Status: NDA filed, on long-term hold

Organizations involved:

AGI DERMATICS, Inc. – Manuf.; R&D; Tech.; World mark.

Applied Genetics Inc. – Former

Stanford University – R&D; Tech.

University of Texas – R&D; Tech.

Description: Dimericine is a topical cream formulation of recombinant T4 endonuclease V, a DNA repair enzyme, expressed by transformed Escherichia coli (E. coli) bacteria encapsulated within liposomes (artificial lipid bi-layer membranes similar to cell membranes). The liposomal formulation is optimized for delivery of the DNA repair enzyme to keratinocytes and the stratum corneum below the outer epidermis layer of the skin as a method for reducing ultraviolet light exposure-induced DNA damage (to reverse UV-induced mutations) associated with induction of skin cancer (e.g., basal cell and squamous cell carcinomas and melanomas).

The liposomes are composed of phosphatidylcholine, phosphatidylethanolamine, oleic acid, and cholesteryl hemisuccinate in 2:2:1:5 molar ratio. The liposomes are suspended in a lotion consisting of 1% Hypan SS 201 hydrogel (from Hymedix International Inc.; Dayton, NJ) in phosphate buffered saline at a concentration of 1 µg of T4 endonuclease V encapsulated in liposomes.

Nomenclature: T4 endonuclease, liposomal [BIO]; Dimericine [TR]; T4N5 Liposome Lotion [SY]; T4 endonuclease V, recombinant liposome encapsulated [SY]; denV endonuclease V [SY]; T4-pyrimidine dimer glycosylase [SY]; T4-PDG [SY]; T4 PDG [SY]; endodeoxyribonuclease (pyrimidine dimer) [SY]; T4endV [SY]; E.C.3.1.25.1 [EC]

Biological.: Simply stated, this enzyme can identify, snip away, and repair fused pyrimidine base pairs in DNA, which frequently result from exposure to ultraviolet (UV) light. Dimericine works by delivering a purified DNA repair enzyme to the cell’s nucleus, speeding the removal of sun-induced DNA damage. Studies in cell cultures and animals indicate that T4 endonuclease V liposomes (Dimericine) increased repair of DNA damage caused by UV light. Dimericine has been shown in clinical studies to reduce the incidence of pre-malignant actinic keratosis and basal cell carcinoma (The Lancet, 357:926, 2001).

T4 endonuclease V is a DNA glycosylase with a molecular weight = ~16 kDa. The endonuclease V enzyme is produced by the denV (deny) gene of the bacteriophage T4. The first report of the purification of T4 endonuclease V and the naming of the enzyme was in 1970 (Proc. Nat. Acad. Sci., vol. 67, p. .1839-1845). The enzyme has both N-glycosylase and apurinic/apyrimidinic lyase (AP lyase) activity, and specifically nicks DNA containing pyrimidine dimers (apparently, the source for the name Dimericine).

T4 endonuclease V locates and binds to pyrimidine dimers in double-stranded DNA. The enzyme then cleaves the N-glycosylic bond of the 5’-pyrimidine of the dimer (pyrimidine dimer DNA glycosylase activity), and the endonucleolytic activity breaks the phosphodiester bond 3’ to the resulting abasic site (3’ AP lyase activity). AP lyase activity is specific for cis-syn isomers of cyclobutane pyrimidine dimers of nucleic acids induced by UV irradiation, i.e., it acts on covalently linked dimers of nucleic acids (a type of mutation) caused by UV light, including the UV component of sunlight. T4 Endonuclease V is free of detectable nonspecific exo- and endonuclease activities and RNase activity.

The target for ultraviolet light damage leading to cancer is DNA. UV light exposure results in covalently-linked photoproducts in DNA, the most prevalent being a cis-syn cyclobutane pyrimidine dimer. Dimer accumulation in skin cells causes mutations that lead to premalignant skin lesions, such as actinic keratoses, and to tumors of the skin, such as basal cell and squamous cell carcinomas and melanomas. The casual link between non-melanoma skin cancer and UV exposure from the sun has been clearly established, and sun exposure is an important causative factor in melanoma. Wavelengths of UV radiation in the middle, or UV-B (280-320 nm), range can impair immune responses in humans and animals both locally, within UV-irradiated skin, and systemically, at distant sites.

Exposure of mice to UV-B radiation interferes with the rejection of UV-induced skin cancers and the induction of delayed and contact hypersensitivity (DHS, CHS) responses initiated at unirradiated sites. These forms of immune suppression are associated with the induction of antigen-specific suppressor T lymphocytes. How UV-B radiation exerts its systemic immunosuppressive effects remains unknown.

Bacterial DNA repair systems differ significantly from repair in human cells. However, the T4 endonuclease V enzyme has the ability to enhance DNA repair in human cells as evidenced by increased UV-specific incision of cellular DNA, increased DNA repair replication, and increased UV survival after treatment with the enzyme. The enzyme catalyzes the rate limiting, first step in the removal of UV-induced DNA damage involving single strand incision of DNA at the site of damage (UVinduced pyrimidine dimers). The enzyme is commonly used as a reagent for DNA repair studies and single cell gel eletrophoresis (comet assay).

One unit of enzyme is the amount required to completely relax (convert to nicked form) 250 ng of UV-irradiated supercoiled (covalently closed circles) plasmid DNA in 30 minutes at 37°C. Another source defines one enzyme unit as the amount of enzyme that catalyzes the conversion of 0.5 µg of UV irradiated supercoiled pUC19 DNA to > 95% nicked plasmid in a total reaction volume of 20 µl in 30 minutes at 37°C. Nicking is assessed by agarose gel electrophoresis.

In Feb. 2007, AGI reported new data indicating that treatment of UVB-induced DNA damage with T4N5 enzyme significantly decreases MMP-1 secretion and increases repair of corresponding UVB-induced DNA damage to keratinocytes. The study examined T4N5’s ability to simultaneously increase repair of cyclobutane pyrimidine dimmers (CPD) in irradiated keratinocytes (NHEK) and reduce MMP-1 secretion from fibroblasts (NHDF) exposed to irradiated NHEK media. CPDs are the most common cause of UV-induced damage to skin DNA. NHEK media were exposed to 500J/m2 UVB then treated with 1 µg/mL T4N5 for 24 hours. DNA was extracted for measurement of CPD repair. T4N5 was shown to have a significant 21% increase in repair of UVB-induced CPD. Concurrently, media from the irradiated NHEK were transferred to NHDF for 48 hours. Production of MMP-1 was measured by ELISA. RNA was also extracted from the cells for determination of MMP-1 mRNA by RT PCR. T4N5 significantly reduced MMP-1 by 96% resulting in a reduction in MMP-1 expression in the NHDF exposed to the media from irradiated NHEK. The study indicates that the DNA repair enzyme T4N5 is effective in treating the MMP-1 secretions and UVB-induced DNA damage which contributes to the collagen degradation found in photodamaged and photoaged skin.

Companies.: AGI DERMATICS, Inc., originally Applied Genetics Inc., is developing, manufactures and hold worldwide marketing rights to Dimericine.

Companies selling T4 endonuclease V for reagent/experimental purposes include Trevigen, Inc. and Epicentre Biotechnologies.

Manufacture: The enzyme is purified from cultured E. coli containing a recombinant plasmid harboring the T4 phage DenV gene. As described in U.S. 5,272,079, manufacture involves culture of transformed E. coli strain SR1268 harboring the plasmid pTACdenV obtained from Dr. E. Friedberg, Stanford University, School of Medicine.

As described at small-scale in patents, E. coli harboring a plasmid with the denV structural gene under the control of the TAC promoter are grown to log phase, with denV gene expression induced by the addition of isopropyl-thiogalactopyranoside. The purification process takes advantage of specific characteristics of most DNA repair enzymes—their small size and their affinity for nucleic acids, in particular, single-stranded DNA (ssDNA). A cell lysate is prepared, concentrated and dialyzed against the buffer subsequently used in both gel filtration and DNA affinity chromatography. The pore diameter of the gel filtration media was selected to exclude from the gel most contaminating proteins, but retain the desired T4 endonuclease V protein. The cell lysate is applied to the gel filtration column and eluted isocratically. The retained proteins are applied directly to a single-stranded DNA agarose column (i.e., ssDNA covalently bound to agarose chromatography medium). This is prepared by boiling calf thymus DNA and covalently linking it to cyanogen bromide (CNBr)-activated Sepharose (Pharmacia). The column is washed and developed with a salt gradient. The eluent is monitored for optical density at 280 nm and the peak of optical density is pooled. This peak comprised the desired enzyme purified to homogeneity.

As described in example 3 of U.S. 5,272,079, purified enzyme is encapsulated in liposomes using standard methods involving egg yolk-derived phosphatidylcholine with either dicetyl phosphate or stearylamine, followed by air and vacuum drying. Other less-technical company sources, e.g., its Web site, report the liposomes are primarily composed of lecithin.

Status: An NDA was filed, apparently in 2002, seeking approval to reverse the damage to skin DNA caused by exposure to ultraviolet light. Dimericine has received orphan status for treatment of xeroderma pigmentosum. FDA requested further studies, including clinical trials in other skin cancer-susceptible groups, such as kidney transplant patients. The application remains on hold, awaiting results from further trials, and now is more targeted,seeking approval for protection against actinic keratoses in patients with xeroderma pigmentosa.

Dimericine can be used after, as well as before and during, sun exposure. This has resulted in the popular press calling it a “morningafter cream” for undoing sun damage. AGI DERMATICS has filed a trademark application for the phrase “morning-after” to describe Dimericine.

As the product nears and after its approval, many in the medical community and others can be expected to protest that prevention of skin cancer best involves avoiding sun exposure, and that Dimericine could send the wrong message that excessive sun exposure no longer needs to be avoided.

AGI expects, with FDA approval, to initiate a compassionate-use protocol to provide Dimericine at no cost to seriously ill patients with xeroderma pigmentosum.

Tech. transfer: AGI Dermatics holds a portfolio of over two dozen patents and applications covering DNA repair enzyme technology, skin therapy and liposomal dermatics. Prior to inventions by AGI, clinical applications of DNA repair enzymes were not feasible, since there was no effective way of purifying commercial quantities of enzyme, and no effective non-toxic way of administering DNA repair enzymes to living cells, e.g., using liposomes.

The broadest U.S. patent appears to be 5,190,762, “Method of administering proteins to living skin cells,” March 2, 1993, originally assigned to Applied Genetics, Inc.. This covers biologically active proteins in liposomes delivered into interior cells of the skin. Claims include, a method for administering a protein, e.g., T4 endonuclease V, into the interior of living skin cells below the skin’s stratum corneum layer using enzyme(s) encapsulated in pH-sensitive liposomes applied to the outer surface of the skin (epidermis). Pending U.S. patent applications include US 2004248230, “Stabilization of T4 Endonuclease V.”

U.S. 5,077,211; 5,272,079; and 5,296,231, each entitled “Purification and Administration of DNA Repair Enzymes”, are assigned to Applied Genetics, Inc. These claim methods for purifying DNA repair enzymes and encapsulating them in pH-sensitive liposomes for topical delivery along the lines discussed in the manufacture section above. Claims include use of molecular sieves for purification of T4 endonuclease V. The process involves culture of the transformed host cells, centrifuging the disrupted cells, collecting the supernatant, precipitating the proteins in the supernatant, chromatography using appropriately-sized molecular sieve media, contacting the resulting solution with chromatography medium with immobilized (attached) single-stranded nucleic acids (DNA), resulting in the formation of complexes with the DNA repair enzyme; and eluting the immobilized enzyme.

U.S. 5,302,389, “Method for Treating UV-Induced Suppression of Contact Hypersensitivity by Administration of T4 Endonuclease”, assigned to the University of Texas, with Dr. D.B. Yarosh, current CEO of AGI DERMATICS, as an inventor, relates to the use of DNA repair liposomes encapsulating T4 endo V to protect the immune system, particularly for prevention or treatment of UV-induced contact hypersensitivity, including sunscreens. AGI likely has exclusively licensed this and related Univ. of Texas patents.

E. coli strain SR1268 containing plasmid pTACdenV has, presumably, been licensed from Stanford University.

Trials: A Phase III study in xeroderma pigmentosum patients has been completed, with results published in Lancet (vol. 357, p. 926-9, 2001). The incidence of basal cell carcinomas was reduced by 30% and that of actinic keratoses by 68%, compared with placebo. The annualized rate of new actinic keratoses was 8.2% among the patients assigned T4N5 liposome lotion and 25.9% among those assigned placebo (difference 17.7%; p = 0.004 by Poisson modelling). For basal-cell carcinoma, the annualized rates of new lesions were 3.8% in the treatment group and 5.4% in the placebo group. No significant adverse effects were reported, and the cream was not systemically absorbed.     

Ongoing, post-NDA filing, clinical studies are concentrating on reversing skin damage in highly susceptible populations. A Phase IIb study started in Nov. 2003 is ongoing for prevention of skin cancer in renal/kidney transplant patients, who are susceptible to and have elevated rates of skin cancer, with results expected in mid-late 2006. These and other transplant recipients are at greatly increased risk of skin cancer due to the immunosuppressive therapeutics they receive. Dimericine is expected in mid-2006 to enter Phase II studies in another at-risk population – patients with a history of skin cancer or pre-skin cancer. Dimericine is expected to be effective after, as well as before and during, sun exposure, but clinical trials are not testing all of these Indications:.

Placebo lotion used in clinical trials consists of Dimericine lotion boiled to inactivate the enzyme.

Disease: Skin cancer is a serious human health problem. About 58 million Americans have skin precancers (actinic keratosis), and 1.3 million new cases of skin cancer are diagnosed each year. Most of these are directly related to sun exposure. Although sunscreen preparations are available, even the best do not fully block ultraviolet light. Furthermore, most people do not apply sunscreens correctly, sufficiently or frequently enough for them to be effective.

The incidence of non-melanoma skin cancer in the U.S is 500,000 per year, and 23,000 per year for melanoma. A fifth of Americans are expected to develop skin cancer at some point in their lifetime. Annual deaths are 2,000 and 6,000 or more, respectively. If caught early, all types of skin cancer are curable by local tumor excision. Melanomas are rarer but more deadly than other types of skin cancer—with 8,000 new cases and about 7000 deaths in each year the U.S. The incidence of all types of skin cancer is rising.

Xeroderma pigmentosum (XP) is a rare genetic (hereditary) disorder in which innate enzymes that excise and repair damaged DNA are disabled. Patients with this condition have severe photosensitivity, and develop solar damage, pigmentation abnormalities, and malignancies in sun-exposed skin at a young age. The disease occurs in 1 of 250,000 worldwide. Cells from XP patients are deficient in functional enzymes to repair ultraviolet damage to DNA. Skin cancer incidence in XP patients is 4,800 times the frequency in the general U.S. population. There is no cure, and treatment consists of avoiding sun exposure and excising of skin lesions. Death typically occurs 30 years earlier in these patients than among the general U.S. population.

Market: The current suncare product market, including sunscreens, is $4.7 billion and growing at 13% per year. An effective post-exposure prophylactic treatment, particularly if approved for over-the-counter (OTC; nonprescription) use, could capture a significant share of the market.