Ornithine decarboxylase
Ornithine decarboxylase (ODC). L‑Eflornithine binds irreversibly to the active site of ODC, forming a stable enzyme‑inhibitor complex. The KD is 1.3+/-0.3 microM and the Kinact is 0.15+/-0.03 min-¹.

Eflornithine (D/L-DFMO) is an inhibitor of ODC, the first enzyme in eukaryotic polyamine production. Both enantiomers of eflornithine (DFMO) irreversibly inactivate ODC. Two eflornithine enantiomers (L-eflornithine and D-eflornithine) decrease ODC activity in a time- and concentration-dependent manner. The inhibitor dissociation constant (KD) values of D-eflornithine, L-eflornithine and eflornithine producing enzyme-inhibitor complexes are 28.3±3.4, 1.3±0.3 and 2.2± respectively. 0.4 µM. The inhibitor deactivation constants (Kinact) for the irreversible phase were 0.25±0.03, 0.15±0.03 and 0.15±0.03 min-1 for D-Eflornithine, L-Eflornithine and Eflornithine, respectively. Treatment of human colon tumor-derived HCT116 cells with L-eflornithine or D-eflornithine lowers cellular polyamine levels in a concentration-dependent manner [1]. Enantiomers exhibit varied potencies in vitro, with the L-enantiomer having a 20-fold greater affinity for the target enzyme ornithine decarboxylase [2]. L-eflornithine also appears to be more efficient against cultured parasites of B. gambiae [2].

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In vitro, L‑eflornithine irreversibly inhibits ODC activity, leading to a rapid decrease in intracellular polyamine levels. This inhibition blocks cell cycle progression and induces apoptosis in rapidly dividing cells, including cancer cells and parasites. The L‑enantiomer shows stereospecific inhibition; the D‑enantiomer is significantly less potent. The compound is used to study the role of polyamines in cell growth, differentiation, and apoptosis. It also affects the expression of genes regulated by polyamine levels, such as c‑Myc and p53.

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].

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In vivo, eflornithine (as the racemate) is an approved drug for the treatment of West African trypanosomiasis (sleeping sickness) and for reducing unwanted facial hair in women (hirsutism). The L‑enantiomer is not used therapeutically alone but is studied for its pharmacological effects. In animal models, L‑eflornithine depletes polyamines in tissues, inhibits tumor growth, and shows activity against parasites. It is often used in combination with other agents to enhance efficacy.

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.

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ODC enzyme activity is measured using a radiometric assay. Cell lysates or purified ODC are incubated with L‑ornithine (containing [14C]‑ornithine) and pyridoxal phosphate in Tris‑HCl buffer (pH 7.5) at 37 degC. L‑Eflornithine is added at various concentrations (0.01-1000 microM) and pre‑incubated with the enzyme for 0-60 min to allow irreversible inhibition. The reaction is initiated by adding substrate, and the released 14CO2 is trapped on filter paper soaked in hyamine hydroxide. Radioactivity is counted, and the remaining enzyme activity is calculated. The Kinact and Ki are determined from time‑dependent inactivation curves. For binding studies, radiolabeled eflornithine may be used, but typically enzyme activity assays are sufficient.

Cells (e.g., cancer cell lines, parasites) are cultured in appropriate medium and treated with L‑eflornithine at concentrations ranging from 0.1 to 1000 microM for 24-72 h. Polyamine levels are measured by HPLC after dansylation or by LC‑MS/MS. Cell viability is assessed by MTT or CellTiter‑Glo assays. Cell cycle analysis is performed by flow cytometry after propidium iodide staining. Apoptosis is evaluated by Annexin V/PI staining or caspase‑3/7 activity. ODC activity in cell lysates is measured as described above. The effects on downstream targets such as c‑Myc and p53 are examined by Western blot or qPCR.

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]

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In vivo efficacy is evaluated in mouse xenograft models or in models of trypanosomiasis. L‑Eflornithine is administered orally or intraperitoneally at doses of 100-500 mg/kg, daily or twice daily, for 7-21 days. Tumor volume or parasitemia is monitored. Polyamine levels in tissues (liver, tumor, brain) are measured at sacrifice. Pharmacokinetic studies involve collecting blood samples at various time points to measure drug concentrations by LC‑MS/MS. For trypanosomiasis, survival and parasitemia are the primary endpoints.

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.

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L‑Eflornithine monohydrochloride has molecular formula C₆H13ClF2N2O2 and molecular weight 218.63. It is highly water‑soluble. The compound is orally bioavailable, though absorption is variable and may be affected by food. It is primarily excreted unchanged in the urine, with a half‑life of 3-4 h in humans. The L‑enantiomer may have similar pharmacokinetics to the racemate, but stereoselective differences are minimal. The compound is stable under recommended storage conditions (2-8 degC).

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.

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Eflornithine is generally well‑tolerated, but common adverse effects include gastrointestinal disturbances (nausea, vomiting, diarrhea), anemia, and thrombocytopenia at high doses. Reversible hearing loss has been reported in some patients. The L‑enantiomer is expected to have a similar safety profile. The compound is not approved for use as the single enantiomer; it is for research purposes only. Standard precautions for handling cytotoxic agents should be followed.

[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.

L‑Eflornithine monohydrochloride is the L‑enantiomer of the ODC inhibitor eflornithine (DFMO). Eflornithine is an approved drug for African trypanosomiasis and hirsutism, but the L‑form is used exclusively as a research tool to investigate stereospecific enzyme inhibition and polyamine biology. It irreversibly inactivates ODC with a KD of 1.3 microM and a Kinact of 0.15 min-¹, leading to polyamine depletion and subsequent antiproliferative effects. The compound is available as a high‑purity research reagent for studying cancer, parasitic diseases, and cellular metabolism.

C6H15CLF2N2O3

236.644707918167

218.063

69955-42-6

Eflornithine;70052-12-9;Eflornithine hydrochloride hydrate;96020-91-6;Eflornithine hydrochloride;68278-23-9;L-Eflornithine;66640-93-5

16048568

Light yellow to yellow solid

4

6

5

13

166

1

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

VKDGNNYJFSHYKD-FYZOBXCZSA-N

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

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

L-DFMO monohydrochloride ; L-RMI71782 monohydrochloride; L-Eflornithine (monohydrochloride); 69955-42-6; L-Eflornithine monohydrochloride; (2S)-2,5-diamino-2-(difluoromethyl)pentanoic acid;hydrochloride; (S)-2,5-diamino-2-(difluoromethyl)pentanoic acid hydrochloride; L-DFMO (monohydrochloride); SCHEMBL1322403; L-α-difluoromethylornithine monohydrochloride

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, avoid exposure to moisture.

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

DMSO : ~200 mg/mL (~914.79 mM)
H2O : ~50 mg/mL (~228.70 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (22.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 50.0 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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Solubility in Formulation 2: ≥ 5 mg/mL (22.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 5 mg/mL (22.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 100 mg/mL (457.39 mM) (saturation unknown) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution.

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