HSP90 (IC50 = 62 nM); GRP94 (IC50 = 65 nM)
Heat Shock Protein 90 (HSP90) - inhibitor [2]
IKKα (I-κB kinase α) - depleted via HSP90 inhibition [2]
IKKβ (I-κB kinase β) - depleted via HSP90 inhibition [2]
NF-κB (Nuclear factor-κB) signaling pathway - indirect inhibitor [2]

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].

---

Cytotoxicity in CLL Cells: 17-DMAG demonstrated dose- and time-dependent cytotoxicity against primary chronic lymphocytic leukemia (CLL) cells. At 1 μM, it significantly reduced viability as measured by MTT assay and annexin V/PI staining. [2]
Selective Toxicity to Normal Lymphocytes: 17-DMAG showed minimal toxicity to normal T cells (P = 0.26) and NK cells (P = 0.86) from healthy volunteers after 24-48 hours of treatment, even when stimulated with CpG (B cells, P = 0.32) or CD3 (T cells, P = 0.63). [2]
Caspase-Dependent Apoptosis: 17-DMAG (1 μM) induced mitochondrial membrane depolarization (detected by JC-1 staining) and PARP cleavage, which was prevented by the pan-caspase inhibitor z-VAD-fmk (100 μM). Cell death was significantly rescued by z-VAD (interaction contrast, P < 0.001). [2]
Depletion of IKKα and IKKβ: Unlike 17-AAG, which only depleted IKKβ, 17-DMAG (100 nM - 1 μM, 24 h) led to strong depletion of both IKKα and IKKβ in CLL cells. AKT was depleted by both inhibitors. [2]
Inhibition of NF-κB Signaling: 17-DMAG (1 μM, 24 h) reduced phosphorylation of IκBα, maintained total IκBα levels, and prevented nuclear localization of p65 and p50. It also reduced NF-κB DNA binding in EMSA assays. [2]
Downregulation of NF-κB Target Genes: 17-DMAG treatment significantly reduced transcript levels of BCL2 (P = 0.02) and MCL1 (P < 0.001), and decreased their protein expression. The decrease in MCL1 transcript was not significantly affected by z-VAD (P = 0.21), indicating it was not a consequence of apoptosis. [2]
Blockade of Microenvironment-Induced NF-κB Activation: Pretreatment with 17-DMAG (1 μM) blocked CD40L- and CpG-induced p65 nuclear localization and MCL1 upregulation. CD40L did not rescue 17-DMAG-induced cell death and actually enhanced its cytotoxic effect (P < 0.001). [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].

---

Survival Prolongation in TCL1-SCID Mouse Model: In a TCL1-SCID transplant mouse model of CLL, treatment with 17-DMAG (10 mg/kg i.p., 5 times/week) significantly prolonged survival compared to vehicle control (median survival 75 days vs 66 days, P = 0.027, n = 10/group). [2]
Reduction of Leukemic Burden: 17-DMAG treatment significantly reduced white blood cell counts in treated mice compared to vehicle control (P = 0.007, n = 20/group) at day 55 after treatment initiation. [2]

Competition Binding Assay. [1]
\nNative 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].

---

\n\nDissociation of 17-AAG from Hsp90 (Complex). [1]
\nThe 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].

---

Cell Viability Assay (MTT): CD19-selected B cells from CLL patients (1×10⁶ cells) were incubated with 17-DMAG or vehicle for 24-48 hours. MTT reagent was added, and plates were incubated for an additional 24 hours before spectrophotometric measurement. [2]
Apoptosis Assay (Annexin V/PI): After drug exposure, cells were washed, stained with annexin V-FITC and propidium iodide in binding buffer, and analyzed by flow cytometry (10,000 cells/sample). [2]
Mitochondrial Membrane Potential (JC-1 Staining): Cells were stained with the lipophilic cationic dye JC-1 and analyzed by flow cytometry to detect depolarized mitochondria. [2]
Immunoblot Analysis: Nuclear and cytoplasmic lysates were prepared using NE-PER kit. Proteins (50 μg/lane) were separated on polyacrylamide gels, transferred to nitrocellulose, probed with antibodies against AKT, p-IκBα, PARP, p65, p50, RELB, MCL1, BCL2, IκBα, actin, IKKα, IKKβ, and BRG1, and detected by chemiluminescence. [2]
Electrophoretic Mobility Shift Assay (EMSA): Nuclear protein (5 μg) was incubated with ³²P-labeled probe containing an NF-κB consensus binding site. Complexes were separated on polyacrylamide gels, dried, and autoradiographed. Antibody supershift experiments were performed with anti-p65 or anti-p50 antibodies. [2]
Real-Time PCR: RNA was extracted with TRIzol and purified using Qiagen RNeasy columns. cDNA was synthesized using SuperScript First-Strand Synthesis System. Real-time PCR was performed with commercial or custom-designed primers. Data were normalized to TBP or 18S internal control genes and analyzed using linear mixed-effects models. [2]
Stimulation Experiments: For CD40L stimulation, cells were treated with 17-DMAG (1 μM) for 0-24 hours, then stimulated with CD40L (500 ng/mL) for 1 hour. For CpG stimulation, cells were treated with 17-DMAG (1 μM) for 0-24 hours, then stimulated with CpG oligodeoxynucleotides (3.2 μM) for 3 hours. [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.

---

TCL1-SCID Transplant Mouse Model:** SCID mice were engrafted with CD19-selected transformed B cells (1×10⁶) from the spleen of a TCL1 transgenic mouse with active leukemia via tail vein injection. Two weeks after engraftment, mice were treated with 17-DMAG (10 mg/kg) or DMSO vehicle control by intraperitoneal injection 5 times per week. Blood was collected at various time points for analysis. Mice were euthanized upon development of hind-limb paralysis or other disease criteria causing discomfort. Overall survival was determined. White blood cell counts were determined at day 55 after treatment initiation by hematoxylin and eosin-stained peripheral blood smear. All experiments were approved by the Ohio State University Institutional Animal Care and Use Committee. [2]

---

TCL1-SCID Transplant Mouse Model: SCID mice were engrafted with CD19-selected transformed B cells (1×10⁶) from the spleen of a TCL1 transgenic mouse with active leukemia via tail vein injection. Two weeks after engraftment, mice were treated with 17-DMAG (10 mg/kg) or DMSO vehicle control by intraperitoneal injection 5 times per week. Blood was collected at various time points for analysis. Mice were euthanized upon development of hind-limb paralysis or other disease criteria causing discomfort. Overall survival was determined. White blood cell counts were determined at day 55 after treatment initiation by hematoxylin and eosin-stained peripheral blood smear. All experiments were approved by the Ohio State University Institutional Animal Care and Use Committee. [2]

Absorption, Distribution and Excretion
Increased drug concentration leads to a dose-proportional increase in plasma concentration. At the maximum tolerated dose of 80 mg/m², plasma concentrations in all patients exceeded 63 nM (mean IC50 of 17-DMAG in 60 human tumor cell lines of the NCI) in less than 24 hours. At this dose, the mean peak concentration (Cmax) reached 2680 nmol/L. Excretion is primarily via the kidneys and biliary tract. In mouse studies, the recovery rate of the drug excreted in urine 24 hours after administration was 10.6%–14.8% of the administered dose, with no metabolism observed. At the maximum tolerated dose of 80 mg/m², the mean volume of distribution (Vd) was 385 L. At a dose of 80 mg/m², the mean clearance was 18.9 L/hr.

---

Metabolism/Metabolites
Avespicycin undergoes a redox cycle catalyzed by purified human cytochrome P450 reductases (CYP3A4/3A5) to produce quinones and hydroquinones. It can also form a glutathione conjugate at position 19 of the quinone ring. However, in vivo and in vitro studies have shown that avespicycin is poorly metabolized in humans.

---

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

---

Solubility and Formulation: 17-DMAG exhibits better solubility than 17-AAG and is available in an oral formulation, which facilitates administration and may improve patient compliance. [2]
Pharmacokinetic Properties: In phase 1 studies in hematologic malignancies, intravenous 17-DMAG demonstrated a half-life of approximately 24 ± 15 hours. The maximum concentration (Cmax) of 291 ng/mL (approximately 471 nM) was achieved at doses below the maximum tolerated dose, corresponding to concentrations showing cytotoxicity in this study. [2]
Oral Formulation: An oral formulation of 17-DMAG has demonstrated similar pharmacologic properties to the intravenous compound and can provide continuous HSP90 inhibition. At the recommended phase 2 dose, 2-fold HSP90 inhibition was observed, similar to the intravenous formulation. [2]

Protein binding is reported to be extremely rare.

---

Dose-Limiting Toxicities: In phase 1 studies in acute myelogenous leukemia, dose-limiting toxicities included acute myocardial infarction and elevation of troponin at the highest dose level (32 mg/m²). These toxicities were not observed at doses achieving the maximum concentration of 291 ng/mL (approximately 471 nM), which corresponds to concentrations showing significant CLL cytotoxicity in this study. [2]
Selective Toxicity Profile: 17-DMAG demonstrates selective cytotoxicity toward CLL cells with minimal toxicity to normal T cells and NK cells, suggesting it may be less immunosuppressive than other currently used CLL therapies. [2]

[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 macrocyclic compound, a derivative of geldanamycin, in which the methoxy group on the benzoquinone moiety is replaced by a 2-(N,N-dimethylamino)ethylamino group. It is an Hsp90 inhibitor. It is a secondary amino compound, a tertiary amino compound, an anisomycin class compound, a 1,4-benzoquinone class compound, and a carbamate compound. Functionally, it is related to geldanamycin. Alvespimycin is a derivative of geldanamycin and also a heat shock protein (HSP) 90 inhibitor. It has been used in clinical trials for the treatment of various solid tumors as an antitumor drug. Compared to tanspimycin, the first HSP90 inhibitor, alvespimycin exhibits several pharmacologically desirable properties, such as reduced metabolic stability, decreased plasma protein binding, increased water solubility, higher oral bioavailability, reduced hepatotoxicity, and stronger antitumor activity.
Avespicycin has been reported to be found in Cullen corylifolium and Trichosanthes kirilowii, and relevant data exist.
Avespicycin is an analogue of the antitumor benzoquinone antibiotic geldmycin. Avespicycin binds to HSP90, a molecular chaperone protein that assists in protein assembly, maturation, and folding. Subsequently, the function of Hsp90 is inhibited, leading to the degradation and depletion of its substrate proteins, such as kinases and transcription factors involved in cell cycle regulation and signal transduction.
Drug Indications

Its use as an antitumor drug for the treatment of solid tumors, advanced solid tumors, or acute myeloid leukemia is under investigation.
Mechanism of Action

Avespicycin inhibits HSP90 and its regulation of the proper folding and function of many cell signaling proteins (called Hsp90 substrate proteins). These substrate proteins, also known as oncoproteins, include Her-2, EGFR, Akt, Raf-1, p53, Bcr-Abl, Cdk4, Cdk6, and steroid receptors. They are involved in cellular signaling pathways that drive cell proliferation and inhibit apoptosis. They are frequently overexpressed or mutated in tumors, promoting cancer progression and treatment resistance. Avesmycin exerts its anticancer activity by disrupting the molecular chaperone function of Hsp90 and inducing proteasome degradation of oncoproteins. Studies have shown that it reduces the levels of CDK4 and ERBB2.

---

Pharmacodynamics
Avesmycin exerts its antitumor activity by inhibiting HSP90, thereby targeting proteasome-degrading substrate proteins, including oncogenic kinases such as BRAF. Studies have shown that administration of this drug leads to the depletion of oncogenic substrate proteins and may induce the expression of HSP70 (HSP72). It exhibits higher selectivity for tumor tissues than normal tissues. A study also reported that, in a preclinical model of human osteosarcoma, avesmycin enhanced the inhibitory effect of imetelstat on telomerase.

---

Background and Development: 17-DMAG (alvespimycin) is a derivative of geldanamycin and a water-soluble analog of 17-AAG (tanespimycin). It was developed to overcome the poor solubility and delivery difficulties of 17-AAG while maintaining HSP90 inhibitory activity. [2]
Mechanism of Action - NF-κB Inhibition: 17-DMAG is the first therapeutic agent shown to target both NF-κB activating kinases IKKα and IKKβ in primary CLL cells. By depleting these HSP90 client proteins, it inhibits both the classic (IKKβ-mediated) and alternative (IKKα-mediated) NF-κB signaling pathways, leading to downregulation of antiapoptotic target genes (BCL2, MCL1) and caspase-dependent apoptosis. [2]
Clinical Relevance in CLL: The constitutive activation of NF-κB in CLL cells, coupled with overexpression of BCL2 and MCL1, may create "oncogene addiction" that renders CLL cells particularly sensitive to 17-DMAG compared to normal lymphocytes. The ability to block microenvironmental survival signals (CD40L, CpG) further supports its clinical potential. [2]
Clinical Development: Based on the data presented, a clinical study of 17-DMAG in CLL was initiated through the Cancer Therapy Evaluation Program of the National Cancer Institute. The oral formulation offers a more convenient route of administration while maintaining biologic activity. [2]

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.

---

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