) Drugs

[0033] Kainic acid (KA, CAS No.: 487-79-6) and (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA, CAS No.: 140158-50-5) were purchased from Tocris Cookson (Bristol, UK) and dissolved in normal saline (0.9% sodium chloride injection). Complete Freund's adjuvant (CFA, CAS No.: 9007-81-2) was purchased from Sigma-Aldrich (USA) and dissolved in normal saline when used.

[0034] 2. Experimental methods

[0035] (1) Preparation of ACC brain slices in vitro

[0036] Reference: Koga, K., G. Descalzi, T. Chen, H. G. Ko, J. S. Lu, S. Li, J. Son, T. Kim, C. Kwak, R. L. Huganir, M. G. Zhao, B. K. Kaang, G. L. Collingridge and M. Zhuo (2015). "Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain (vol 85, pg 377, 2015)." Neuron 86(4): 1109-1109.

[0037] First, prepare 50 mL of artificial cerebrospinal fluid (ACSF) and high-sugar slice solution each and oxygenate (oxygenate with mixed gas 95% O2 and 5% CO2) for 30 min. When slicing, anesthetize the mouse with isoflurane and execute by cervical dislocation, acutely dissociate the brain tissue and immerse it in oxygenated high-sugar slice solution, cool for 1 min, then remove the whole brain and trim it. Fix the front end upwards with glue on the tray of a vibrating microtome (Leica, VT1200S). Cut the ACC-containing coronal brain slice from the isolated brain slice, and the brain slice thickness is 300 μm. Then transfer the brain slice to ACSF, incubate at room temperature for at least 1 h before recording.

[0038] (2) Multi-channel field potential recording

[0039] 1) Preparation of microelectrode array probe

[0040] This example uses a MED64 multi-channel recording system (P515A, Panasonic Alpha-Med Sciences). The MED64 probe has a 64-channel planar microelectrode array arranged in an 8 × 8 pattern with an electrode spacing of 150 μm. In order for the brain slice to better adhere to the MED64 probe, the MED64 probe needs to be hydrophilic before use, soaked in 0.1% polyethyleneimine (P-3143, Sigma-Aldrich) borate buffer (pH 8.4) at room temperature overnight in the dark. Then rinse the probe surface with sterile distilled water at least three times.

[0041] 2) Multi-channel field potential recording

[0042] After incubation for 1 hour, place the ACC-containing brain slice in the MED64 probe (Figure 1 A), after proper positioning, the grid and anchors were carefully placed on the brain slice to ensure stability of the slice during the experiment. Fresh ACSF bubbled with a mixture of 95% 02 and 5% C02 was continuously perfused through the slice at a rate of 2-3 mL / min using a perfusion pump throughout the experiment. The slice was incubated on the probe for at least 1 hour before recording. A channel in the deep layer (V-VI layer) was chosen as the stimulation site. Through the Mobius software, the stimulation site was continuously stimulated with a biphasic constant current pulse (10-20 μΑ, 0.2 ms) and evoked excitatory field excitatory postsynaptic potentials (fEPSPs) in the surrounding area. The stimulation was recorded at 1 min intervals, and the analysis was averaged every 5 min. The "slope" parameter represents the average slope of each fEPSP recorded at the activation channel. After 0.5 hours of stable baseline recording, KA was added to the perfusion solution, and after 20 min, it was washed. Antagonists or inhibitors were administered 10 min before the administration of KA and were washed together with KA. Then the fEPSP response was recorded for 2.5 h.

[0043] (3) Chronic neuropathic pain model (SNI)

[0044] Chronic neuropathic pain was induced by ligating the sciatic nerve. Mice were anesthetized with isoflurane. The left hind limb was shaved and iodophor was used for disinfection in the surgical area. A longitudinal incision was made in the middle of the femur, and the muscle layer was bluntly separated to expose the sciatic nerve and its three branches. The common peroneal nerve (CPN) and tibial nerve (TN) were cut and tightly ligated with 5-0 silk. The sural nerve (SN) was left intact Figure 1 B), then the muscle and skin were sutured layer by layer. The sham group (Sham) only exposed the sciatic nerve, but did not damage any nerve. After the operation, all animals were placed on a warming pad for at least 30 minutes.

[0045] (4) Chronic Complete Freund's Adjuvant-induced Inflammatory Pain Model (CFA)

[0046] Complete Freund's adjuvant (CFA; Sigma-Aldrich, USA) was mixed with 0.9% sterile normal saline at a volume ratio of 1:1 to prepare a homogeneous emulsion by vortex shaker. A microsyringe was used to accurately extract 10 μL of CFA emulsion, and the needle was inserted from the central area of the mouse left hind paw, slowly injected into the subcutaneous tissue, and the needle was stopped for 20 s after injection to ensure the deposition of the emulsion, and then the needle was slowly rotated to avoid liquid extravasation.

[0047] (5) ACC cannula implantation and microinjection of drugs

[0048] Cannula implantation and microinjection references: Zhao, M. G., H. Toyoda, Y. S. Lee, L. J. Wu, S. W. Ko, X. H. Zhang, Y. Jia, F. Shum, H. Xu, B. M. Li, B. K. Kaang and M. Zhuo (2005). "Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory." Neuron 47(6): 859-872.

[0049] After isoflurane anesthesia, the mice were fixed on a stereotaxic apparatus, ensuring the head was stable, the head hair was shaved and iodophor was sterilized. The scalp was cut open, the skull was leveled, and the Bregma point was exposed. According to the brain atlas, the coordinates of the mouse ACC (AP +0.9 mm, ML ± 0.3 mm, DV -1.5 mm) were determined with the Bregma point as the origin, a hole was drilled at the positioning point, the cannula was slowly inserted to the target depth, and the dental cement was fixed. After the dental cement was solidified (about 15 minutes), the clamping device was removed, the catheter cap was inserted and tightened, and it was placed on the warming pad until the mouse was completely awake.

[0050] Microinjection of drugs and behavioral testing were performed 7 days after SNI. ATPA (dissolved in normal saline, Tocris Cookson, Bristol, UK) solutions with concentrations of 10, 50 and 150 ng / kg were prepared. Microinjection was performed using an injection pump (Razel Scientific Instruments, Stamford, CT, USA) and a Hamilton syringe (Hamilton, Reno, NY, USA), and the injection inner tube and syringe were connected by a polyethylene tube. The drug was injected at a slow speed (0.5 μL / min), 0.5 μL was injected on each side, and the needle was left for 1 minute after injection to ensure drug diffusion. Behavioral testing was performed 5 minutes after microinjection was completed.

[0051] (5) Behavioral testing

[0052] 1) Mechanical withdrawal threshold test

[0053] The mouse left hind paw withdrawal threshold (PWT) was detected by the Up-Down method using Von Frey filaments. A series of filaments with different bending forces were used, including 0.008 g, 0.02 g, 0.04 g, 0.16 g, 0.4 g, 0.6 g, 1 g, 1.4 g and 2.0 g. The mice were placed in a Plexiglas cage with a metal grid at the bottom, and after they were calm (no exploratory behavior or grooming action), the test began, starting with a 0.4 g filament, which was gently touched vertically to the central area of the mouse hind paw sole (avoiding the edge of the footpad), until it was slightly bent and remained for 3-6 seconds. If there was no response, the next thicker filament was used until a positive response occurred, which included licking, biting or sudden withdrawal of the hind leg. If a positive response occurred at 0.4 g, the next thinner filament was used until no response occurred. The test was repeated 5 times to obtain the results, and there needed to be a 1 minute interval between each positive result.

[0054] 2) Hot plate test

[0055] The mice were placed on a hot plate tester with a temperature of 55 ± 1 °C. Reference: Zhou, Z., W. Shi, K. Fan, M. Xue, S. Zhou, Q. Y. Chen, J. S. Lu, X. H. Li and M. Zhuo (2021). "Inhibition of calcium-stimulated adenylyl cyclase subtype 1 (AC1) for the treatment of neuropathic and inflammatory pain in adult female mice." Mol Pain 17: 17448069211021698. The latency of the first positive response of the mouse hind paw was recorded. Positive responses included rapid lifting of the paw, licking of the paw and jumping. To avoid burning the mouse, the test time was a maximum of 30 seconds.

[0056] 2. Experimental results

[0057] (1) GluK1 receptor-mediated LTD in ACC is impaired after SNI model

[0058] To explore the changes of GluK1 receptor-mediated LTD in chronic pain, this embodiment recorded the excitatory postsynaptic field potential (fEPSP) of ACC slices of sham operation group and SNI model group mice by MED64 multi-channel system. The results showed that the ACC slices of sham operation group mice could induce stable long-term depression LTD under KA (5 μM) stimulation (baseline slope decreased to 58.19 ± 8.23%, n = 5 slices / 5 mice,p <0.001), while LTD induction was significantly impaired in the SNI group mice (baseline slope maintained at 93.49 ± 15.08%, n=5 brain slices / 5 mice, p >0.05)( Figure 1 E- Figure 1 F). This result indicates that chronic nerve injury leads to abnormal synaptic plasticity mediated by GluK1 receptors within the ACC, suggesting that GluK1 receptor-mediated LTD dysregulation may be a key mechanism of pain sensitization.

[0059] (2) Upregulation of GluK1 receptor expression after chronic pain model

[0060] To further verify whether the function of the GluK1 receptor changes after chronic pain, we used Western blotting to examine the expression of GluK1 protein after spontaneous intravascular coagulation (SNI). Figure 2 As shown, the expression of both GluK1 and GluK2 / 3 proteins was upregulated after the SNI chronic pain model (n = 3), suggesting that the function of the GluK1 receptor is enhanced after chronic pain.

[0061] (3) In the SNI model, ACC microinjection of ATPA produced an analgesic effect in a dose-dependent manner.

[0062] In SNI model mice, bilateral microinjection of the GluK1 receptor-specific agonist ATPA into the ACC significantly improved mechanical aberrant pain. Figure 3 A) and thermal hyperalgesia ( Figure 3 B), compared with the saline control group (PWT: 0.051 ± 0.0067 g), the mechanical withdrawal threshold (PWT) in the 50 ng / kg ATPA group was significantly increased to 0.291 ± 0.0538 g ( p <0.001, Tukey test), while the 10 ng / kg and 150 ng / kg groups reached 0.065 ± 0.005 g 5 minutes after administration ( p <0.05) and 0.32 ± 0.0506 g ( p <0.01)( Figure 3 (C) indicates that the analgesic effect of ATPA is dose-dependent. In the hot plate test, the latency of the response in the 50 ng / kg ATPA group was prolonged from 6.249 ± 0.4442 s in the saline group to 8.955 ± 0.5114 s (C). p <0.01). Further analysis showed that the analgesic effect began 5 minutes after a single injection of 50 ng / kg ATPA, and lasted for approximately 20 minutes. Figure 3 D), indicating its rapid and reversible action.

[0063] (4) ACC microinjection of ATPA dose-dependently produced anti-inflammatory pain effect in CFA model

[0064] In CFA-induced inflammatory pain model, after ACC injection of different concentrations of ATPA (10, 50, and 150 ng / kg) in mice, the mechanical paw withdrawal threshold (PWT) of CFA-induced inflammatory pain mice was significantly increased. Compared with the saline control group (Saline group, 0.07 ± 0.01 g), the PWT of 10, 50, and 150 ng / kg ATPA groups was increased to 0.28 ± 0.06 g ( p <0.01), 0.9 ± 0.06 g ( p <0.001), and 0.95 ± 0.05 g ( p <0.001), respectively, indicating that ATPA has a concentration-dependent analgesic effect ( Figure 4 B). In addition, after a single ACC injection of ATPA, the analgesic effect showed a time-dependent change. The onset of drug effect was 5 minutes after injection, and the analgesic effect lasted for about 20 minutes, and then gradually subsided ( Figure 4 C). In summary, ATPA can effectively relieve CFA-induced hyperalgesia, and the analgesic effect has a concentration and time-dependent effect.

[0065] It should be noted that when the present application claims involving numerical ranges, it should be understood that each numerical range of two endpoints and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes the preferred embodiments.

[0066] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

Claims

1. Use of a GluKl receptor agonist for the manufacture of a medicament for the treatment of chronic pain, characterized in that, The GluK1 receptor agonist is ATPA; the chronic pain is neuropathic pain; the ATPA is (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid, and its structural formula is: The drug is a drug for injection into the prefrontal cingulate cortex.

2. Use of a GluKl receptor agonist according to claim 1 for the manufacture of a medicament for the treatment of chronic pain, characterized in that, The medicament has a GluK1 receptor agonist as the sole active ingredient.

3. Use of a GluKl receptor agonist according to claim 2 for the manufacture of a medicament for the treatment of chronic pain, characterized in that, The medicament is in a liquid dosage form.

4. Use of a GluKl receptor agonist according to claim 3 for the manufacture of a medicament for the treatment of chronic pain, characterized in that, The liquid dosage form is a solution, suspension or emulsion.

5. Use of a GluKl receptor agonist according to claim 3 for the manufacture of a medicament for the treatment of chronic pain, characterized in that, The medicament comprises suitable pharmaceutical adjuvants.

6. Use of a GluKl receptor agonist according to claim 5 for the manufacture of a medicament for the treatment of chronic pain. The adjuvants are selected from one or more of the group consisting of preservatives, stabilizers, wetting agents, emulsifying agents, osmotic pressure adjusting agents and buffers. The medicament has a GluK1 receptor agonist as the sole active ingredient. The medicament is in a liquid dosage form. The liquid dosage form is a solution, suspension or emulsion. The medicament comprises suitable pharmaceutical adjuvants. The adjuvants are selected from one or more of the group consisting of preservatives, stabilizers, wetting agents, emulsifying agents, osmotic pressure adjusting agents and buffers.

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