Ornithine decarboxylase

Eflornithine is a specific, irreversible inhibitor of the enzyme ornithine decarboxylase which is thought to slow hair growth by inhibiting this enzyme in hair follicles[2].

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In vitro permeation study of eflornithine was performed using Franz diffusion cell. The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles.[3]

The only novel drug approved in the last fifty years for the treatment of human African trypanosomiasis is eflornithine. It is mostly used as a stand-by medication for Trypanosoma brucei gambiense infections that do not respond to melarsoprol [1]. When it came to decreasing the growth of facial hair in participants with excess hair, eflornithine 15% cream outperformed a placebo. Following a 24-week course of treatment, face hirsutism improved at least somewhat in 58% of eflornithine patients and 34% of placebo subjects [2]. When eflornithine cream was administered to mouse skin areas that had been microneedled beforehand, the hair growth inhibitory activity of eflornithine was markedly increased [3]. After 14 days of hypertension, eflornithine treatment of constrictive hypertensive rats led to the normalization of KCI and norepinephrine contractile strength as well as the relaxing of acetylcholine [4].

In in vitro studies using Salmonella and two strains of Saccharomyces, eflornithine did not induce mutagenic changes [1].

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In vitro permeation of eflornithine hydrochloride through mouse skin[3]
In vitro permeation assay using Franz diffusion cell apparatus was completed as previously described (Kumar et al. 2012; Kumar et al. 2011; Naguib, Kumar, & Cui 2014) using the lower dorsal skin of C57BL/6 mice. Hair was trimmed using an electric clipper 24 h before the collection of the skin. Skin was harvested, wrapped in aluminum foil, and stored at −20°C for a maximum period of one month and used whenever needed. Freezing of the skin at −20°C (without a cryo-protectant) is commonly applied in literature, and such skin samples have been used frequently for permeability studies (Stahl, Wohlert, & Kietzmann 2012). Dennerlein et al. showed that freezing and storing of freshly excised human skin for up to 30 days at −20°C does not affect the skin permeability (Dennerlein et al. 2013). Other researchers showed that when human skin was wrapped in aluminum foil and stored at −26°C, the skin retained its barrier properties for up to 6 months (Badran, Kuntsche, & Fahr 2009). After the fat layer was removed, the skin was mounted onto the Franz diffusion cells with dorsal side facing upward. The receiver compartment contained 5 ml of water and was maintained at 37°C with a Haake SC 100 Water Circulator (ThermoScientific, Wellington, NH). The hair-trimmed skin was treated with a Dermaroller® microneedle roller as previously described before it was mounted onto the Franz diffusion cells (Kumar et al. 2011; Naguib, Kumar, & Cui 2014). The skin sample was placed onto the flat surface of a balance, and the microneedle roller was rolled in four perpendicular directions over the skin surface, 5 times each for a total of 20 times, with an applying pressure of 350–400 g, which was constantly measured using the balance while the roller was rolled. The diffusion area of the skin was 0.64 cm2. The donor compartment was loaded with 4 mg of eflornithine hydrochloride in 500 μl water and covered with parafilm to prevent evaporation. After 0, 1, 3, 6, 8, and 24 h, samples (150 μl) were withdrawn from the receiver compartment and immediately replenished with fresh medium. The samples were analyzed using HPLC following a method described previously with modifications (Saravanan et al. 2009). Chromatographic analysis was carried out with an Agilent 1260 Infinity HPLC station equipped with ZORBAX Eclipse Plus C18 (5 μm, 4.6 × 150 mm) column using a acetonitrile-buffer mixture (70%:30%, v/v) as the mobile phase. The buffer was prepared by dissolving 0.68 g of potassium phosphate monobasic in 1 l of water. The flow rate was 0.8 ml/min. The detector wavelength was 210 nm.

Skin tissues were fixed with a buffered formalin (10%) solution for 24 h, washed with 0.1 M of sodium phosphate buffer (pH 7.4), dehydrated in graded ethanol, embedded in paraffin, and sectioned vertically. The sections were stained using hematoxylin-eosin (H&E) or an antibody against 5-bromo-2′-deoxyuridine (BrdU) in the Histology and Tissue Processing facility in the Dell Pediatric Research Institute at the University of Texas at Austin. Mice were injected intraperitoneally with BrdU in phosphate buffered saline (PBS, pH 7.4, 10 mM) at the dose of 100 μg/g body weight, 30 min prior to euthanasia. All skin sections were examined under an Olympus BX53 microscope [3].

In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically[3].
Female C57BL/6 mice (8–10 weeks old) were are ideal for examining the physiological actions during different hair cycle phases due to the occurrence of naturally synchronized hair cycles with cyclic pigmentation (Slominski, Paus, & Costantino 1991). Each experimental group was composed of 3–4 mice. Hair in the lower dorsal skin of anesthetized mice was either trimmed using an electric clipper, plucked using GiGi® Honee warm wax as previously described (Xiao et al. 2012), or chemically removed using Nair® lotion. The skin area where the hair was removed was then treated with the eflornithine hydrochloride 13.9% cream (~50 mg per mouse per treatment) using a spatula two times a day in an interval of at least 8 h for a maximum period of 36 days. A group of mice whose hair in the application site was trimmed using a clipper were also treated with the microneedle roller every time before the application of eflornithine cream as previously described (Kumar et al. 2012). Briefly, mice were placed onto the flat surface of a balance, and the microneedle roller was rolled over the marked skin surface, 10 times parallel to mouse length, with an applying pressure of 350–400 g as indicated on the balance. In control groups, the hair in mouse dorsal skin was removed by trimming, plucking, or chemical depilation with Nair®, but the area was not treated with the eflornithine cream. The hair re-growth was evaluated by taking digital photographs of the mouse skin areas for a maximum period of 36 days after the first application of the eflornithine cream. On the last day of the study, animals were euthanized, and skin samples were collected from the treated areas for immunohistochemical studies.[3]

Absorption, Distribution and Excretion
Following oral administrations of eflornithine, peak plasma concentrations of eflornithine (Cmax) were achieved (Tmax) 3.5 hours post-dosing. The Cmax and AUC (area under the concentration-time curve) of eflornithine were not affected by food (high fat and high calories). Administration of crushed tablets in a standard pudding admixture had no effect on eflornithine exposure (Cmax and AUC6h). The mean percutaneous absorption of eflornithine in women with unwanted facial hair, from a 13.9% w/w cream formulation, is < 1% of the radioactive dose, following either single or multiple doses under conditions of clinical use, that included shaving within 2 hours before radiolabeled dose application in addition to other forms of cutting or plucking and tweezing to remove facial hair. Steady state was reached within four days of twice-daily application. Following twice-daily application of 0.5 g of the cream (total dose 1.0 g/day; 139 mg as anhydrous eflornithine hydrochloride), under conditions of clinical use in women with unwanted facial hair (n=10), the steady-state Cmax, Ctrough and AUC12hr were approximately 10 ng/mL, 5 ng/mL, and 92 ng hr/mL, respectively, expressed in terms of the anhydrous free base of eflornithine hydrochloride. At steady state, the dose-normalized peak concentrations (Cmax) and the extent of daily systemic exposure (AUC) of eflornithine following twice-daily application of 0.5 g of the cream (total dose 1.0 g/day) is estimated to be approximately 100- and 60-fold lower, respectively, when compared to 370 mg/day once-daily oral doses.
This compound is not known to be metabolized and is primarily excreted unchanged in the urine.
Eflornithine volume of distribution (Vz/F) is 24.3 L.
The clearance (CL/F) of eflornithine is 5.3 L/h.
The mean percutaneous absorption of eflornithine in women with unwanted facial hair, from a 13.9% w/w cream formulation, is < 1% of the radioactive dose, following either single or multiple doses under conditions of clinical use, that included shaving within 2 hr before radiolabeled dose application in addition to other forms of cutting or plucking and tweezing to remove facial hair.
Following twice daily application of 0.5 g of the cream (total dose 1.0 g/day; 139 mg as anhydrous eflornithine hydrochloride), under conditions of clinical use in women with unwanted facial hair (n=10), the steady-state Cmax, Ctrough and AUC12hr were approximately 10 ng/mL, 5 ng/mL, and 92 nghr/mL, respectively, expressed in terms of the anhydrous free base of eflornithine hydrochloride.
At steady state, the dose-normalized peak concentrations (Cmax) and the extent of daily systemic exposure (AUC) of eflornithine following twice-daily application of 0.5 g of the cream (total dose 1.0 g/day) is estimated to be approximately 100- and 60-fold lower, respectively, when compared to 370 mg/day once-daily oral doses.
Eflornithine is not metabolized and is excreted unchanged in urine.
For more Absorption, Distribution and Excretion (Complete) data for Eflornithine (8 total), please visit the HSDB record page.

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

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

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Maternal intravenous eflornithine 400 mg/kg daily for 7 days did not cause any adverse serious effects in breastfed infants. After topical application, eflornithine is poorly absorbed so it is not likely to reach the bloodstream of the infant or cause any adverse effects in breastfed infants.
◉ Effects in Breastfed Infants
A cohort of 33 infants who were breastfed (extent not stated) by hospitalized mothers taking nifurtimox was followed in the Democratic Republic of the Congo. Thirty mothers took a full course of 30 doses of oral nifurtimox 15 mg/kg daily and all received 14 doses of intravenous eflornithine 400 mg/kg daily for 7 days for human African trypanosomiasis. (sleeping sickness). Nursing mothers also took a median of 4 other concomitant medications, including amoxicillin, ciprofloxacin, metronidazole, trimethoprim-sulfamethoxazole, aspirin, and diclofenac (1 patient each); hydrocortisone, promethazine and quinine (2 patients each); levamisole (6 patients); sulfadoxine-pyrimethamine (8 patients); dipyrone (13 patients); acetaminophen (16 patients); and mebendazole (17 patients). No serious adverse events were reported in any of the breastfed infants.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. Eflornithine for the treatment of human African trypanosomiasis. Parasitol Res. 2003 Jun;90 Supp 1:S49-52.

[2]. Topical eflornithine. Am J Clin Dermatol. 2001;2(3):197-201; discussion 202.

[3]. A method to improve the efficacy of topical eflornithine hydrochloride cream. Drug Deliv. 2016 Jun;23(5):1495-501.

[4]. Eflornithine alters changes in vascular responsiveness associated with coarctation hypertension. Clin Exp Hypertens. 1997 Apr;19(3):297-312.

Eflornithine hydrochloride monohydrate is the hydrochloride and hydrate of the trypanocidal drug eflornithine. It is a hydrochloride and a hydrate. It contains an eflornithine.
Eflornithine Hydrochloride is the hydrochloride form of eflornithine, a difluoromethylated ornithine compound with antineoplastic activity. Eflornithine irreversibly inhibits ornithine decarboxylase, an enzyme required for polyamine biosynthesis, thereby inhibiting the formation and proliferation of tumor cells. Polyamines are involved in nucleosome oligomerization and DNA conformation, creating a chromatin environment that stimulates neoplastic transformation of cells. (NCI04)
An inhibitor of ornithine decarboxylase, the rate limiting enzyme of the polyamine biosynthetic pathway.
See also: Eflornithine (has active moiety).

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Drug Indication
Treatment of facial hirsutism in women.

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Eflornithine is the only new molecule registered for the treatment of human African trypanosomiasis over the last 50 years. It is the drug used mainly as a back-up for melarsoprol refractory Trypanosoma brucei gambiense cases. The most commonly used dosage regimen for the treatment of T. b. gambiensesleeping sickness consists of 100 mg kg(-1) body weight at intervals of 6 h for 14 days (150 mg kg(-1) body weight in children) of eflornithine given as short infusions. Its efficacy against Trypanosoma brucei rhodesiense is limited due to the innate lack of susceptibility of this parasite based on a higher ornithine decarboxylase turnover. Adverse drug reactions during eflornithine therapy are frequent and the characteristics are similar to other cytotoxic drugs for the treatment of cancer. Their occurrence and intensity increase with the duration of treatment and the severity of the general condition of the patient. Generally, adverse reactions to eflornithine are reversible after the end of treatment. They include convulsions (7%), gastrointestinal symptoms like nausea, vomiting and diarrhea (10%-39%), bone marrow toxicity leading to anemia, leucopenia and thrombocytopenia (25-50%), hearing impairment (5% in cancer patients) and alopecia (5-10%). The drug arrests embryonic development in mice, rats and rabbits but the extent of excretion into breast milk is unknown. The mean half-life is around 3-4 h and the volume of distribution in the range of 0.35 l kg(-1). Renal clearance is about 2 ml min kg(-1) (i.v.) and accounts for more than 80% of drug elimination. Bioavailability of an orally administered 10 mg kg(-1) dose was estimated at 54%. One of the major determinants of successful treatment seems to be the cerebrospinal fluid drug level reached during treatment, and it was shown that levels above 50 micro mol l(-1) must be reached to attain the consistent clearance of parasites. Based on its trypanostatic rather than trypanocidal mode of action, it is a rather slow-acting drug.[1]

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Eflornithine is a specific, irreversible inhibitor of the enzyme ornithine decarboxylase which is thought to slow hair growth by inhibiting this enzyme in hair follicles. Percutaneous absorption of eflornithine in women with unwanted facial hair (hirsutism) was < 1% when the 15% cream was applied twice daily to a shaved 50 cm2 area of skin under the chin. In clinical studies in women with excessive, unwanted facial hair, eflornithine 15% cream was superior to placebo in reducing hair growth, as demonstrated by objective and subjective methods, after 2 to 8 weeks' treatment. After 24 weeks' treatment, 58% of eflornithine and 34% of placebo recipients had at least some improvement in facial hirsutism (for the purposes of this analysis all patients not assessed at week 24 were considered to be worse or to have no improvement). In addition, 32 versus 8% of patients were judged to be successfully treated (at least marked improvement). Hair growth returned to pretreatment rates within 8 weeks of stopping treatment. Use of a self-assessment questionnaire to assess the effect of study treatment on 6 aspects of patient well-being showed that eflornithine reduced the mean level of overall discomfort and bother by 33 versus 15% in placebo recipients. Adverse events mostly affected the skin. Only burning/stinging/tingling was markedly more common with eflornithine than with placebo.[2]

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Context: Facial hirsutism is a cosmetic concern for women and can lead to significant anxiety and lack of self-esteem. Eflornithine cream is indicated for the treatment of facial hirsutism. However, limited success rate and overall patient's satisfaction, even with a long-term and high-frequency application, leave room for improvement. Objective: The objective of this study is to test the effect of microneedle treatment on the in vitro skin permeation and the in vivo efficacy of eflornithine cream in a mouse model. Materials and method: In vitro permeation study of eflornithine was performed using Franz diffusion cell. In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically. Results and discussion: The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles. Conclusion: The integration of microneedle treatment into topical eflornithine therapy represents a potentially viable approach to increase eflornithine's ability to inhibit hair growth. Keywords: Cell proliferation; hair growth inhibition; microneedles; skin permeation; unwanted hair growth.[3]

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This study examined the temporal effects of the polyamine synthesis inhibitor eflornithine (alpha-difluoromethylornithine) on vascular responses to KCI, norepinephrine, sodium nitroprusside and acetylcholine in aortic rings from coarctation hypertensive rats. Coarctation hypertension reduced the contractile response of aortic rings to KCI and norepinephrine, increased sensitivity (reduced the EC50 value) to norepinephrine and attenuated relaxation to acetylcholine by 14 days of hypertension. Treatment of coarctation hypertensive rats with eflornithine resulted in a normalization of the contractile intensity to KCI and norepinephrine and relaxations to acetylcholine by 14 days of hypertension. Responses to sodium nitroprusside were similar in all groups at all time points. Hyperresponsiveness to norepinephrine produced by coarctation of the aorta was not affected by eflornithine. These studies indicate that normalization of vascular function can occur in the presence of significantly elevated blood pressure upon chronic administration of eflornithine. This functional normalization correlates with eflornithine-mediated regression of structural abnormalities normally associated with pressure overload hypertension.[4]

C6H15CLF2N2O3

236.6447

236.074

C, 30.45; H, 6.39; Cl, 14.98; F, 16.06; N, 11.84; O, 20.28

96020-91-6

Eflornithine;70052-12-9;L-Eflornithine monohydrochloride;69955-42-6;Eflornithine hydrochloride;68278-23-9;L-Eflornithine;66640-93-5; 96020-91-6 (HCl hydrate) 68278-23-9 (HCl); 70050-55-4 (R-isomer); 69955-42-6 (S-isomer); 66640-93-5 (L-isomer)

441361

White to off-white solid powder

1.293g/cm3

347ºC at 760mmHg

>210ºC (dec.)

163.7ºC

1.91

5

7

5

14

166

0

Cl.O=C(C(C(F)F)(CCCN)N)O.O

FJPAMFNRCFEGSD-UHFFFAOYSA-N

InChI=1S/C6H12F2N2O2.ClH.H2O/c7-4(8)6(10,5(11)12)2-1-3-9/h4H,1-3,9-10H2,(H,11,12)1H1H2

2,5-diamino-2-(difluoromethyl)pentanoic acid hydrochloride hydrate

Ornidyl; CPP-1X; RMI71782; Eflornithine hydrochloride hydrate; Eflornithine hydrochloride monohydrate; Vaniqa; Eflornithine HCl; dfmo; MDL 71,782 A; Eflornithine HCl hydrate; RMI-71782; RMI 71782; DL-Ornithine hydrochloride;

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)

H2O : ~83.33 mg/mL (~352.14 mM)

Solubility in Formulation 1: 100 mg/mL (422.58 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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