HSP90 (IC50 = 62 nM); GRP94 (IC50 = 65 nM)

Alvespimycin (17-DMAG) has an EC50 of 62 nM, making it a potent inhibitor of Hsp90. The human cancer cell lines SKBR3 and SKOV3, which overexpress the Hsp90 client protein Her2, are inhibited in their growth by alvespimycin (17-DMAG). This results in the down-regulation of Her2 and the induction of Hsp70, which is consistent with Hsp90 inhibition. In SKBR3 and SKOV3 cells, the EC50 values for Her2 degradation are 8 ± 4 nM and 46 ± 24 nM, respectively, while the EC50 values for Hsp70 induction are 4 ± 2 nM and 14 ± 7 nM, respectively[1]. Alvespimycin (17-DMAG) showed dose-dependent apoptosis (P<0.001 averaged over 24- and 48-hour time points) when compared to the vehicle control at concentrations ranging from 50 nM to 500 nM, which correspond to pharmacologically achievable doses. Alvespimycin (17-DMAG) exhibits time-dependent apoptosis (P <0.001, averaged across all doses) in chronic lymphocytic leukemia (CLL) cells after an extended exposure period of 24 to 48 hours, which is comparable to that of many other agents. Alvespimycin (17-DMAG) also has significantly greater potency than 17-AAG after 24 and 48 hours of treatment[2].

Tumors are grown for two months prior to the initiation of intraperitoneal injections (i.p.) every four days for a month, using either 0, 5, 10, and 20 mg/kg Alvespimycin (17-DMAG) or 0, 50, 100, and 200 mg/kg Dipalmitoyl-radicicol. The animals receiving HSP90 inhibitor treatment had much smaller tumor volumes than the animals receiving vehicle control treatment, despite sample heterogeneity. In a gastrointestinal cancer animal model, HSP90 inhibitors have been demonstrated to cause liver toxicity. Yet, at 100 mg/kg, dipalmitoyl-radicicol reduces tumor size in a statistically significant way, whereas at 10 or 20 mg/kg, alvespimycin (17-DMAG) also significantly reduces tumor size[3].

Competition Binding Assay. [1]
Native human Hsp90 protein (α+β isoforms) isolated from HeLa cells (SPP-770) and recombinant canine Grp94 (SPP-766) were purchased from Stressgen Biotechnologies. The procedures of the FP-based binding assay were adapted from those described by Chiosis and colleagues.42,43 BODIPY-AG solution was freshly prepared in FP assay buffer (20 mM HEPES−KOH, pH 7.3, 1.0 mM EDTA, 100 mM KCl, 5.0 mM MgCl2, 0.01% NP-40, 0.1 mg/mL fresh bovine γ-globulin (BGG), 1.0 mM fresh DTT, and Complete protease inhibitor) from stock solution in DMSO. Binding curves were obtained by mixing equal volume (10 μL) of the BODIPY-AG solution and serially diluted human Hsp90 (or Grp94) solution in a 384-well microplate to yield 10 nM BODIPY-AG, varying concentration of Hsp90 (0.10 nM-6.25 μM monomer), and 0.05% DMSO. After 3 h incubation at 30 °C, fluorescence anisotropy (λEx = 485 nm, λEm = 535 nm) was measured on an EnVision 2100 multilabel plate reader. Competition curves were obtained by mixing 10 μL each of a solution containing BODIPY-AG and Hsp90 (or Grp94), and a serial dilution of each compound freshly prepared in FP assay buffer from stock solution in DMSO. Final concentrations were 10 nM BODIPY-AG, 40 or 60 nM Hsp90 (or Grp94), varying concentration of each compound (0.10 nM−10 μM), and ≤0.25% DMSO. Because compounds (1−3)a oxidize easily at neutral pH, assays of these compounds were performed in parallel with the quinone compounds (1−3)b under nitrogen atmosphere in a LabMaster glovebox (M. Braun, Stratham, NH). Typically, Hsp90 protein solution and compound stock solutions were brought into the glovebox as frozen liquid, and binding mixtures were prepared in FP assay buffer deoxygenated by repeated cycles of evacuation and flushing with argon. After incubation, the microplate was removed from the glovebox and fluorescence anisotropy was immediately measured. Interestingly, binding of BODIPY-AG to Hsp90 results in simultaneous increases in fluorescence anisotropy (FA) and intensity, whereas binding to Grp94 gives relatively little change in fluorescence intensity. Triplicate data points were collected for each binding or competition curve. Competition binding curves were fitted by a four-parameter logistic function[1].

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Dissociation of 17-AAG from Hsp90 (Complex). [1]
The dissociation rate of 1b from either purified human Hsp90 protein or Hsp90 complex in cell lysates was determined using a spin column assay. [Allylamino-3H]-17-AAG (20 Ci/mmol, ≥97% pure by HPLC) was purchased commercially. 200 μCi (10 nmol) of [3H]-17-AAG in ethanol was dried under vacuum and mixed with 30 nmole of unlabeled 1b in DMSO to give a stock solution of 1 mM [3H]-17-AAG with a SA of 3 × 106−4 × 106 cpm/nmol. The binding reaction contained 400 nM Hsp90, 4.0 μM [3H]-17-AAG, and 0.38 mg/mL BGG in assay buffer (20 mM HEPES−KOH, pH 7.3, 1.0 mM EDTA, 100 mM KCl, 5.0 mM MgCl2, 0.01% NP-40, 1.0 mM fresh DTT, and Complete protease inhibitor). Bovine γ-globulin was included as carrier protein for purified Hsp90 protein only. Alternatively, cell lysates (prepared as described in Kamal et al.20) from normal human dermal fibroblasts (NHDF, 5.0 mg/mL total protein) or the breast cancer cell line SKBR3 (1.5 mg/mL) were used in place of purified Hsp90 protein. After ≥2 h incubation at 37 °C, 65 μL of the binding reaction was passed sequentially through two Zeba desalting spin columns to remove unbound ligand. In the dissociation reaction (650 μL), the desalted protein solution containing bound [3H]-17-AAG was diluted with unlabeled 1b to give final concentrations of ∼40 nM Hsp90, 40 μM 17-AAG, and 0.48 mg/mL BGG in assay buffer. Similarly, the desalted cell lysates were diluted 10-fold with 1b at 20 μM final concentration. Unlabeled 17-AAG was present at ≥1000-fold excess to Hsp90 to ensure that dissociation of [3H]-17-AAG was practically irreversible. At various times of incubation (37 °C), 60 μL of the dissociation reaction was withdrawn and passed sequentially through two Zeba spin columns. The flow-through fractions were analyzed on a MicroBeta microplate scintillation counter. Dissociation kinetics was fitted with a single-exponential function A = A0 × exp-kt + A∞ to derive the first-order rate constant k.

The MTT assay is used to measure cytotoxicity. Alvespimycin, 17-AAG, or a vehicle are incubated for 24 or 48 hours with a total of 1×10⁶ CD19-selected B cells from CLL patients. After adding MTT reagent, the plates are incubated for a further twenty-four hours, following which spectrophotometric measurement is performed. Using propidium iodide (PI) and annexin V-fluorescein isothiocyanate staining, apoptosis is identified. Cells are exposed to medications, and then they are cleaned in phosphate-buffered saline and stained once in binding buffer. Using flow cytometry, cell death is evaluated. The System II software package is used to analyze data. For every sample, ten thousand cells are counted. Changes in the potential of the mitochondrial membrane are evaluated by staining with the lipophilic cationic dye JC-1 and analyzing the results using flow cytometry[2].

Mice: The mice used are CB-17/IcrHsd-Prkdc-SCID young male mice. A collagen solution is mixed with 1×105 BPH1 cells and 2.5×10⁵ CAF per graft to create recombinant xenografts, which are then left to gel, covered with medium, and cultured for an entire night. The tumors are given eight weeks to form before being treated for four weeks with intraperitoneal injections of compounds in sesame oil every four days. The three different doses of dipalmitoyl-radicicol (50, 100, and 200 mg/kg) and Alvespimycin (5, 10 and 20 mg/kg) are administered. The mice are killed after a total of 12 weeks, their kidneys removed, the grafts cut in half, and their photos taken before the tissue is processed for histology. The measurements of the graft are taken, and the volume of the resulting tumor is computed using the formula volume=width × length × depth × π/6. This formula understates the volume of large, invasive tumors when compared to smaller, non-invasive tumors, suggesting a cautious approach to evaluating tumor volumes. Grafts that have been removed are embedded in paraffin, fixed in 10% formalin, and then subjected to immunohistochemistry.

Absorption, Distribution and Excretion
Increasing concentration of the drug results in dose-proportional increase in the plasma concentration. At the maximum tolerated dose of 80mg/m^2, the plasma concentration exceeded 63nM (mean IC50 for 17-DMAG in the NCI 60 human tumor cell line panel) for less than 24 hours in all patients. The mean peak concentration (Cmax) reached 2680 nmol/L at this dose.
Mainly renal and biliary elimination pathways. In a mice study, the excreted urine 24 hours post-dose recovered 10.6–14.8% of delivered dose unchanged.
At the maximum tolerated dose of 80mg/m^2, the mean Vd value is 385 L.
The mean clearance is 18.9 L/hr at the dose of 80mg/m^2.

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Metabolism / Metabolites
Alvespimycin demonstrates redox cycling catalyzed by purified human cytochrome P450 reductase (CYP3A4/3A5) to quinones and hydroquinones. It could also form glutathione conjugates at the 19-position on the quinone ring. However in vivo and in vitro studies suggest that weak metabolism of alvespimysin occurs in humans.

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Biological Half-Life
The half-life across all dose levels ranged from 9.9 to 54.1 h (median, 18.2 h).

Protein Binding
Reported to be minimal.

[1]. Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino-17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90. J Med Chem. 2006 Jul 27;49(15):4606-15.

[2]. 17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition. Blood. 2010 Jul 8;116(1):45-53

[3]. Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90. Cancers (Basel). 2016 Aug 24;8(9). pii: E77.

Alvespimycin is a 19-membered macrocyle that is geldanamycin in which the methoxy group attached to the benzoquinone moiety has been replaced by a 2-(N,N-dimethylamino)ethylamino group. It has a role as a Hsp90 inhibitor. It is a secondary amino compound, a tertiary amino compound, an ansamycin, a member of 1,4-benzoquinones and a carbamate ester. It is functionally related to a geldanamycin.
Alvespimycin is a derivative of geldanamycin and heat shock protein (HSP) 90 inhibitor. It has been used in trials studying the treatment of solid tumor in various cancer as an antitumor agent. In comparison to the first HSP90 inhibitor tanespimycin, it exhibits some pharmacologically desirable properties such as reduced metabolic liability, lower plasma protein binding, increased water solubility, higher oral bioavailability, reduced hepatotoxicity and superior antitumor activity.
Alvespimycin has been reported in Cullen corylifolium and Trichosanthes kirilowii with data available.
Alvespimycin is an analogue of the antineoplastic benzoquinone antibiotic geldanamycin. Alvespimycin binds to HSP90, a chaperone protein that aids in the assembly, maturation and folding of proteins. Subsequently, the function of Hsp90 is inhibited, leading to the degradation and depletion of its client proteins such as kinases and transcription factors involved with cell cycle regulation and signal transduction.

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Drug Indication
Investigated for use as an antineoplastic agent for solid tumors, advanced solid tumours or acute myeloid leukaemia.

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Mechanism of Action
Alvespimycin inhibits HSP90 and its regulation of correct folding and function of many cellular signalling proteins, which are referred to as Hsp90 client proteins. These client proteins are also referred to as oncoproteins and include Her-2, EGFR, Akt, Raf-1, p53, Bcr-Abl, Cdk4, Cdk6 and steroid receptors that are involved in cellular signalling pathways that drive cellular proliferation and counteract apoptosis. They are often over-expressed or mutated in tumors, and contribute to cancer progression and therapy resistance. Alvespimycin promotes an anticancer activity by disrupting Hsp90's chaperone function and inducing the proteasomal degradation of oncoproteins. It is shown to reduce the levels of CDK4 and ERBB2.

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Pharmacodynamics
Alvespimycin mediates an antitumor activity through HSP90 inhibition that targets client proteins for proteasomal destruction, including oncogenic kinases such as BRAF. The administration of the drug is shown to result in the depletion of client proteins that have oncogenic activity and potential induction of HSP70 (HSP72). It is more selective for tumors over normal tissue. A study also reports that alvespimycin enhances the potency of telomerase inhibition by imetelstat in pre-clinical models of human osteosarcoma.

C32H48N4O8

616.756

616.347

C, 62.32; H, 7.84; N, 9.08; O, 20.75

467214-20-6

Alvespimycin hydrochloride;467214-21-7

5288674

Pale purple to purple solid powder

1.2±0.1 g/cm3

810.5±65.0 °C at 760 mmHg

444.0±34.3 °C

0.0±6.6 mmHg at 25°C

1.566

2.07

4

10

8

44

1230

6

O(C([H])([H])[H])[C@]1([H])[C@@]([H])([C@@]([H])(C([H])([H])[H])C([H])=C(C([H])([H])[H])[C@@]([H])([C@]([H])(C([H])=C([H])C([H])=C(C([H])([H])[H])C(N([H])C2=C([H])C(C(=C(C2=O)C([H])([H])[C@@]([H])(C([H])([H])[H])C1([H])[H])N([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)=O)OC([H])([H])[H])OC(N([H])[H])=O)O[H] |c:16,31,t:27|

KUFRQPKVAWMTJO-LMZWQJSESA-N

InChI=1S/C32H48N4O8/c1-18-14-22-27(34-12-13-36(5)6)24(37)17-23(29(22)39)35-31(40)19(2)10-9-11-25(42-7)30(44-32(33)41)21(4)16-20(3)28(38)26(15-18)43-8/h9-11,16-18,20,25-26,28,30,34,38H,12-15H2,1-8H3,(H2,33,41)(H,35,40)/b11-9-,19-10+,21-16+/t18-,20+,25+,26+,28-,30+/m1/s1

[(4E,6Z,8S,9S,10E,12S,13R,14S,16R)-19-[2-(dimethylamino)ethylamino]-13-hydroxy-8,14-dimethoxy-4,10,12,16-tetramethyl-3,20,22-trioxo-2-azabicyclo[16.3.1]docosa-1(21),4,6,10,18-pentaen-9-yl] carbamate

17-DMAG; KOS 1022; KOS-1022; KOS1022; Alvespimycin

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ~100 mg/mL (~162.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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