Antiviral; DNA polymerase

CMX001, an orally active lipid conjugate of cidofovir, is 50 times more active in vitro against herpes simplex virus (HSV) replication than acyclovir or cidofovir.[2]
In the 21st century we are faced with the potential use of natural or recombinant VARV and MPXV as biological weapons, and the emergence of human MPXV. Such an occurrences would require therapeutic and prophylactic intervention with antivirals. Cidofovir, an antiviral approved for the treatment of cytomegalovirus retinitis in AIDS patients, has activity against poxviruses, but must be administered intravenously and is associated with nephrotoxicity. An ether-lipid analogue of CDV, CMX001 (HDP-CDV), has potent antiviral activity against a range of DNA viruses including poxviruses, excellent oral bioavailability and minimal nephrotoxicity. [1]

CMX001 and CDV are equally efficacious at protecting mice from mortality following high ectromelia virus doses (10,000 x LD(50)) introduced by the intra-nasal route or small particle aerosol. Using CMX001 at a 10mg/kg dose followed by 2.5mg/kg doses every other-day for 14 days provided solid protection against mortality and weight loss following an intra-nasal challenge of (100-200) x LD(50) of ectromelia virus. Furthermore, complete protection against mortality was achieved when administration was delayed until as late as 5 days post-infection, which is 3-4 days prior to the death of the untreated controls. This therapeutic window would be equivalent to intervening during the rash stage of ordinary smallpox.[1]
These studies compared the efficacy of CMX001 to acyclovir in BALB/c mice inoculated intranasally with HSV types 1 or 2. CMX001 was effective in reducing mortality using doses of 5 to 1.25 mg/kg administered orally once daily, even when treatments were delayed 48-72 h post viral inoculation. Organ samples obtained from mice treated with CMX001 had titers 3-5 log(10) plaque-forming units per gram of tissue lower than samples obtained from mice treated with acyclovir, including 5 different regions of the brain. Detectable concentrations of drug-related radioactivity were documented in the central nervous system of mice after oral administration of (14)C-CMX001. These studies indicate that CMX001 penetrates the blood-brain barrier, is a potent inhibitor of HSV replication in disseminated infections and central nervous system infections, and is superior to acyclovir[2].

The strain of HSV-1 utilized was E-377and the strain of HSV- 2 was MS. The origin of these viruses has been reported previously. HFF cells were prepared as primary cultures from freshly obtained newborn human foreskins. Virus pools were prepared and quantified in primary rabbit kidney cells for use in vitro and in vivo. Culture medium for both cell lines was MEM with Earle’s salts containing 10% FBS and 2μM L-glutamine, 100 units/ml penicillin and 25 mM gentamicin[2].

BSC-1 cells (ATCC CCL 26) were grown in Eagle's minimum essential medium (MEM) containing 10% fetal clone III, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. A plaque-purified isolate of the MOS strain of ECTV (ATCC VR-1374) designated MOS-3-P2 was propagated in an African green monkey kidney cell line, BSC-1 (Chen et al., 1992). Virus was purified through a sucrose cushion as described elsewhere (Moss and Earl, 1998). Virus infectivity was estimated as described previously (Wallace and Buller, 1985). Briefly, virus suspensions were serially diluted in PBS + 1% sera, absorbed to monolayers for 1 h at 37 °C, and overlaid with a suspension of 1% carboxyl methyl cellulose in DMEM + 5% fetal clone III. After 4 days at 37 °C, virus plaques were visualised and virus inactivated by the addition to each well of 0.5 ml of a 0.3% crystal violet/10% formalin solution[1].

Antiviral compounds[1]
Solutions of CDV and CMX001 were prepared fresh prior to each experiment by dissolving the compounds in sterile, distilled water, and stored at 4 °C over the course of the experiment.
Aerosol challenge[1]
Mice were exposed to aerosolized ECTV suspended in MEM using a nose-only inhalation exposure system (NOIES) equipped with a 1-jet BioAerosol Nebulizing Generator, and operated within a class 2 biological safety cabinet. The NOIES was operated with a primary air pressure of 20 psi giving 1.5 l/min flow rate to the aerosol chamber (without secondary air), a virus suspension flow rate of 0.5 ml/min, and a system operating pressure of ∼−0.5 in vacuum relative to the outside atmospheric pressure. The quantity of virus delivered to the mice over the course of exposure was estimated by multiplying the concentration of virus in the aerosol (C A expressed in PFUs) by the total volume (V M) of air respired by a mouse of given body weight over the exposure time using Guyton's formula for minute volumes administered to rodents (Guyton, 1947). This presented virus dose is likely an upper limit as it assumes that the entire virus challenge was optimally aerosolized and completely taken up on inhalation.
Intra-nasal challenge[1]
Mice were anesthetised with 0.1 ml/10 g body weight of ketamine HCl (9 mg/ml) and xylazine (1 mg/ml) by intraperitoneal injections. Anesthetised mice were laid on their dorsal side with their bodies angled so that the anterior end was raised 45° from the surface, a plastic mouse holder was used to ensure conformity. ECTV was diluted in PBS to the required concentration and slowly loaded into each nare (5 μl/nare). Mice were subsequently left in situ for 2–3 min before being returned to their cages.
At indicated times following exposure to ECTV, groups of mice were treated by gavage with 0.1 ml sterile, distilled water (placebo) or water containing the desired concentration of CMX001. CDV was delivered by an intraperitoneal injection at the desired dose. This treatment was repeated as described throughout the results. To determine infectious viral titres, mice were sacrificed at 4, 6, and 8 days post-challenge, and lung, spleen, and liver tissues and nasal wash were isolated. Tissue was ground in PBS (10%, w/v), frozen and thawed three times, and sonicated for 20 s. Virus infectivity (PFU/ml) in tissue homogenates was estimated by titration on BSC-1 monolayers. Arithmetic means were calculated for PFU/ml values above the limit of detection (102 PFU/ml). Remaining mice were observed for clinical signs of disease (morbidity) and mortality. Moribund mice were euthanized.

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Antiviral compounds[2]
CMX001 was suspended in 0.4% carboxymethylcellulose (CMC) for oral delivery to mice. ACV was weighed and suspended in sterile water for oral treatment of mice. Compounds were prepared in a 0.2 ml volume which was administered once daily (CMX001) or twice daily (ACV). Treatments for efficacy evaluations were administered to mice for 7 consecutive days beginning 24-72 h post viral inoculation by oral gavage using doses of 5, 2.5 and 1.25 mg/kg of CMX001 administered once daily or doses of 120, 60 or 30 mg/kg of ACV given twice daily at approximately 12 h intervals. For pathogenesis experiments, treatments began 24 h post viral inoculation and were administered for 7 consecutive days using 5 mg/kg of CMX001 or 100 mg/kg of ACV. These doses were selected based upon the results obtained in the mortality experiments.
Experimental infections[2]
Mice were manually restrained for intranasal inoculations using a total volume of 0.04 ml/mouse containing an approximate LD90 of either HSV-1, E-377 or HSV-2, strain MS. For these studies, the inoculum contained 4.4 × 104 pfu/mouse for HSV-1 or 1.1 × 105 pfu/mouse for HSV-2. For mortality experiments, animals were evaluated at least once daily for 21 days and four times daily during peak occurrence of clinical neurological signs so that mice could be humanely euthanized prior to death. Pathogenesis studies were performed for both HSV-1 and HSV-2 in order to compare the effect of CMX001 and ACV on viral replication of both viral types in target organs of mice. Three mice each from vehicle- and drug-treated groups were euthanized on days 1, 3, 5, 7 or 10 post inoculation for collection of lung, liver, spleen, kidney, olfactory bulbs, cerebral cortex, pons/medulla, diencephalon, cerebellum and trigeminal ganglia. Organ samples were pooled by tissue type and homogenized in a 10% w/v suspension, and frozen until assayed for virus. Virus titers were determined by plating of tissue homogenates on HFF cells and plaques were enumerated after three days incubation. The trigeminal ganglia were collected individually and co-cultured directly on primary rabbit kidney (RK) cells with N’ N’ dimethylbisacetamide for detection of latent virus as described previously. Briefly, the ganglia were minced, placed onto tissue culture cell monolayers and monitored for viral cytopathic effects for 3 weeks. Ganglia were transferred weekly onto fresh RK cells with new media.

[1].Efficacy of therapeutic intervention with an oral ether-lipid\nanalogue of cidofovir (CMX001) in a lethal mousepox model. Antiviral\nRes. 2008 Jan;77(1):39-49.

[2]. Efficacy of CMX001 against herpes simplex virus infections\nin mice and correlations with drug distributionstudies. J Infect Dis.\n2010 Nov 15;202(10):1492-9.

[3]. CMX001 (1-O-hexadecyloxypropyl-cidofovir) inhibits\npolyomavirus JC replication in human brain progenitor-derived\nastrocytes. Antimicrob Agents Chemother. 2011 May;55(5):2129-36.

[4]. CMX001 to prevent cytomegalovirus disease in\nhematopoietic-cell transplantation. N Engl J Med. 2013 Sep\n26;369(13):1227-36

[5].The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus. Antiviral Res. 2016 Jan;125:71-8.

C27H52N3O7P

561.69148

561.354

C, 57.73; H, 9.33; N, 7.48; O, 19.94; P, 5.51

444805-28-1

Solid powder

5.5

156.93

OC[C@H](CN1C=CC(N)=NC1=O)OCP(O)(OCCCOCCCCCCCCCCCCCCCC)=O

WXJFKKQWPMNTIM-VWLOTQADSA-N

InChI=1S/C27H52N3O7P/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-19-35-20-16-21-37-38(33,34)24-36-25(23-31)22-30-18-17-26(28)29-27(30)32/h17-18,25,31H,2-16,19-24H2,1H3,(H,33,34)(H2,28,29,32)/t25-/m0/s1

3-(hexadecyloxy)propyl hydrogen ((((S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl)oxy)methyl)phosphonate

CMX001; BCV; CMX 001; HDP-CDV; CMX-001; HDPCDV

2934.99.9001

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)

Typically soluble in DMSO (e.g. > 10 mM)

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.)