Monitoring the biochemical changes occurring to human keratinocytes exposed to solar radiation by Raman spectroscopy

Solar radiation exposure is recognised to be a significant contributor to the development of skin cancer. Monitoring the simultaneous and consecutive mechanisms of interaction could provide a greater understanding of the process of photocarcinogenesis. This work presents an analysis of the biochemical and morphological changes occurring to immortalised human epithelial keratinocyte (HaCaT) cell cultures exposed to simulated solar radiation (SSR). Cell viability was monitored with the aid of the Alamar Blue assay, morphological examination was done with haematoxylin and eosin staining (H&E) and changes to the biochemical constituents (nucleic acids and proteins) as a result of the radiation insult were demonstrated through a combination of Raman microspectroscopy and multivariate analysis of spectral patterns. The spectral results suggest that SSR induces changes to the conformational structure of DNA as an immediate result of the radiation, whereas alteration in the protein signature is mostly seen as a later response.     

Pathophysiology of photoaging of human skin: focus on neutrophils.

UV-induced skin damage is the result of a complex cascade of events. Many studies have focused on the skin effects induced by UV-B or UV-A separately. Recently a UV-source that emits UV-B and UV-A together in a ratio comparable to daily sunlight has been introduced: i.e. solar simulated radiation (SSR). By exposing human skin type I-III to erythematogenic doses of UV (> or =1 MED) emitted by a SSR source we have noticed that: (a) neutrophils are initially the main infiltrating cell type in the dermis and (b) these infiltrating cells are the a key source of in vivo enzymatically [corrected] active enzymes such as elastase, [corrected] matrix metallo proteinases-1 and -9 (MMPs-1 and -9). These enzymes are relevant to the process of photoaging, as they break down the extracellular matrix. Keratinocytes and fibroblasts also produce matrix degrading enzymes, but to a lesser extent. Our results indicate a primary role for infiltrating neutrophils in the initial steps of photoaging. This is further supported by the observation that after exposure of skin type VI to physical doses of SSR, equivalent to those used for skin types I-III, no neutrophils and neutrophil-derived enzymatic activity were observed, explaining why skin type VI is [corrected] less susceptible to photoaging than skin types [corrected] I-III. Statement: Although most of the data, referred to, have been published, the current perspective in which they are put together is completely novel and has not been published elsewhere.     

Who, what, where and when-influences on cutaneous vitamin D synthesis

The synthesis of vitamin D in skin is a two-stage process that begins with the production of previtamin D after irradiation of 7-dehydrocholesterol by ultraviolet (UV) radiation. A number of personal and environmental factors control the probability of a suitable UV photon reaching a molecule of 7-dehydrocholesterol in the skin. These are astronomical factors that govern the solar zenith angle (SZA), and the local state of the atmosphere, determining the available solar UV radiation; skin pigmentation and age, determining competing absorbers of UV radiation and available 7-dehydrocholesterol; individual behaviour in the local surroundings, determining exposure of unprotected skin to available UV radiation. The only one of these influences that can be determined unequivocally for any situation is the SZA. The other influences must be considered either as individual case studies, or be represented by "typical" and "idealised" situations for the weather, skin and behaviour. At large SZAs there is insufficient solar UV radiation to initiate significant vitamin D synthesis. At smaller SZAs assessment of solar exposure necessary for vitamin D synthesis can only be indicative and application of any such assessment necessarily requires awareness of both self- and the local environment.     

Effect of UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts

During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.     

Epidermal ornithine decarboxylase and polyamines in mice exposed to 50 Hz magnetic fields and UV radiation

We studied the influence of magnetic fields (MFs) and simulated solar radiation (SSR) on ornithine decarboxylase (ODC) and polyamines in mouse epidermis. Chronic exposure to combined MF and SSR did not cause persistent effects on ODC activity or polyamines compared to the animals exposed only to UV, although the same MF treatment was previously found to accelerate skin tumor development. In an acute 24-h experiment, an elevation of putrescine and down-regulation of ODC activity was observed in the animals exposed to a 100-μT MF. No effect was seen 24 h after a single 2-MED (minimal erythemal dose) exposure to SSR. The results indicate that acute exposure to 50 Hz MF does exert distinctive biological effects on epidermal polyamine synthesis. Bioelectromagnetics 19:388–391, 1998. © 1998 Wiley-Liss, Inc.     

Most common skin tumours in correlation with solar ultraviolet radiation in the area of West Herzegovina

Incidence rate of skin tumours, both, non-melanoma and melanoma, is increasing nowadays. Various etiological factors are of relevance for the occurrence of the diseases. The solar radiation, as well, long-term exposure to ultraviolet (UV) radiation, have the greatest impact on development of these skin tumours. Non-melanoma skin tumours, Basal Cell Carcinoma (BCC) and Squamous Cell Carcinoma (SCC), are the most common skin tumours in humans, and usually develop on the chronically photo-exposed areas. As for the Malignant Melanoma (MM), one of the most aggressive skin tumours, the exposure to solar radiation also plays an important role. This study investigates the correlation between the skin tumours and UV radiation in the area of West Herzegovina, on the sample of 1676 patients. It presents the occurrence of skin tumours in the period from 1997 to 2003. The study investigates the incidence and the risk factors separately for every skin tumour which can be etiologically related to the occurrence of skin tumours and UV radiation: occupation, exposure to UV radiation, skin type, and family history on malignan tumours within the patient's family. The exact incidence rate of non-melanoma and melanoma skin tumours in Bosnia and Herzegovina is still unknown, for the reason that the united National Cancer Register does not exist yet.     

A novel automatic 3D stitching algorithm for optical coherence tomography angiography and its application in dermatology

Optical coherence tomography (OCT) and OCT angiography (OCTA) techniques offer numerous advantages in clinical skin applications but the field of view (FOV) of current commercial systems are relatively limited to cover the entire skin lesion. The typical method to expand the FOV is to apply wide field objective lens. However, lateral resolution is often sacrificed when scanning with these lenses. To overcome this drawback, we developed an automated 3D stitching method for creating high-resolution skin structure and vascular volumes with large field of view, which was realized by montaging multiple adjacent OCT and OCTA volumes. The proposed stitching method is demonstrated by montaging 3 × 3 OCT and OCTA volumes (nine OCT/OCTA volumes as one data set with each volume covers 2.5 cm × 2.5 cm area) of healthy thin and thick skin from six volunteers. The proposed stitching protocol achieves high flexibility and repeatable for all the participants. Moreover, according to evaluation of structural similarity index and feature similarity index, our proposed stitched result has a superior similarity to single scanning protocol in large-scaled. We had also verified its improved performance through assessing metrics of vessel contrast-noise-ratio (CNR) from 2.07 ± 0.44 (single large-scaled scanning protocol) to 3.05 ± 0.51 (proposed 3 × 3 sub-volume stitching method).     

p53-independent apoptosis in UV-irradiated mouse skin: possible inhibition by 50 Hz magnetic fields

Our recent results suggest that 50 Hz magnetic fields (MF) enhance ultraviolet (UV)-induced tumorigenesis in mouse skin. The aim of the present experiment was to study suppression of apoptosis as a possible mechanism for MF effects on skin tumorigenesis. Another aim was to test the importance of a UV and MF exposure schedule, particularly the role of MF exposure prior to UV irradiation. Female mice were exposed to a UV dose of 2 human MED and to 100 microT MF of 50 Hz, using the following exposure schedules: group 1 sham MF 24 h, UV 1 h, sham MF 24 h; group 2 sham MF 24 h, UV 1 h, MF 24 h; group 3 MF 24 h, UV 1 h, MF 24 h. Lamps emitting simulated solar radiation (SSR) were used for UV irradiation. Skin samples were analysed for apoptosis, expression of the p53 gene, activity of the enzyme ornithine decarboxylase (ODC) and polyamine concentrations. A significantly (p = 0.017) lower number of apoptotic cells was measured in group 2 compared to group 1. A similar but not statistically significant (p = 0.064) decrease was also detected in group 3. No p53 expression was detected in any sample. The levels of ODC and putrescine did not differ significantly between the UV-only and UV and MF-exposed groups. Spermidine and spermine levels were significantly (p = 0.014 and 0.014, respectively) lower in group 3 than in group 1, but no decrease was observed in group 2. Our findings suggest that SSR induces p53-independent apoptosis in mouse skin and that the apoptotic response may be inhibited by exposure to MF. The exposure schedule did not alter the MF effect. The results do not support a causal role for polyamines in MF effects on apoptosis.     

Oxidative stress–mediated photoactivation of carbazole inhibits human skin cell physiology

Prolonged exposure of the earth's surface to the sun's ultraviolet radiation may result in various skin diseases and cataract. Carbazole (CBZ), as a polycyclic-aromatic hydrocarbon (PAH), is blended with a five-member nitrogen-containing ring. It is found in cigarette smoke, coal, eye kohl, tattoo ink, and wood combustion and affects various types of flora and fauna. Our findings suggest that CBZ generates reactive oxygen species (ROS) like through type-I photodynamic reaction and causes phototoxicity in the human keratinocyte cell line (HaCaT), which has been proved by mitochondrial dehydrogenase and neutral red uptake assays. CBZ induces single strand DNA damage. We have investigated the involvement of the apoptotic pattern of cell death and confirmed it by cytochrome C release from mitochondria and caspase-9 activation. Similarly, photo-micronuclei formation was associated to CBZ-induced phototoxicity. The results of this study strongly support that the upregulation of bax, cyto-C, apaf-1, casp-9 and down regulation of bcl2, keap-1, nrf-2, and hmox-1 genes cause apoptopic cell death. Downregulation of antioxidant genes showed a significant amount of ROS generation by photosensitized CBZ. Therefore, the current study will be a step forward to safeguard human beings from sunlight-induced photosensitive CBZ prolonged exposure.     

Optical coherence tomography-based non-invasive evaluation of premalignant lesions in SKH-1 mice

Non-invasively diagnosis of actinic keratoses (AK) is important for preventing cutaneous squamous cell carcinoma (cSCC). Optical coherence tomography (OCT) can be used to detect the cross-sectional skin micromorphology with sufficient resolution and imaging depth. It has the capability to reveal the changes in skin microstructure during the development of AK. Therefore, OCT can serve as a tool for diagnosing AK. This study explores the feasibility of OCT in evaluating the structural changes in mouse skin at the different stages following exposure to ultraviolet radiation. The performance of OCT is compared with histology, the gold standard in this context. The imaging results demonstrate that a wave-shaped irregular dermo-epidermal junction (DEJ), as well as the continuous thickening of the epidermis, are useful diagnostic parameters for diagnosing AK. Histological examinations confirm these observations. These findings emphasize the need for effective skin protection or medical treatment once changes in the DEJ and epidermis are detected. OCT shows strong potential for non-invasive evaluation of such changes and AK development, and can be used for the prevention of cSCC without the necessity of taking biopsies.     

Hyperspectral imaging and characterization of allergic contact dermatitis in the short‐wave infrared

Short‐wave infrared hyperspectral imaging is applied to diagnose and monitor a case of allergic contact dermatitis (ACD) due to poison ivy exposure in one subject. This approach directly demonstrates increased tissue fluid content in ACD lesional skin with a spectral signature that matches the spectral signature of intradermally injected normal saline. The best contrast between the affected and unaffected skin is achieved through a selection of specific wavelengths at 1070, 1340 and 1605 nm and combining them in a pseudo‐red‐green‐blue color space. An image derived from these wavelengths normalized to unaffected skin defines a “tissue fluid index” that may aid in the quantitative diagnosis and monitoring of ACD. Further clinical testing of this promising approach towards disease detection and monitoring with tissue fluid content quantification is warranted.     

Inhibition of UV radiation-induced DNA damage by a 5-methoxypsoralen tan in human skin

Previously untanned buttock skin of 4 volunteers (skin type II; tan with difficulty as they sunburn easily) was treated with various sunscreen preparations and solar--simulated radiation (SSR) or SSR alone for 2 weeks. One week later, the treatment sites were challenged with a DNA-damaging dose of SSR--twice the minimal erythema dose (2 MED). Skin biopsy samples were assayed for the levels of unscheduled DNA synthesis (a measure of DNA damage), melanin distribution, and skin thickening. 5-Methoxypsoralen-containing sunscreen preparations plus SSR or SSR alone induced melanogenesis and increased the stratum corneum thickness, but only the former regimen afforded a high degree of protection against subsequent SSR-induced DNA damage. 5-Methoxypsoralen-free sunscreen preparations plus SSR induced negligible tanning, skin thickening, and photoprotection. These findings are relevant to the risk-benefit analysis of sunscreen preparations, especially in skin type II, as they provide evidence that a 5-methoxypsoralen-induced tan is protective against the DNA-damaging effects of solar UV radiation, and thus has the potential to reduce the carcinogenic risk of exposure to such radiation.     

Factors that influence the cutaneous synthesis and dietary sources of vitamin D

The major sources of vitamin D for most humans are casual exposure of the skin to solar ultraviolet B (UVB; 290-315 nm) radiation and from dietary intake. The cutaneous synthesis of vitamin D is a function of skin pigmentation and of the solar zenith angle which depends on latitude, season, and time of day. In order to mimic the natural environment of skin to sunlight exposure, we therefore measured serum 25-hydroxyvitamin D levels in volunteers with different skin types following repeated UV irradiation. Because melanin pigment in human skin competes for and absorbs the UVB photons responsible for the photolysis of 7-dehydrocholesterol to previtamin D3, we also studied the effect of skin pigmentation on previtamin D3 production in a human skin model by exposing type II and type V skin samples to noon sunlight in June when the solar zenith angle is most acute. Vitamin D is rare in food. Among the vitamin D-rich food, oily fish are considered to be one of the best sources. Therefore, we analyzed the vitamin D content in several commonly consumed oily and non-oily fish. The data showed that farmed salmon had a mean content of vitamin D that was approximately 25% of the mean content found in wild caught salmon from Alaska, and that vitamin D2 was found in farmed salmon, but not in wild caught salmon. The results provide useful global guidelines for obtaining sufficient vitamin D3 by cutaneous synthesis and from dietary intake to prevent vitamin D deficiency and its health consequences, ensuing illness, especially, bone fractures in the elderly.     

Diseased thyroid tissue classification in OCT images using deep learning: Towards surgical decision support

Intraoperative guidance tools for thyroid surgery based on optical coherence tomography (OCT) could aid distinguish between normal and diseased tissue. However, OCT images are difficult to interpret, thus, real-time automatic analysis could support the clinical decision-making. In this study, several deep learning models were investigated for thyroid disease classification on 2D and 3D OCT data obtained from ex vivo specimens of 22 patients undergoing surgery and diagnosed with several thyroid pathologies. Additionally, two open-access datasets were used to evaluate the custom models. On the thyroid dataset, the best performance was achieved by the 3D vision transformer model with a Matthew's correlation coefficient (MCC) of 0.79 (accuracy = 0.90) for the normal-versus-abnormal classification. On the open-access datasets, the custom models achieved the best performance (MCC > 0.88, accuracy > 0.96). Results obtained for the normal-versus-abnormal classification suggest OCT, complemented with deep learning-based analysis, as a tool for real-time automatic diseased tissue identification in thyroid surgery.     

Adaptation to the UV-induced suppression of phagocytic activity in murine peritoneal macrophages following chronic exposure to solar simulated radiation.

Exposure of certain strains of mice to ultraviolet radiation (UVR) is known to suppress both local and systemic immune responses, including a reduction in the phagocytic activity of peritoneal macrophages. However, in many instances, the immunological effects have been observed following a single or a limited number of doses of UVR from sources containing a higher proportion of UVB than that emitted by the sun. The first aim of the present study was to establish whether a single exposure of C3H/HeN mice to solar simulated radiation (SSR) suppressed the ability of the peritoneal macrophages to phagocytose opsonised sheep red blood cells. The mice were irradiated with SSR from Cleo Natural lamps and a single dose of 31.9 J cm(-2) was found to be the minimal dose for significant suppression of macrophage phagocytic activity. Such a dose did not modulate the surface expression of I-A(k), CD11b, CD86 or FcgammaRII/III (CD32/16) on the macrophages. The second aim was to assess whether repeated SSR exposures with a dose below the minimal immunosuppressive dose affected macrophage activity and, if so, to test for photoadaptation by repeated exposures followed by a single, normally immunosuppressive dose of SSR, and then assaying the macrophage activity. Groups of mice were irradiated on each of 2, 10 and 30 days with 14.9 J cm(-2) SSR, followed in some instances by a single additional exposure of 31.9 J cm(-2) on the same day as the last irradiation. The phagocytic activity of the peritoneal macrophages was tested 24 h later. It was reduced by 32%, 18% and 4% respectively after 2, 10 and 30 repeated exposures to SSR, and by 39%, 21% and 7% respectively after 2, 10 and 30 repeated exposures plus the additional higher dose at the end. Thus, although the macrophage activity was initially suppressed by the SSR, photoadaptation of this immune parameter occurred following repeated exposures.     

UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G₁-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer.     

Drugs that target early stages of Onchocerca volvulus: A revisited means to facilitate the elimination goals for onchocerciasis

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of “prophylactic” drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These “prophylactic” drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC₅₀ and IC₉₀ that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

Onchocerca volvulus is an obligate human parasite and the causative agent for onchocerciasis, which is a chronic neglected tropical disease prevalent mostly in the sub-Saharan Africa. In 2017, 20.9 million people were infected, with 14.6 million having skin pathologies and 1.15 million having vision loss [1]. The socioeconomic impact of onchocerciasis and the debilitating morbidity caused by the disease prompted the World Health Organization (WHO) to initiate control programs that were first focused on reducing onchocerciasis as a public health problem, and since 2012, the ultimate goal is to eliminate it by 2030 [2]. Over the years, WHO sponsored and coordinated 3 major programs: The Onchocerciasis Control Programme (OCP), the African Programme for Onchocerciasis Control (APOC), and the Onchocerciasis Elimination Program of the Americas (OEPA). Since 1989, the control measures depended on mass drug administration (MDA) annually or biannually with ivermectin, which targets the transmitting stage of parasite, the microfilariae [3–5]. However, several issues have been identified with the current MDA programs including the need to expand the treatment to more populations depending on baseline endemicity and transmission rates [2,6]. Moreover, it became apparent that alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed [2]. Ivermectin has only mild to moderate effects on the adult stages of the parasite [7–9], and there are communities in Africa where the effects of ivermectin are suboptimal [10]. It is also contraindicated in areas of Loa loa co-endemicity [11], as well as in children under the age of 5 and in pregnant women. By relying only on MDA with ivermectin, the most optimistic mathematical modeling predicts that elimination will occur only in 2045 [12].To support the elimination agenda, much of the recent focus has been on improving efficacy outcomes through improved microfilariae control with moxidectin and the discovery of macrofilaricidal drugs that target the adult O. volvulus parasites [13–18]. We posit that the addition of “prophylactic” drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may also hasten the time for elimination and support achieving a sustained elimination of onchocerciasis. These “prophylactic” drugs will target the infective third- (L3) and fourth-stage (L4) larvae of O. volvulus and consequently prevent the establishment of new infections not only in the uninfected individuals but also in the already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin) [19,20], and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development [21].Prophylaxis of onchocerciasis is not a new concept. In the 1980s, once ivermectin was introduced as a “prophylactic” drug against the filarial dog heartworm, Dirofilaria immitis [22], its prophylactic effects were also examined in Onchocerca spp. In chimpanzees, a single dose of ivermectin (200 μg/kg) was highly protective (83% reduction in patent infections) when given at the time of the experimental infection and tracked for development of patency over 30 months. It was, however, much less effective (33% reduction in patent infections) when given 1 month postinfection with the L3s, at which time the L4s had already developed [23]. Moreover, monthly treatment with ivermectin at either 200 μg/kg or 500 μg/kg for 21 months completely protected naïve calves against the development of O. ochengi infection as compared to untreated controls, which were 83% positive for nodules and 100% positive for patency [24]. When naïve calves exposed to natural infection were treated with either ivermectin (150 μg/kg) or with moxidectin (200 μg/kg) monthly or quarterly, none of the animals developed detectable infections after 22 months of exposure, except 2 animals in the quarterly ivermectin treated group which had 1 nodule each; in the non-treated control group, the nodule prevalence was 78.6% [25]. These prophylactic studies in calves exposed to natural infections clearly demonstrated that monthly or quarterly treatments with ivermectin and/or moxidectin over 22 months were highly efficacious against the development of new infections. When ivermectin was administered in a highly endemic region of onchocerciasis in Cameroon every 3 months over a 4-year period, it resulted in reduced numbers of new nodules (17.7%) when compared to individuals who were treated annually. This recent study suggests that ivermectin may have also a better prophylactic effect in humans when administered quarterly [26].Importantly, moxidectin, a member of the macrocyclic lactone family of anthelmintic drugs, also used in veterinary medicine like ivermectin [20], was recently approved for the treatment of onchocerciasis as a microfilaricidal drug in individuals over the age of 12 [20]. In humans, a single dose of moxidectin (8 mg) appeared to be more efficacious than a single dose of ivermectin (150 μg/kg) in terms of lowering microfilarial loads [17]. Modeling has shown that an annual treatment with moxidectin and a biannual treatment with ivermectin would achieve similar reductions in the duration of the MDA programs when compared to an annual treatment with ivermectin [27].In our efforts to identify macrofilaricidal drugs, we tested a selection of drugs for their ability to inhibit the molting of O. volvulus L3 to L4 as part of the in vitro drug screening funnel [13,28–31]. With some being highly effective, we decided to also examine the effects of the known MDA drugs and those already in clinical development for macrofilaricidal effects on molting of L3 and the motility of L4 (S1 Text) as potential “prophylactic” drugs. When ivermectin and moxidectin were evaluated, we found that both drugs were highly effective as inhibitors of molting: IC₅₀ of 1.048 μM [918.86 ng/ml] and IC₉₀ of 3.73 μM [2,949.1 ng/ml] for ivermectin and IC₅₀ of 0.654 μM [418.43 ng/ml] and IC₉₀ of 1.535 μM [985.3 ng/ml] for moxidectin (Table 1 and S1 Fig), with moxidectin being more effective than ivermectin. When both drugs were tested against the L4, we found that both drugs inhibited the motility of L4s after 6 days of treatment: Ivermectin had an IC₅₀ of 1.38 μM [1,207.6 ng/ml] and IC₉₀ of 31.45 μM [27,521.9 ng/ml] (Table 1 and S1 Fig), while moxidectin had an IC₅₀ of 1.039 μM [665.4 ng/ml] and IC₉₀ of approximately 30 μM [approximately 19,194 ng/ml] (Table 1 and S1 Fig). Interestingly, when the treatment of L4 with both drugs was prolonged, the IC₅₀ values for the inhibition of L4 motility on day 11 with ivermectin and moxidectin were 0.444 μM and 0.380 μM, respectively. Significantly, from the prospect of employing both drugs for prophylaxis against new infections with O. volvulus, moxidectin (8 mg) has an advantage as it achieves a maximum plasma concentration of 77.2 ± 17.8 ng/ml, is metabolized minimally, and has a half-life time of 40.9 ± 18.25 days with an area under the curve (AUC) of 4,717 ± 1,494 ng*h/ml in healthy individuals [32], which covers the experimental IC₅₀ achieved by moxidectin for inhibiting both L3 molting and L4 motility, and the IC₉₀ for L3s. In comparison, ivermectin reaches a maximum plasma concentration of 54.4 ± 12.2 ng/ml with a half-life of 1.5 ± 0.43 days and an AUC of 3,180 ± 1,390 ng*h/ml in healthy humans [33], which only covers the IC₅₀ for inhibiting molting of L3 and motility of L4. We therefore reason that based on the significantly improved pharmacokinetic profile of moxidectin and its efficacy against both L3 and L4 larvae in vitro (Table 1), it might have a better “prophylactic” profile than ivermectin for its potential to interrupt the development of new O. volvulus infections, and thus ultimately affect transmission and further support the elimination of onchocerciasis. Adding to moxidectin’s significance, in dogs, it is a highly effective prophylactic drug against ivermectin-resistant D. immitis strains [19], an important attribute in the event that a suboptimal responsiveness to ivermectin treatment becomes more widespread in the onchocerciasis endemic regions of Africa. Testing the potential effect of moxidectin on the viability or development of transmitted L3 larvae was already recommended by Awadzi and colleagues in 2014 [34], when the excellent half-life of moxidectin in patients with onchocerciasis was realized. We have to acknowledge, however, that the key parameters that can predict the potency of a drug is actually a combination of exposure (drug concentrations) at the site of action and the duration of that exposure that is above the determined IC₅₀/IC₉₀. As we have access to only the AUC, half-life, and Cmax data for each of the in vitro–tested drugs, the use of plasma concentrations for predicting the anticipated potency of these putative “prophylactic” drugs in vivo has to be further assessed with care during clinical trials.Table 1Inhibition of O. volvulus L3 molting and L4 motility in vitro by the prospective prophylactic drugs and their essential pharmacokinetic parameters at doses currently used or deemed safe for use in humans.