Trypanosoma; Ornithine decarboxylase Ornithine decarboxylase

Eflornithine (D/L-DFMO) is an inhibitor of the first enzyme involved in the manufacture of eukaryotic polyamines, ODC. ODC is irreversibly rendered inactive by eflornithine (DFMO) in both enantiomers. ODC activity is inhibited by two eflornithine enantiomers, L-eflornithine and D-eflornithine, in a concentration- and time-dependent manner. The enzyme-inhibitor complexes formed by D-eflornithine, L-eflornithine, and eflornithine have inhibitor dissociation constants (KD) values of 28.3±3.4, 1.3±0.3, and 2.2±, respectively. 0.4 micrograms. For D-eflornithine, L-eflornithine, and Eflornithine, the inhibitor deactivation constants (Kinact) for the irreversible process were 0.25±0.03, 0.15±0.03, and 0.15±0.03 min-1, respectively. L-or D-eflornithine treatment of human colon tumor-derived HCT116 cells lowers cellular polyamine levels in a concentration-dependent manner [1]. The L-enantiomer has a 20-fold greater affinity for the target enzyme ornithine decarboxylase [2]. Enantiomers have varied potencies in vitro. Additionally, it seems that L-eflornithine is more efficient against B. gambiae parasites grown in culture [2].

Compared to D-eflornithine, the more potent L-eflornithine is found in significantly lower amounts in plasma and cerebrospinal fluid (CSF). L-eflornithine's typical plasma concentration is 52% of the D-enantiomer's concentration. L-eflornithine and D-eflornithine had typical oral clearance rates of 17.4 L/h and 8.23 L/h, respectively [2].

Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clinical applications.[1]

This study aimed to characterize the stereoselective pharmacokinetics of oral eflornithine in 25 patients with late-stage Trypanosoma brucei gambiense sleeping sickness. A secondary aim was to determine the concentrations of L- and D-eflornithine required in plasma or cerebrospinal fluid (CSF) for an efficient eradication of the T. brucei gambiense parasites. Patients were randomly allocated to receive either 100 (group I, n=12) or 125 (group II, n=13) mg/kg of body weight of drug every 6 h for 14 days. The concentrations of L- and D-eflornithine in the plasma and CSF samples were measured using a stereospecific liquid chromatographic method. Nonlinear mixed-effects modeling was used to characterize the plasma pharmacokinetics. The plasma concentrations of L-eflornithine were on average 52% (95% confidence interval [CI], 51, 54%; n=321) of the D-enantiomer concentrations. The typical oral clearances of L- and D-eflornithine were 17.4 (95% CI, 15.5, 19.3) and 8.23 (95% CI, 7.36, 9.10) liters/h, respectively. These differences were likely due to stereoselective intestinal absorption. The distributions of eflornithine enantiomers to the CSF were not stereoselective. A correlation was found between the probability of cure and plasma drug exposure, although it was not more pronounced for the L-enantiomer than for that of total eflornithine. This study may explain why oral treatment for late-stage human African trypanosomiasis (HAT) patients with racemic eflornithine has previously failed; the more potent L-enantiomer is present at much lower concentrations in both plasma and CSF than those of the D-enantiomer. Eflornithine stereoselective pharmacokinetics needs to be considered if an oral dosage regimen is to be explored further.[2]

Absorption, Distribution and Excretion
Following oral administration of efornithine, the peak plasma concentration (Cmax) of efornithine is reached at 3.5 hours post-administration (Tmax). Food (high-fat and high-calorie) does not affect the Cmax and AUC (area under the concentration-time curve) of efornithine. Crushing the tablet and adding it to a standard pudding mixture has no effect on the exposure to efornithine (Cmax and AUC6h). Under clinical use conditions, for women with excess facial hair, the mean transdermal absorption of the 13.9% (w/w) efornithine cream formulation, after single or multiple administrations, is less than 1% of the radioactive dose. Clinical use conditions include shaving within 2 hours prior to administration of the radiolabeled dose, as well as other forms of facial hair removal such as shaving, plucking, or tweezing. Steady state is reached within four days with twice-daily administration. Under clinical use conditions, in 10 women with excess facial hair (n=10), applying 0.5 g of cream twice daily (total dose 1.0 g/day; equivalent to 139 mg of anhydrous efornithine hydrochloride) resulted in steady-state Cmax, Ctrough, and AUC12hr of approximately 10 ng/mL, 5 ng/mL, and 92 ng hr/mL, respectively, expressed as anhydrous free base of efornithine hydrochloride. Under steady-state conditions, with twice-daily application of 0.5 g of cream (total dose 1.0 g/day), the dose-normalized peak concentration (Cmax) and daily systemic exposure (AUC) of efornithine are expected to be approximately 100-fold and 60-fold lower, respectively, than with a once-daily oral dose of 370 mg. This compound is not metabolized and is primarily excreted unchanged in the urine. The volume of distribution (Vz/F) of efornithine is 24.3 L. The clearance (CL/F) of efornithine is 5.3 L/h. Under clinical use conditions, in female patients with facial hirsutism, the mean transdermal absorption of efornithine after a single or multiple doses of the 13.9% (w/w) cream formulation was less than 1% of the radioactive dose. Clinical use conditions included shaving within 2 hours prior to application of the radiolabeled drug. Apart from other methods of facial hair removal such as cutting, plucking, or tweezing, under clinical use conditions, in women (n=10) with excess facial hair, after twice-daily application of 0.5 g of the cream (total dose 1.0 g/day; equivalent to 139 mg of anhydrous efornithine hydrochloride), the steady-state Cmax, Ctrough, and AUC12hr, expressed as free base of anhydrous efornithine hydrochloride, were approximately 10 ng/mL, 5 ng/mL, and 92 ng/mL, respectively.
At steady state, compared to 370 mg daily, the dose-normalized peak concentration (Cmax) and daily systemic exposure (AUC) of efflunitine were estimated to be reduced by approximately 100-fold and 60-fold, respectively, by twice-daily application of 0.5 g of cream (total dose 1.0 g/day). Oral administration once daily.
Eflunitine is not metabolized and is excreted unchanged in the urine.
For more complete data on the absorption, distribution, and excretion of efflunitine (8 metabolites), please visit the HSDB record page.

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Metabolism/Metabolites
This compound is not metabolized and is primarily excreted unchanged in the urine.

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Biological Half-Life
The terminal plasma elimination half-life of efflunitine is 3.5 hours, and the apparent steady-state plasma half-life is approximately 8 hours.
The apparent steady-state plasma t1/2 of efflunitine is approximately 8 hours.

Effects During Pregnancy and Lactation
◉ Overview of Lactation Use
Mothers received daily intravenous infusions of 400 mg/kg efflunitine for 7 days without any serious adverse effects on breastfed infants. Topical efflunitine is poorly absorbed and therefore unlikely to enter the infant's bloodstream, and is unlikely to cause any adverse effects on breastfed infants.
◉ Effects on Breastfed Infants
In the Democratic Republic of Congo, a cohort study of 33 infants followed hospitalized mothers taking nifurulimus who were breastfeeding (feeding extent not specified). 30 mothers completed 30 doses of oral nifurulimus (15 mg/kg/day), and all mothers received 14 intravenous infusions of efflunitine (400 mg/kg/day) for 7 days for the treatment of human African trypanosomiasis (sleeping sickness). On average, breastfeeding mothers were concurrently taking four other medications, including amoxicillin, ciprofloxacin, metronidazole, sulfamethoxazole/trimethoprim, aspirin, and diclofenac (1 case each); hydrocortisone, promethazine, and quinine (2 cases each); levamisole (6 cases); sulfadoxine-pyrimethamine (8 cases); aminopyrine (13 cases); acetaminophen (16 cases); and mebendazole (17 cases). No serious adverse events were reported in any of the breastfed infants.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.
Protein binding
Effornithine does not specifically bind to human plasma proteins.

[1]. Qu N, et al. Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine. Biochem J. 2003 Oct 15;375(Pt 2):465-70.
[2]. Jansson-Löfmark R, et al. Enantiospecific reassessment of the pharmacokinetics and pharmacodynamics of oral eflornithine against late-stage Trypanosoma brucei gambiense sleeping sickness. Antimicrob Agents Chemother. 2015 Feb;59(2):1299-307.

See also: Eflornithine (note moved to).

C6H12F2N2O2

182.16848

182.086

C, 39.56; H, 6.64; F, 20.86; N, 15.38; O, 17.56

66640-93-5

Eflornithine;70052-12-9;Eflornithine hydrochloride hydrate;96020-91-6;L-Eflornithine monohydrochloride;69955-42-6;Eflornithine hydrochloride;68278-23-9

6992039

solid powder

1.3±0.1 g/cm3

347.0±42.0 °C at 760 mmHg

163.7±27.9 °C

0.0±1.6 mmHg at 25°C

1.462

0.29

3

6

5

12

166

1

C(C[C@@](C(F)F)(C(=O)O)N)CN

VLCYCQAOQCDTCN-ZCFIWIBFSA-N

InChI=1S/C6H12F2N2O2/c7-4(8)6(10,5(11)12)2-1-3-9/h4H,1-3,9-10H2,(H,11,12)/t6-/m1/s1

(S)-2,5-diamino-2-(difluoromethyl)pentanoic acid

L-Eflornithine; L-alpha-Difluoromethylornithine; L-EFLORNITHINE; 66640-93-5; (S)-2,5-diamino-2-(difluoromethyl)pentanoic acid; EFLORNITHINE, (S)-; DTXSID00880061; (2S)-2,5-diamino-2-(difluoromethyl)pentanoic acid; 66640-93-5 (L-isomer); (-)-2-Difluoromethylornithine; (-)-2-Difluoromethylornithine

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.)