Ignite Creation Date:
2024-05-06 @ 8:15 AM
Last Modification Date:
2024-10-26 @ 11:58 AM
Study NCT ID:
NCT02698735
Status:
COMPLETED
Last Update Posted:
2019-10-29
First Post:
2016-02-19
Brief Title:
Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa RDEB Nonsense Mutation Patients
Sponsor:
University of Southern California
Organization:
University of Southern California
Study Overview
Official Title:
None
Status:
COMPLETED
Status Verified Date:
2019-10
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Recessive dystrophic epidermolysis bullosa RDEB is an incurable devastating inherited skin disease for which there is only supportive care RDEB is due to mutations in COL7A1 gene that encodes for type VII collagen C7 the major component of anchoring fibrils AFs mediating epidermal-dermal adherence Approximately 20 of COL7A1 mutations are nonsense mutations leading to premature stop codons and a truncated C7 with diminished function The investigators demonstrated that aminoglycosides such as gentamicin readily induce premature termination codon PTC read through and produce biologically functional C7 in 22 reported COL7A1 nonsense mutations Importantly aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction Herein the investigators propose the first clinical trial of gentamicin topical and intradermal in RDEB patients with nonsense mutations that the investigators have fully characterized The milestones include increased C7 and AFs at the patients dermal-epidermal junction and absence of significant gentamicin side effects
Detailed Description:
Evaluate C7 and AF expression with topical and intradermal gentamicin treatment Topical application of gentamicin to skin lesion We will apply commercially available gentamicin 01 ointment three times a day under Tegaderm occlusion for two weeks to a target RDEB skin erosion Likewise we will apply the ointment control vehicle three times a day to a similarly sized RDEB erosion This dosage schedule is based on the successful clinical trial in CF patients that resulted in clinical efficacy without side effects19 In our in vitro cell culture studies we showed that a single dose of gentamicin 400 ugml induced C7 expression at a level of 20-40 of that seen in normal cells Figure 1A and 1B23 In our clinical trial the experimental ointment will contain 1 milligram of gentamicin per milliliter of ointment vehicle
Injection of gentamicin into the high dermis of RDEB patient skin In addition we will identify target 20 cm x 20 cm areas of unwounded intact skin within areas prone to blister formation and intradermally inject commercially available sterile gentamicin solution 40 mgml in saline A similar control area will be injected with equal volumes of saline We will inject 200 ul 8 milligrams into each site once on day 0 and once again on day 1 A single injection will be administered to the site on each of the two days The total dose will be 16 milligrams injected into the upper dermis where it will contact the patients dermal fibroblasts and basal keratinocytes In our published in vitro read-through study each 1 cm2 of cultured cells was exposed to 400 ugsml of gentamicin that showed read-through activity with no cytoxocity For the proposed intradermal study we will be using a total dose approximately 5-fold greater than the in vitro dose
Initial and follow-up Parameters Prior to any treatment the RDEB patients will be subjected to a 9 mm shave biopsy of intact skin that will be divided into 3 parts and evaluated for HE histology transmission electron microscopy and direct immunofluorescence for C7 expression At 1 and 3 months after gentamicin treatment of RDEB erosions or intact skin in blister prone areas we will biopsy the treated sites and repeat the histological ultrastructural and C7 expression evaluations For the assessment of C7 expression at the DEJ by immunofluorescence IF 5 micron cryosections will be probed with anti-C7 polyclonal antibodies to the NC1 and NC2 domains of C7 The increased expression of the NC2 domain of C7 at the DEJ of gentamicin-treated skin or erosions will serve as one major milestone in this study since it would indicate PTC read-through and restoration of a full-length C7 The third part of the biopsy will be evaluated ultrastructurally and AFs will be enumerated by computer-assisted morphometry These studies will assess if there is restoration of normal AFs Skin sections from normal human subjects will serve as positive controls while skin sections from the vehicle control site will serve as negative controls
Patient clinical assessment Skin Erosion Sites Patients will be blinded to the gentamicin and vehicle treatments of the Experimental Site and Control Site Each week the patients will assess the sites and grade their healing as follows - 1 enlargement of the erosion compared to its initial size 0 no change in the size of the erosion 1 partial healing and a smaller erosion than its initial size and 2 complete closure of the wound
Secondly baseline photographs of the erosions will be generated and the area of the erosions calculated by computer-assisted planimetry Identical assessments will be made at 1 and 3 months post treatment Therefore a second milestone for this study will be decreased surface areas of gentamicin-treated erosions compared with vehicle control-treated erosions
Evaluation of RDEB intact skin treated by intradermal injections of gentamicin Patients and Investigators will be blinded to the treatments of the Experimental Area and Control Area Each week the patient will evaluate the areas and grade them as follows - 1 new blister or erosion formation in the site and 1 no new blisters or erosions At USC visits at 1 and 3 months biopsies will be obtained from the sites and evaluated as above for the expression of C7 at the DEJ and enumeration of AFs
Evaluation of Patients Safety Patients will have baseline histories review of systems ROS vital signs including weight and physical examinations on Day 0 before treatment and at Day 1 one day after treatment and then at 1 and 3 months during their visits to USC At these same time points blood tests will be performed and include a complete blood count electrolytes liver enzymes erythrocyte sedimentation rate creatinine and BUN Creatinine clearances will be also calculated and the patients treatment sites will be evaluated for erythema edema blistering and erosions At Day 0 1 month and 3 months after treatment audiometry evaluations will be done Patients will complete a ROS questionnaire daily at home and be telephoned weekly by a USC study member inquiring about any new signs symptoms or ROS changes
E Characterization of Immune Responses to Gentamicin-Induced C7 Our study patients all express lower levels of C7 including the NC1 domain which is the most antigenic domain of C7 Therefore with the exception of patient B we doubt that reading through the patients PTC will induce a protein that is viewed as antigenic by the patients immune system Nevertheless we hope that gentamicin will generate a functional rather than non-functional species of C7 To evaluate if this change triggers an immune response and generates anti-C7 antibodies patient serum will be obtained at baseline 1 month and 3 months for evaluation of anti-C7 antibodies by salt-split IIF and ELISA If a patient develops antibodies to C7 we will then examine their skin for C7 antibody deposits by DIF
Injection of gentamicin into the high dermis of RDEB patient skin In addition we will identify target 20 cm x 20 cm areas of unwounded intact skin within areas prone to blister formation and intradermally inject commercially available sterile gentamicin solution 40 mgml in saline A similar control area will be injected with equal volumes of saline We will inject 200 ul 8 milligrams into each site once on day 0 and once again on day 1 A single injection will be administered to the site on each of the two days The total dose will be 16 milligrams injected into the upper dermis where it will contact the patients dermal fibroblasts and basal keratinocytes In our published in vitro read-through study each 1 cm2 of cultured cells was exposed to 400 ugsml of gentamicin that showed read-through activity with no cytoxocity For the proposed intradermal study we will be using a total dose approximately 5-fold greater than the in vitro dose
Initial and follow-up Parameters Prior to any treatment the RDEB patients will be subjected to a 9 mm shave biopsy of intact skin that will be divided into 3 parts and evaluated for HE histology transmission electron microscopy and direct immunofluorescence for C7 expression At 1 and 3 months after gentamicin treatment of RDEB erosions or intact skin in blister prone areas we will biopsy the treated sites and repeat the histological ultrastructural and C7 expression evaluations For the assessment of C7 expression at the DEJ by immunofluorescence IF 5 micron cryosections will be probed with anti-C7 polyclonal antibodies to the NC1 and NC2 domains of C7 The increased expression of the NC2 domain of C7 at the DEJ of gentamicin-treated skin or erosions will serve as one major milestone in this study since it would indicate PTC read-through and restoration of a full-length C7 The third part of the biopsy will be evaluated ultrastructurally and AFs will be enumerated by computer-assisted morphometry These studies will assess if there is restoration of normal AFs Skin sections from normal human subjects will serve as positive controls while skin sections from the vehicle control site will serve as negative controls
Patient clinical assessment Skin Erosion Sites Patients will be blinded to the gentamicin and vehicle treatments of the Experimental Site and Control Site Each week the patients will assess the sites and grade their healing as follows - 1 enlargement of the erosion compared to its initial size 0 no change in the size of the erosion 1 partial healing and a smaller erosion than its initial size and 2 complete closure of the wound
Secondly baseline photographs of the erosions will be generated and the area of the erosions calculated by computer-assisted planimetry Identical assessments will be made at 1 and 3 months post treatment Therefore a second milestone for this study will be decreased surface areas of gentamicin-treated erosions compared with vehicle control-treated erosions
Evaluation of RDEB intact skin treated by intradermal injections of gentamicin Patients and Investigators will be blinded to the treatments of the Experimental Area and Control Area Each week the patient will evaluate the areas and grade them as follows - 1 new blister or erosion formation in the site and 1 no new blisters or erosions At USC visits at 1 and 3 months biopsies will be obtained from the sites and evaluated as above for the expression of C7 at the DEJ and enumeration of AFs
Evaluation of Patients Safety Patients will have baseline histories review of systems ROS vital signs including weight and physical examinations on Day 0 before treatment and at Day 1 one day after treatment and then at 1 and 3 months during their visits to USC At these same time points blood tests will be performed and include a complete blood count electrolytes liver enzymes erythrocyte sedimentation rate creatinine and BUN Creatinine clearances will be also calculated and the patients treatment sites will be evaluated for erythema edema blistering and erosions At Day 0 1 month and 3 months after treatment audiometry evaluations will be done Patients will complete a ROS questionnaire daily at home and be telephoned weekly by a USC study member inquiring about any new signs symptoms or ROS changes
E Characterization of Immune Responses to Gentamicin-Induced C7 Our study patients all express lower levels of C7 including the NC1 domain which is the most antigenic domain of C7 Therefore with the exception of patient B we doubt that reading through the patients PTC will induce a protein that is viewed as antigenic by the patients immune system Nevertheless we hope that gentamicin will generate a functional rather than non-functional species of C7 To evaluate if this change triggers an immune response and generates anti-C7 antibodies patient serum will be obtained at baseline 1 month and 3 months for evaluation of anti-C7 antibodies by salt-split IIF and ELISA If a patient develops antibodies to C7 we will then examine their skin for C7 antibody deposits by DIF
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None