Central complement component C3 (KD = 0.5 nM)

A new generation of highly selective and potent C3 inhibitors, termed compstatins Cp40/AMY-101, are clinically developed by Amyndas Pharmaceuticals for various complement-mediated indications. These small-sized peptidic C3 inhibitors are primate/human-specific and display more favorable pharmacological profiles and a greater tissue-penetrating capacity than larger biologics, such as the complement inhibitor TP-10, previously evaluated as a treatment option for ARDS [2].

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Given that C3 interception with compstatin-based inhibitors (such as AMY-101) may offer broader therapeutic coverage than anti-C5 or anti-C5a agents by blocking simultaneously generation of all downstream proinflammatory mediators involved in SARS-CoV-2-induced ARDS and thrombotic microangiopathies, AMY-101 is well poised for clinical evaluation as an anti-inflammatory agent in severe cases of COVID-19 infection [2].

For NHPs with naturally occurring chronic periodontitis, AMY-101 can help with periodontal health [1]. Long-lasting anti-inflammatory effects can be produced by AMY-101 [1]. Subcutaneous administration of AMY-101 (4 mg/kg body weight) every 24 hours for 28 days results in a notable and long-lasting decrease in PPD, a marker of gradual tissue deterioration [1]. AMY-101 (Cp40, 1 mg/kg, sc, injected once daily for 7 or 14 days, every 12 hours)

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Locally Administered AMY-101 (0.1 mg/Site) Does Not Cause Irritation in Healthy Gingiva [1]
To determine possible local gingival irritation after administration of the peptidic C3 inhibitor AMY-101 in NHPs, a therapeutic dose of AMY-101 (50 μL of 2 mg/mL solution corresponding to 0.1 mg/site)30 was injected in healthy gingiva of posterior teeth in five animals. Each animal received a total of four injections, one per quadrant; two injections were with AMY-101 and the other two injections involved water for injection (WFI) containing 5% dextrose (control). In each animal, AMY-101 was administered on both maxillary and mandibular quadrants (2 sites total), whereas the control solution was injected on the two contralateral sites. AMY-101 and control solution were injected a total of three times, at days 0, 7, and 14, followed by a 2-week observation period without further injections. Intraoral photographs were taken at baseline (day 0) and every 2–3 days to document the gingival condition around injection sites. Careful daily clinical examination revealed no signs of irritation after injection of AMY-101 or control solution throughout the observation period (Figure 1). Blood samples were collected at day −1 and day 15 and were processed for hematology and biochemistry analysis. All measurements were within the normal range for all animals (not shown).

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AMY-101 Confers Protection, Even when Administered Once Every 3 Weeks [1]
We have previously shown that weekly intragingival injections of AMY-101 can improve the periodontal condition of NHPs with natural chronic periodontitis.30 A less frequent but nevertheless successful administration would facilitate the application of AMY-101 for human use. To explore this possibility, we tested whether AMY-101 can be efficacious also when administered less frequently. To this end, a 2-mg/mL solution of AMY-101 was administered once every 2 weeks in 5 animals or once every 3 weeks in another 5 animals. Specifically, AMY-101 was injected locally into the gingiva of anterior and posterior teeth on both sides of the maxilla (17 sites total; palatal papilla between the teeth [15 sites], and distal gingiva of third molars [2 sites]). Clinical examinations were performed at baseline and 1, 2, 4, 6, 7, 8, 10, and 12 weeks throughout the study to determine the progression of the disease and the potential beneficial effects of AMY-101. Clinical readings made before AMY-101 injection served as baseline controls. The mandible was not treated but was monitored by clinical periodontal examination throughout the study for comparative purposes. The study consisted of 6 weeks of AMY-101 treatments (treatment period), followed by 6 weeks without AMY-101 treatment (follow-up period).

Regardless of the frequency of administration, AMY-101 caused a significant reduction in clinical indices that measure periodontal inflammation (GI and bleeding on probing [BOP]) or tissue destruction (PPD and CAL) (Figures 4 and 5). Interestingly, differences between baseline and subsequent readings reached statistical significance at or after 6 weeks (i.e., at the time point when the treatments with AMY-101 were discontinued). Many of the differences observed at 6 weeks remained statistically significant even at 12 weeks (BOP, PPD, and CAL) (Figures 4B–4D and 5B–5D). The aforementioned clinical indices were also monitored in the untreated jaw (mandible) during the same 12-week interval. In contrast to the improved clinical condition in the AMY-101-treated maxillae, the clinical indices in the mandibles did not show significant differences in the course of the study as compared to their baseline values (Figures 4 and 5). In conclusion, AMY-101 can induce a long-lasting clinical anti-inflammatory effect.

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Systemic Administration of AMY-101 Can Improve the Periodontal Condition of NHPs [1]
Given that AMY-101 is also being considered for systemic disorders and periodontitis is a highly prevalent disease,34 we tested whether AMY-101 can be effective when administered systemically. AMY-101 was administered in 10 animals via subcutaneous injection at a concentration of 4 mg/kg bodyweight, once per 24 hr for a total of 28 days. To determine the progression of the disease and the potential beneficial effects of AMY-101, clinical examinations were performed at baseline (week 0) and throughout the study (at the 1-, 2-, 3-, 4-, and 11-week time points). Additionally, biopsies were taken from the gingiva and bone at baseline, 4 weeks, and 11 weeks.

Systemically administered AMY-101 caused a significant and long-lasting reduction in PPD, an index that measures tissue destruction (Figure 6A). The protective effect was first observed at week 4. Strikingly, the protective effect persisted without decline for at least another 7 weeks (week 11) (Figure 6A), even though the drug was discontinued after week 4. Improvement of BOP, which assesses periodontal inflammation, was also observed; differences relative to the baseline reached statistical significance at weeks 2 and 3 (Figure 6B). Histological observations at 4 weeks showed that AMY-101 caused decreased expression of pro-inflammatory and pro-osteoclastogenic cytokines (interleukin [IL]-17 and receptor activator of nuclear factor-κB ligand [RANKL]) and elevated expression of osteoprotegerin (OPG; a natural inhibitor of RANKL) in the connective tissue adjacent to the alveolar bone, as compared to their baseline expression (Figure 7). Moreover, AMY-101 treatment caused a decrease in the complement cleavage fragments C3d and C5a, further confirming its ability to inhibit complement activation. In conclusion, systemic AMY-101 improves the periodontal condition of NHPs, which is stably maintained for at least 7 weeks after drug withdrawal.

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C3 deficiency attenuates fibrosis and infiltration of inflammatory cells in UUO-induced renal fibrosis [3]
To investigate the role of C3 in the pathogenesis of UUO, we used a peptidic C3 inhibitor, Compstatin analog AMY-101/Cp40, to block C3 activation. Masson's staining and α-SMA expression analysis showed that UUO mice injected with 1 mg/kg Cp40 had much less severe interstitial fibrosis than control peptide-injected mice (Supplement Figures 3A–D). Western blot analysis also indicated that the α-SMA and PDGFR-β levels were decreased in the Cp40-injected UUO mice (Supplement Figures 3E,F). In addition to the attenuated tubulointerstitial fibrosis, renal infiltration of F4/80+ macrophages, CD3+T cells, CD4+T cells, and CD8+T cells was significantly reduced in Cp40-injected UUO mice compared with peptide-injected mice (Figures 5A–E). Meanwhile, elevated MCP-1, IL-6, IL-1β, and TNF-α mRNA expression in UUO mice was markedly limited by Cp40 (Figure 5F). These data indicate that C3 mediates the infiltration of T cells and macrophages into the kidney in response to obstructive injury.

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Blocking C3-C3aR signaling attenuates renal fibrosis by inhibiting IL-17A production in UUO mice [3]
As shown in Figure 7A, renal mRNA levels of IL-17A were substantially increased in UUO mice compared with sham control mice. In addition, IL-17A levels in the serum of UUO mice were significantly increased in the early and late stages compared with those in the serum of sham control mice (Figure 7B). Consistent with the ELISA and mRNA data, the FACS results revealed that 8.48 and 10.9% of CD4+ renal cells in obstructed kidneys expressed IL-17A following UUO, respectively, whereas ≤5.3% were IL-17A+ in sham-operated mice, and this effect was strongly inhibited by AMY-101/Cp40 and SB290157 (Figures 7C–H). In addition, we performed analysis of CD11b+F4/80+IL-17+ cells ratio in kidney from C3 blockade UUO mice and UUO mice. The results showed that CD11b+F4/80+IL-17+cells were around 1% in mononuclear cells in two groups, and only slightly changed after blockade C3 with CP40 (Supplement Figures 4A,B). Similar results were confirmed in 14 days of UUO mice (Supplement Figures 4C,D). Thus, we identified that the main producer of IL-17A in the UUO mice were T cells, which were strikingly increased after unilateral ureteral ligation.

PBMC isolation [3]
Patients and normal subjects donated 5 ml of blood collected in heparinized tubes. Blood was diluted 1:1 with PBS and overlaid onto lymphocyte separation medium. After centrifugation, 3 ml of the interface containing the PBMCs was collected and diluted to 6 ml with PBS, then washed twice with cold PBS and counted. The PBMCs were collected for flow cytometric analysis.

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CFSE labeling [3]
A CFSE stock solution (5 mM) was prepared fresh by dissolving lyophilized CFSE in DMSO. Splenocytes were obtained from the spleens of naïve mice, and labeled with CFSE at 5 μM in PBS for 15 min at 37°C. Excess CFSE was quenched by adding three volumes of ice-cold FBS and incubating the cells for 5 min on ice. CFSE labeled cells were then washed three times with PBS and cultured with or without stimulation.

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T cell activation [3]
The 96-well assay plate precoated with anti-CD3 and anti-CD28 Ab was incubated at 37°C for 4 h. Splenocytes (1 ×106 cells/well) were obtained from the spleens of naïve mice and were cultured for 3 days in 96-well plates in medium containing IL-2 (10 ng/mL) as well as IL-12 (10 μg/mL), or IL-4 (4 ng/mL).

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Flow cytometric analysis [3]
A single renal cell suspension was prepared and stimulated with PMA/Ionomycin/Golgi-plug for 4 h. The cells were incubated with different primary antibodies or the appropriate isotype control antibodies at 4°C for 30 min. The following antibodies were used PerCP/Cy5.5-conjugated anti-human CD14; PerCP/Cy5.5-conjugated anti-mouse CD4 ; APC-conjugated anti-mouse F4/80; and PerCP/Cy5.5-conjugated anti-mouse CD11b. After cellular surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm Soln Kit for intracellular staining with Alexa Fluor 488-conjugated anti-human C3 and PE-conjugated anti-mouse IL-17A. All flow cytometric analyses were performed using an LSR II Flow Cytometer and Flowjo software.

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ELISA [3]
To quantify IL-17A levels in the kidney, samples were analyzed using a mouse IL-17A ELISA according to the manufacturer's instructions. All measurements were performed in duplicate.

Animal/Disease Models: 15 adult male cynomolgus monkeys (Macaca fascicularis) (7-15 years old; body weight 5.0- 7.6 kg)[1].
Doses: 0.1 mg/site; 50 μL of 2 mg/mL solution.
Route of Administration: local injection. (3 times a week or 1 time a week for 6 weeks, then 6 weeks of follow-up, no treatment required.)
Experimental Results: No irritation to healthy gums.

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Animal/Disease Models: UUO and sham-operated mice [3].
Doses: 1 mg/kg.
Route of Administration: Inject subcutaneously (sc) (sc) every 12 hrs (hrs (hours)) one time/day for 7 or 14 days.
Experimental Results: 1 mg/kg Cp40 produced Dramatically less severe interstitial fibrosis than control mice injected with the peptide.

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C3 Inhibitor AMY-101 [1]
The 14-residue compstatin analog AMY-101 [(D)Tyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH2, where Sar is sarcosine/N-methyl glycine and mIle is N-methyl isoleucine] was produced as a disulfide-bridged, cyclic peptide by solid-phase peptide synthesis methodology as previously described. AMY-101 was injected locally into the gingiva (50 μL volume) at different concentrations (2–200 mg/mL) using a 30G short needle. Alternatively, for systemic administration, AMY-101 was given by subcutaneous injection (4 mg/kg bodyweight) using a 1-mL insulin safety syringe with a 28G × 1/2-inch needle.

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Clinical Examination and Observation [1]
Clinical periodontal examinations were performed and the diagnosis was established according to the criteria of the American Academy of Periodontology for human periodontal disease. Examinations using a periodontal probe were performed at baseline and throughout the study to monitor the progression of the disease and the effect of AMY-101 treatment. The examinations included determination of PPD (by measuring the distance [in millimeters] from the gingival margin to the base of the pocket), CAL (distance from the cementoenamel junction to the base of the pocket), GI (using a scale of 0–3, according to Löe), BOP (percentage of positive sites), and plaque index (PI; scale of 0–3 according to Löe). PPD, CAL, and BOP were measured at six sites: mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual aspects of each tooth. GI and PI were assessed at four sites (buccal, lingual, mesial, and distal). GI and BOP are measures of periodontal inflammation, while CAL and PPD assess tissue destruction. The PI is a clinical measure of biofilm accumulation on tooth surfaces. In the irritation study, injection sites were clinically observed daily for signs of inflammation or the formation of an abscess, redness, itching, hematoma, bruising, bleb, or nodules. The degree of gingival inflammation was assessed as healthy, mild (slight change in color, no BOP), moderate (redness, BOP), or severe (marked redness, tendency to spontaneous bleeding).

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Animal model [3]
Unilateral ureteral obstruction (UUO) is a popular experimental model of renal injury. Mice aged 6–8 weeks were anesthetized followed by a lateral incision on the back of the mouse. Subsequently, the left ureter was exposed and tied off with two 4.0 silk suture. Sham-operated mice underwent an identical procedure but without ureteric ligation. The therapeutic experiment was performed with the Compstatin analog AMY-101/Cp40 (dTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-His-Arg-Cys]-mIle-NH2;1.7kDa) which was produced by solid-phase peptide synthesis, and SB290157, a C3a receptor antagonist, which was purchased from Sigma-Aldrich. UUO and sham-operated mice were treated with Cp40 (1 mg/kg) via subcutaneous injection every 12 h and SB290157 (30 mg/kg) via intraperitoneal injection daily. After 7 or 14 days, the mice were sacrificed by cervical vertebra dislocation, and then, peripheral blood, spleen, and renal tissues were collected. The mouse kidneys were fixed in 4% formalin for 24 h, processed through dehydration in a graded series of alcohol and embedded in paraffin The remaining sample was frozen in liquid nitrogen for later use.

Dose-Escalation Study for Local Injection of AMY-101 [1]
This study was designed to determine possible local gingival irritation after local injection of increasing concentrations of AMY-101 in NHPs with naturally occurring periodontitis. Escalating doses of AMY-101 tested were 2, 10, 50, 100, and 200 mg/mL in a total volume of 50 μL, thus corresponding to 0.1, 0.5, 2.5, 5, and 10 mg/site, respectively. The injected sites involved posterior teeth on both sides of the maxilla (palatal interdental papillae) and mandible (buccal interdental papillae). Five animals were used and all injections were given in a single session followed by a 2-week observation period. The animals were examined daily for the possible presence of local gingival irritation in response to AMY-101 injections. Doses equal to or higher than 10 mg/mL caused mild to moderate inflammation, which was observed more often with the highest doses (100 and 200 mg/mL) (Figure 2). No irritation was observed with the 2-mg/mL dose at any treated site, consistent with the data discussed above. Clinical examinations to determine periodontal disease activity and intraoral photography were performed at baseline and after 1 and 2 weeks. In terms of efficacy, doses up to 50 mg/mL caused a reduction in periodontal clinical parameters (probing pocket depth [PPD], clinical attachment level [CAL], and gingival index [GI]) (Figure 3). In contrast, the highest doses (100 and 200 mg/mL) caused deterioration in the same clinical parameters (Figure 3). Therefore, among the different AMY-101 concentrations tested, the 2-mg/mL dose appears to be an optimal dose fulfilling both safety and protection requirements.

[1]. Safety and Efficacy of the Complement Inhibitor AMY-101 in a Natural Model of Periodontitis in Non-human Primates. Mol Ther Methods Clin Dev. 2017 Aug 18;6:207-215.

[2]. The first case of COVID-19 treated with the complement C3 inhibitor AMY-101. Clin Immunol. 2020 Apr 29:108450.

[3]. Complement C3 Produced by Macrophages Promotes Renal Fibrosis via IL-17A Secretion. Front Immunol. 2018 Oct 22;9:2385.

AMY-101, also known as compstatin 40, is a peptidic inhibitor of the central complement component C3. AMY-101 is under investigation in clinical trial NCT03694444 (A Study of the C3 Complement Inhibitor AMY-101 in Adults With Gingivitis).
C3 Complement Inhibitor AMY-101 is a compstatin-based inhibitor of human complement component C3, with potential use as a treatment for various diseases in which excessive complement activation plays a key role, including paroxysmal nocturnal hemoglobinuria (PNH) and complement 3 glomerulopathy (C3G). Upon administration, C3 complement inhibitor AMY-101 selectively binds to C3 and inhibits C3 activity. This prevents complement pathway activation, and inhibits complement-mediated inflammation and cell lysis. Excessive complement activation plays a key role in various inflammatory and autoimmune diseases, and leads to tissue destruction. C3 is a crucial and central component of the complement system, and the complement system is an integral component of the innate immune response.

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Periodontitis is a chronic inflammatory disease associated with overactivation of the complement system. Recent preclinical studies suggest that host-modulation therapies may contribute to effective treatment of human periodontitis, which may lead to loss of teeth and function if untreated. We previously showed that locally administered AMY-101 (Cp40), a peptidic inhibitor of the central complement component C3, can inhibit naturally occurring periodontitis in non-human primates (NHPs) when given once a week. This study was undertaken to determine the local safety of increasing doses of the drug as well as its efficacy when given at a reduced frequency or after systemic administration. Our findings have determined a local dose of AMY-101 (0.1 mg/site) that is free of local irritation and effective when given once every 3 weeks. Moreover, a daily subcutaneous dose of AMY-101 (4 mg/kg bodyweight) was protective against NHP periodontitis, suggesting that patients treated for systemic disorders (e.g., paroxysmal nocturnal hemoglobinuria) can additionally benefit in terms of improved periodontal condition. In summary, AMY-101 appears to be a promising candidate drug for the adjunctive treatment of human periodontitis, a notion that merits investigation in human clinical trials. [1]

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Acute respiratory distress syndrome (ARDS) is a devastating clinical manifestation of COVID-19 pneumonia and is mainly based on an immune-driven pathology. Mounting evidence suggests that COVID-19 is fueled by a maladaptive host inflammatory response that involves excessive activation of innate immune pathways. While a "cytokine storm" involving IL-6 and other cytokines has been documented, complement C3 activation has been implicated as an initial effector mechanism that exacerbates lung injury in preclinical models of SARS-CoV infection. C3-targeted intervention may provide broader therapeutic control of complement-mediated inflammatory damage in COVID-19 patients. Herein, we report the clinical course of a patient with severe ARDS due to COVID-19 pneumonia who was safely and successfully treated with the compstatin-based complement C3 inhibitor AMY-101. [2]

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Complement synthesis in cells of origin is strongly linked to the pathogenesis and progression of renal disease. Multiple studies have examined local C3 synthesis in renal disease and elucidated the contribution of local cellular sources, but the contribution of infiltrating inflammatory cells remains unclear. We investigate the relationships among C3, macrophages and Th17 cells, which are involved in interstitial fibrosis. Here, we report that increased local C3 expression, mainly by monocyte/macrophages, was detected in renal biopsy specimens and was correlated with the severity of renal fibrosis (RF) and indexes of renal function. In mouse models of UUO (unilateral ureteral obstruction), we found that local C3 was constitutively expressed throughout the kidney in the interstitium, from which it was released by F4/80+macrophages. After the depletion of macrophages using clodronate, mice lacking macrophages exhibited reductions in C3 expression and renal tubulointerstitial fibrosis. Blocking C3 expression with a C3 and C3aR inhibitor provided similar protection against renal tubulointerstitial fibrosis. These protective effects were associated with reduced pro-inflammatory cytokines, renal recruitment of inflammatory cells, and the Th17 response. in vitro, recombinant C3a significantly enhanced T cell proliferation and IL-17A expression, which was mediated through phosphorylation of ERK, STAT3, and STAT5 and activation of NF-kB in T cells. More importantly, blockade of C3a by a C3aR inhibitor drastically suppressed IL-17A expression in C3a-stimulated T cells. We propose that local C3 secretion by macrophages leads to IL-17A-mediated inflammatory cell infiltration into the kidney, which further drives fibrogenic responses. Our findings suggest that inhibition of the C3a/C3aR pathway is a novel therapeutic approach for obstructive nephropathy. [3]

C85H118F3N23O20S2

1903.11374616623

1789738-04-0

AMY-101 acetate;AMY-101;1427001-89-5

168013663

H-D-Tyr-xiIle-Cys(1)-DL-Val-DL-Trp(Me)-DL-Gln-Asp-DL-Trp-Sar-Ala-His-Arg-DL-Cys(1)-N(Me)Ile-NH2.TFA; {D-Tyr}-Ile-Cys-Val-{Trp(Me)}-Gln-Asp-Trp-{Sar}-Ala-His-Arg-Cys-{N(Me)Ile}-NH2 (Disulfide bridge:Cys3-Cys13); D-tyrosyl-(3xi)-L-isoleucyl-L-cysteinyl-DL-valyl-N1-methyl-DL-tryptophyl-DL-glutaminyl-L-alpha-aspartyl-DL-tryptophyl-sarcosyl-L-alanyl-L-histidyl-L-arginyl-DL-cysteinyl-N2-methyl-L-isoleucinamide (3->13)-disulfide trifluoroacetic acid

YXCVXQDWGAHRCI; {D-Tyr}-ICV-{Trp(Me)}-QDW-{Sar}-AHRC-{N(Me)Ile}-NH2 (Disulfide bridge:Cys3-Cys13)

White to off-white solid powder

22

28

30

133

3860

9

S1C[C@@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@@H](CC(=O)O)C(N[C@H](C(N(C)CC(N[C@@H](C)C(N[C@@H](CC2=CN=CN2)C(N[C@H](C(N[C@@H](CS1)C(N(C)[C@H](C(N)=O)[C@@H](C)CC)=O)=O)CCCNC(=N)N)=O)=O)=O)=O)CC1=CNC2C=CC=CC1=2)=O)=O)CCC(N)=O)=O)CC1=CN(C)C2C=CC=CC1=2)=O)C(C)C)=O)NC([C@H]([C@@H](C)CC)NC([C@@H](CC1C=CC(=CC=1)O)N)=O)=O.FC(C(=O)O)(F)F

GDSMXERXVAAZHM-GQPSZVCTSA-N

InChI=1S/C83H117N23O18S2.C2HF3O2/c1-11-43(5)68(103-72(114)53(84)30-46-23-25-50(107)26-24-46)80(122)100-61-39-125-126-40-62(82(124)106(10)69(70(86)112)44(6)12-2)101-73(115)55(21-17-29-90-83(87)88)94-76(118)58(33-49-36-89-41-92-49)96-71(113)45(7)93-65(109)38-105(9)81(123)60(31-47-35-91-54-20-15-13-18-51(47)54)99-77(119)59(34-66(110)111)97-74(116)56(27-28-64(85)108)95-75(117)57(98-79(121)67(42(3)4)102-78(61)120)32-48-37-104(8)63-22-16-14-19-52(48)63;3-2(4,5)1(6)7/h13-16,18-20,22-26,35-37,41-45,53,55-62,67-69,91,107H,11-12,17,21,27-34,38-40,84H2,1-10H3,(H2,85,108)(H2,86,112)(H,89,92)(H,93,109)(H,94,118)(H,95,117)(H,96,113)(H,97,116)(H,98,121)(H,99,119)(H,100,122)(H,101,115)(H,102,120)(H,103,114)(H,110,111)(H4,87,88,90);(H,6,7)/t43?,44-,45-,53+,55-,56?,57?,58-,59-,60?,61-,62?,67?,68-,69-;/m0./s1

2-[(7S,10S,13S,22S,34R)-34-[[(2S)-2-[[(2R)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-4-[[(2S,3S)-1-amino-3-methyl-1-oxopentan-2-yl]-methylcarbamoyl]-25-(3-amino-3-oxopropyl)-7-(3-carbamimidamidopropyl)-10-(1H-imidazol-5-ylmethyl)-19-(1H-indol-3-ylmethyl)-13,17-dimethyl-28-[(1-methylindol-3-yl)methyl]-6,9,12,15,18,21,24,27,30,33-decaoxo-31-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29,32-decazacyclopentatriacont-22-yl]acetic acid;2,2,2-trifluoroacetic acid

AMY101 TFA; AMY-101 TFA; 1789738-04-0; AMY 101 TFA

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 50 mg/mL
DMSO : ≥ 50 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)