NIAID’s Evaluation of PoxiDerm against Cowpox (CV) and Vaccinia (VV) Virus
The National Institute of Allergy and Infectious Diseases (NIAID) established the AACF (Antimicrobial Acquisition and Coordinating Facility) to provide free and confidential services for suppliers interested in submitting compounds to be evaluated for antiviral activity. The purpose of this program is to accelerate new antiviral drug development. The program is designed to examine pure compounds, but on occasion will also examine extracts. Researchers nationwide submit compounds for antiviral testing to the AACF.
Viruses are a problem across the world, causing many of the diseases that afflict individuals today. Some of the most heinous and recalcitrant viruses belong to the pox viridae family of viruses. Three particularly troublesome pox viruses are: smallpox (vaccinia), which has produced significant death in the human populus in the past, but is now confined to licensed laboratory analysis (and suspected to still be a potential bioterrorism agent); cowpox (and other animal pox viruses), which afflict the large animal populations and cause much loss of viable livestock animals; and molluscum contagiosum, which is a self-limiting but highly undesirable virus that resides primarily in pediatric and AIDS patients, thought to be responsible for 5% of all skin diseases affecting children and to infect 20% of AIDS patients. The product tested here, PoxiDerm, is has been shown clinically to have activity in treating Molluscum contagiosum, but to date no antiviral confirmation of its activity against pox viruses has been confirmed.
General Approach for Determining Antiviral Activity and Toxicity for Orthopoxviruses
The approach of NIAID’s AACF for determining antiviral efficacy and toxicity of antiviral agents is to gain enough definitive information such that a compound can be taken into animal efficacy and toxicology studies and then into Phase I/II Clinical Studies. The experimental approach is based upon the following: 1) previous experience at AACF has found that a high percentage of samples submitted will not have activity or will be too toxic to evaluate. Therefore, an inexpensive, rapid assay such as a cytopathic effect inhibition assay (CPE-inhibition assay) that is semi-automated is used initially to screen out the negatives; 2) all screening assays are conducted in low-passaged human cells; 3) each assay system contains a positive control (CDV) and a negative control (ACV); 4) efficacy is demonstrated by at least two different assay systems that detect functional biologic activity; 5) efficacy against VV and CV should be confirmed using other isolates; 6) toxicity is determined using both resting and proliferating human fibroblast cells; and 7) for selected compounds, toxicity in rodent myeloid and erythroid progenitor cells is assessed.
Screening Assay Systems for Determining Antiviral Activity Against VV and CV
Compounds are initially screened for activity using the CPE assay in human foreskin fibroblast (HFF) cells. The screening assay systems utilized have been selected to show specific inhibition of a biologic function, i.e., cytopathic effect in susceptible human cells. In the CPE-inhibition assay, drug is added 1 hr prior to infection so the assay system will have maximum sensitivity and detect inhibitors of early replicative steps such as adsorption or penetration as well as later events. To rule out non-specific inhibition of virus binding to cells all compounds that show reasonable activity in the CPE assay are confirmed using a classical plaque reduction assay in which the drug is added 1 hr after infection. These assay systems also can be manipulated by increasing the pre-treatment time in order to demonstrate antiviral activity with oligodeoxynucleotides and/or peptides. By delaying the time of addition of drug after infection, information regarding which step in the virus life cycle is inhibited (i.e., early vs. late functions) can be gained. It is also important to determine the toxicity of new compounds on dividing cells at a very early stage of testing. Past research has found that a cell proliferation assay using HFF cells is a very sensitive assay for detecting drug toxicity to dividing cells and the drug concentration that inhibits cell growth by 50% (IC50).
HFF cells are the most sensitive and predictive of toxicity for bone marrow cells, thus they are the preferred cell type to be used in these assays.
Efficacy, Toxicity, and Drug Activity
In all the assays used for primary screening, a minimum of six drug concentrations is normally used covering a range of 100μg/ml to 0.03μg/ml, in 5-fold increments to obtain good dose response curves. From these data, the dose that inhibited viral replication by 50% (effective concentration 50; EC50) was calculated. The same drug concentrations used to determine efficacy were also used on uninfected cells in each assay to determine toxicity of PoxiDerm. The drug concentration that was cytotoxic to cells as determined by their failure to take up a vital stain, neutral red, (cytotoxic concentration 50; CC50) was determined as above. To determine if PoxiDerm has sufficient antiviral activity that exceeds its level of toxicity, a selectivity index (SI) was calculated according to CC50/EC50. This index, also referred to as a therapeutic index, was used to determine if a compound warrants further study.
Results were reported by Cecil D. Kwong, Ph.D., AACF Compound Screening Program Medicinal Chemist
The concentrations in the reported CPE assay were 50, 10, 2, 0.4, 0.08 and 0.016%. The reported EC50 values for both VV and CV was <0.016%. The optical density readings were higher for all concentrations tested for this compound compared to the average cell control readings in the neutral red uptake and crystal violet cytotoxicity assays, as well as the VV and CV virus controls. The CC50 for both VV and CV was >50% and the SI (selectivity index) was >3125 for each (see Table 1 and 2 below).
Table 1.CPE efficacy, toxicology, and drug activity results of PoxiDerm against Cowpox and Vaccinia virus.
Table 2. Neutral red uptake results for the toxicity of PoxiDerm in uninfected HFF cells.
PoxiDerm appears to be very active against both cowpox and vaccinia (smallpox) virus. Low doses of PoxiDerm inhibit viral replication and have not been found to be toxic to uninfected cells. The effective concentration of PoxiDerm was much lower than that of the control, indicating that this product is very effective as a treatment for cowpox and vaccinia virus.
This confirms what has been seen in the clinical use of PoxiDerm when treating Molluscum contagiosum. One of the mechanisms of action of PoxiDerm appears to be its innate ability to inhibit the pox virus, which would account at least partly for the clinical results. Future tests are recommended to examine the effectiveness of PoxiDerm against other similar viruses, as well as other, non pox-type viruses.
A Novel Topical Product, Flavokine, for the Relief of Molluscum Contagiosum Lesions in Children
Judith V. Williams, MD(1), Aimee A. Byman, MPA(1), Margene Tranter, MPA(1), Autumn N. Karwiel(2) & Gary M. Pekoe, PhD(2)
1 Division Dermatology, Children's Specialty Group, Children’s Hospital of The King’s Daughters, Norfolk, Virginia and 2 Dalos BioPharma, Virginia Beach, Virginia
Molluscum contagiosum (MC) is a superficial viral infection of skin and mucous membranes affecting about 1 in 6 children with outbreaks lasting from 0.5-5 years. Autoinnoculation may result in widespread lesion clusters. Patients with atopic dermatitis1 and immunocompromised patients are more susceptible to MC infection. Current destructive methods for treatment, such as curettage, cautery and cryotherapy, are not tolerated well by children2. Most topical products (acids, retinoids, blistering agents) act by causing local irritation in hopes of stimulating an immune response. To date, no acceptable topical product has existed for the improvement of MC lesions. Flavokine [Dalos BioPharma] is a novel botanical substance containing over 270 flavonoids and other substances. Flavokine has been shown to enhance wound healing as well as have strong anti-inflammatory, antiviral, and antibacterial abilities. Flavokine was tested by the NIAID and shown to be active against the pox family of viruses. Ten patients, ages 3-13 years, were treated for up to 12 weeks with Flavokine in a non- controlled pilot study. Patients/parents were instructed to rub the liquid onto each lesion twice daily. Subjects returned at weeks 1, 2, 4, 6, 8, 10, 12, and at 4 weeks post- treatment for lesion counts and treatment area evaluation. All patients experienced a Complete or Partial Response. In four patients, the lesions cleared completely. The six remaining patients were equally divided between lesions clearing greater than or less than 50% as compared to baseline. No unusual adverse events were reported or observed. Flavokine appears to represent an active, non-blistering, well-tolerated topical product for relief of MC lesions.
1. Dohil MA, Lin P, Lee J, Lucky AW, Paller AS, Eichenfield LF. The epidemiology of molluscum contagiosum in children. J Am Acad Dermatol 2006;54(1):47-54.
2. Scheinfeld N. Treatment of molluscum contagiosum. Dermatol Online J 2007;13(3):15. Support was provided by Dalos BioPharma.
Flavokine, a Novel Topical Antiviral for Use Against Molluscum Contagiosum in Children and Adults.
Arnold R. Oppenheim, MD(1), Amanda K. Houck-Miller, MS(2), M. Kirby Query, MS(2), Lindsey Nathaniel(2), and Gary M. Pekoe, PhD(2).
1 Virginia Beach Dermatology, Virginia Beach, VA 2 Dalos BioPharma, Virginia Beach, VA
Flavokine [Dalos BioPharma] is a novel complex botanical entity containing over 270 flavonoids and other components. Different fractions of flavokine have been associated with antiviral, antibacterial, antitumor, anti-inflammatory and enhanced wound healing activity. Mechanisms of action include direct activity against viruses and bacteria including MRSA, as well as stimulation of factors and cells within the skin through a variety of biologic pathways. Molluscum contagiosum (MC) is a common infection worldwide, and accounts for about 1% of all diagnosed skin disorders in the US. Children, most commonly under 5 years old, become infected through direct skin-to-skin contact or by touching MC-contaminated objects. In adults, MC can be a sexually transmitted disease. It is estimated that 5% of children and up to 20% of AIDS patients have MC. In healthy children and adults, the MC rash will eventually clear, but it can last 18 months or longer, and is bothersome for parents and children due to its appearance and contagiousness. MC causes a characteristic lesion/rash with one or more round, dome-shaped pink, waxy papules, with a small central indentation. They are usually 2.5 mm in diameter but can be as large as 1 centimeter in diameter. Current treatments for MC include curettage, cautery, cryotherapy, or blistering agents.
Eighty-two patients aged 18 months to 62 years who had used flavokine twice daily for MC were evaluated for their response to the agent. The mean time of application was 6 weeks. Thirty-seven patients (45%) had complete clearing of their lesions. Twenty-four patients (30%) experienced partial clearing of their lesions. Twenty-one patients (25%) had no clearing of their lesions. Two patients reported mild skin irritation that may or may not have been related to flavokine. A partial or complete response rate of 75% of MC to flavokine is very positive, as no other topical antiviral agent demonstrates this response in this troublesome albeit benign skin disease. Flavokine may have potential across a broad range of topical conditions due to its complex nature and biologic activity.
Support was provided by Dalos BioPharma.