BARRIERS AGAINST DRUG DELIVERY TO THE FOLLICLES | Kickoff

BARRIERS AGAINST DRUG DELIVERY TO THE FOLLICLES

15 May

Several  sites  are  present within the  follicle  that  represent potentially significant targets for  drug delivery, but  these  may  be  limited by  the  structural  aspects of the  hair  follicle  and  its  chemical  environment (14,15). Getting the  drug to these sites is also limited by the chemical  nature of the drug, for example, hydrophobic or hydrophilic. Aspects of the hair follicle structure that may impede drug delivery are the following.

1.    The glass membrane surrounding the entire follicle and the keratinous layers of the  inner  and  outer  sheaths may  physically restrict the  passage of chemical agents deep  within the follicle.

2.    Sebum  flow  into  the  hair  follicle  may  represent a  physical barrier to  drug delivery from some formulations. The effect of sebum composition on delivery of compounds into  the follicles is not known. It is, however, likely  that  in the sebum-filled  follicle,  efficient   drug  delivery, and   pharmacological  activity would depend on the interaction of drug and  sebum and  the physicochemical properties of the vehicle.

3.    Optimum particle size requirements may  also be required to traverse the hair follicle. It was found (16) that delivery of polystyrene beads  into the hair follicle was  size-dependent. Beads  between 5 and  7 mm in diameter were  optimally deposited  deep   within the  follicle,  whereas  smaller or  larger   beads   were more  likely to be localized in the SC and  skin surface.

ROLE OF SEBUM IN THE FOLLICULAR ROUTE

Sebaceous  glands connected to  the  hair  follicle  by  ducts  release  sebum into  the upper third of the  follicular  canal,  creating an  environment enriched in neutral, nonpolar lipids.  Among mammals, sebum composition varies  considerably. It has been reported that the sebum-enriched environment of the hair follicle that invagi- nates  the epidermis into the dermis could  provide significant lipoidal pathway for the  transport of  large  lipophilic molecules. Chapter 15 shows   a  comparison  of sebum  composition among  different species   commonly  used   in  percutaneous absorption studies (Table 2 in that chapter). Wide variations in sebum composition among  species   should  be  considered  before   extrapolating  data   from   animal to  human  studies. However, knowledge of  sebum lipid  components and  their physical chemical   interactions helps   us  to  consider how  a  penetrating  vehicle will react upon entrance into the follicular  canal.

The use  of vehicles  that  are compatible with  sebum to selectively transport drug via the transfollicular pathway is certainly not a new  concept (17). The basis for  understanding a  vehicle’s  effect  on  the  delivery into  the  sebum-rich areas, such  as the  hair  follicle, appears to be fully  explained by conventional solubility properties. The  Hildebrand coefficients  for  model   sebum compositions demon- strate  that sebum is overall  a nonpolar, oily material, with  a Hildebrand coefficient of approximately 7.5 to 8.0 cal1/2/cm3/2. Some authors believe  (6) that  many  polar vehicles   used   in  topicals, vehicles   such   as  water, propylene  glycol  (PG),  and ethanol would be too polar  to be readily soluble  in sebum. Hence,  to deliver rela- tively  polar  drugs, if the  vehicle  is not  specifically  designed to solubilize sebum, there  would be little  chance  to effectively  deliver the drug to the deeper portions of the  hair  follicle.  Hydrophilic or  marginally hydrophobic drugs would not  be

expected to  be  highly  compatible with  the  sebum. For  hydrophobic drugs, the transport of the drug is predominantly follicular  and  is brought about  by cotran- sport  of the drug dissolved in the oil phase  into and  across  the PSUs (17). The effi- ciency  of such  a follicular  enhancement in drug transport would depend on the solubility of the drug in the vehicle  and  the compatibility of the vehicle,  with  the sebum-rich lipid  environment within the  follicles.  The deposition of a drug into sebaceous glands follows  the  rank  order  suggested in Table  1 (17). Thus,  sebum therefore may  represent a significant physical barrier to drug delivery from  some formulations.

It has been reported that the highest pilosebaceous penetration occurred with vehicles  that  were  mixtures of various solvents, interface active  agents, coupling agents, and  solubilizers (18). Studies  identified isopropyl myristate, glyceryl  dilau- rate  (GDL),  and  polyoxyethylene-10-stearyl ether  (POE-10)  as  being  effective  at delivering drugs into  the  pilosebaceous duct  because  of their  compatibility with sebum. Studies  have  suggested that  the  follicular  delivery may  be dependent on the  physicochemical properties  of the  drug and/or vehicle  (19,20). The  lipoidal environment of  the  follicular   canal  may  favor  certain   drugs and  manipulating formulation factors,  and  thereby may  enhance delivery of vehicles  and  drugs by the  follicular   delivery  route.   Solvents,   which   interact  with   sebum,  thus   were opening the passageway for drug deposition within the follicle.

Since sebum is a mixture of different components, it is important to character- ize it. It is useful  to know  how  variations in the  components, their  carbon  chain length, and  the ratios  of unsaturation to saturation affect the physical but  thermal behavior of sebum. This will provide direction on how to manipulate these proper- ties with  topical  delivery systems. The thermal behavior of sebum in response to these  variables has  been  reviewed in Chapter 15. Vehicles  that  alter  this  behavior have  also been reviewed in that  chapter and  in Motwani et al. (20). These are dis- cussed later  in this chapter in relation to drug delivery.

MODELS USED TO STUDY FOLLICLES

A major  limitation in elucidating the  follicular pathway is the  current lack of an adequate pharmacokinetic model  that  can clearly  distinguish transfollicular from transepidermal percutaneous absorption. Animal models and  human models can be used  to study follicular  delivery. The  experiments can  be in vivo  or  in vitro. These  models can  further be  divided into  models for  studies of  diseases and models to investigate follicular delivery. To date,  the  following models (Table  2) have  been  used  and  details on  each  model  are  given  after  the  table,  along  with studies using  these  models in the next section.

Rabbit Ear  Model

Kligman and Kwong (21) proposed this model  for studying the comedogenic poten- tial of substances commonly found in cosmetic  formulations. This model  is very well-suited for assessing comedogenic compounds that are encountered in the etio- pathogenesis of acne  vulgaris, acne  cosmetica, chloracne, cutting oil acne,  pitch acne, pomade acne, and  tar acne (22). The procedure involves applying a comedo- lytic substance using  a glass rod over the undersurface during a two-week period. After the animal is sacrificed,  skin is sampled up to the level of the cartilage and  is immersed in 608C water for two  minutes, after  which  the epidermis is peeled off, with   the  follicular   extensions  still  attached. The  comedones  can  be  observed under the microscope. For quantitative studies, the number of comedones can be counted and  compared to comedones induced by other  comedogenic substances.

Rhino Mouse Model

This model  was  used  extensively by Kligman and  Kligman (23). The skin  of the rhino   mouse, which   is  an  allelic  of  the  hairless mouse, contains deep   dermal cysts  and  a huge  number of utriculi filled  with  keratinous debris that  resembles comedones. The horny cells do  not  contain P. acnes, but  like in human acne,  the sebaceous glands shrink as the  pseudocomedones  enlarge. They  concluded that the  rhino  mouse is a suitable model  for assessing chemicals that  effect epithelial differentiation (retinoids) or that promote loss of cohesion between horny cells (des- caling agents).  In the procedure, formulations are applied to the dorsal trunk of the mouse for a specified period and then the skin sections  are histologically examined. The procedure involves application of the test substances to the entire  dorsal trunk for a specified time.

Macaque Monkey Model

This  model   has  been  used   to  study drugs affecting   hair  growth. This  species exhibits  baldness due  to hormonal and  genetic  factors,  which  is similar to andro- genic  alopecia (19). The stumptailed macaque monkey model  exhibits  a species- specific  frontal   scalp  baldness that  coincides with   puberty at  a  rate  of  nearly

100% in both  sexes.

Uno  and  Kurata (24) investigated the  effects  of topically applied minoxidil and  diazoxide on  hair  growth in  bald  macaque monkeys and  they  studied the activity   of  5-a  reductase  inhibitors in  nonbald  monkeys.  Hair   and   follicular growth were  evaluated by  obtaining phototrichograms (gross  photographs and images  of frontal  scalps)  after closely  clipping the hairs  and  folliculograms (4 mm punch biopsies embedded in paraffin, serially  sectioned and  stained). The rate  of DNA  synthesis in the follicular  cells was  also determined. The phototrichograms revealed a conversion of short  vellus  hair to long terminal hairs,  and  the folliculo- grams  showed enlarged follicles. The DNA synthesis studies indicated an increased rate of follicular  cell proliferation. This model  therefore appears to have promise for further work  related to human androgenic alopecia.

Hamster Ear  Model

Matias  and  Orentreich (25) examined this model  by planimetry of peeled off skin instead of routine vertical  histological sections.  This model  has  been  proposed as a model  system for human sebaceous glands because of the  similarities in mor- phology and  in turnover time (26). In this case, each sebaceous gland  unit  consists of an infundibular canal,  several  large  lobulated acini with  6 to 20 layers  of sebo- cytes, and  one centrally located  piliary unit.  Male hamsters are large, and  the size of their  sebaceous glands is similar to that  of humans. Microscopic observation of the sebaceous glands from  biopsies detects pilosebaceous drug deposition.

Rat  Model

This model  has  been  used  extensively by Illel et al. (27). The isolation procedure produces both  follicle-free  and  follicular  skin.  The  hairless rat  model  is chosen because the  sebaceous glands  density  and   size  are  closely  related to  those  of human forehead skin.  The  process  involves inducing anesthesia to  the  animals, after  which  the  animals are  treated by immersion of the  back  in 608C  water for one  minute. The  epidermis is  removed from  this  treated area.  The  subsequent healing is monitored histologically and  transepidermal water loss measurements (TEWLs) are done.  After  9 to 10 weeks,  the epidermis and  horny layer  regrow as a continuous layer,  without the  pilosebaceous structures, and  the  TEWL returns to  normal, thereby indicating normal barrier properties. In  this  model, the  SC looks  normal, but  the  epidermis looks  hyperplastic. The  permeation is done  in vitro   to  see  the   differences  between  follicle  and   follicle-free   skin.   However, aside  from  the  pain  and  suffering inflicted  on the  animal and  the  time  involved in  developing the  follicle-skin,   there   is  the  question of  structural composition and   permeability  behavior  of  the   newly   regrown  tissue   compared  with   the normal epidermis of hairless rat  skin  and  whether the  comparison between the two  tissue  types  is valid.  A hairless rat has  also been  used  without the treatment mentioned earlier,  with  autoradiography to give a visual  impression of the follicu- lar route  (28).

Guinea Pig

Wahlberg (29) used  hairy  and  nonhairy skin  of guinea pigs  to  demonstrate the delivery into the follicles. For the hairy  skin, the back of the guinea pig was  used, whereas for the  nonhairy skin,  the  area  behind the  ears  of guinea pigs,  which  is completely devoid of hair  follicles and  of sebaceous and  sweat  glands, was  used.

However, the physiological differences between the two  areas  make  it difficult  to justify  its use. Also, in some  cases, different strains of guinea pigs (hairy  and  hair- less) (30) have been used  to investigate the importance of follicles. However, direct comparison due  to differences in species  is difficult.

Human Model

The human skin contains acne-prone areas  with  numerous huge  sebaceous follicles—an  ideal   site   for   acne   vulgaris  and   other   such   follicular   diseases. The  human model  can  be used  for both  investigating follicular  diseases as well as follicular delivery. The human skin is generally investigated by the noninvasive cyanoacrylate technique or the skin biopsy  technique, the details of which  are given in the next section.

METHODS TO INVESTIGATE DEPOSITION  INTO THE FOLLICLES

The skin is a multilayered organ,  complex in structure and  function, composed of outer  epidermis and  inner  dermis. There  are various potential pathways for per- meation through the  SC, including the  transepidermal (or “bulk”) and  the  trans- appendageal (or “shunt”) routes. The transepidermal route  across  the continuous SC and  the epidermis comprises two routes of entry:  the intercellular spaces,  con- sisting  of bilayers of lipids,  and  the intracellular or transcellular route  through the keratin of the dead cornified cells (corneocytes). The transappendageal route  com- prises   transport via  both  the  sweat   glands, hair  follicles  with   their  associated sebaceous glands. Capillaries closely  surround both  the  follicles  and  the  sweat glands, and  rapid absorption of permeating molecules is assumed once  the  mol- ecules  have  passed through the  follicular walls.  An important aspect  to consider for  transdermal delivery is the  mechanism of penetration, that  is, the  fractional contribution  of  the  bulk   strata  versus  the  appendageal  pathway  to  the  total absorption. Some  of the  techniques, both  qualitative and  quantitative, are  sum- marized next.

Skin  Biopsy Techniques

Noninvasive Cyanoacrylate Technique

Marks and Dawber (31) expanded the ancient technique to remove horny cell layers by  presenting a  very  potent  glue—cyanoacrylate—and termed  it  skin  surface biopsy.  This  technique was  used  for excavation of follicular  contents, analogous to  the  painful squeezing of  sebaceous filaments. It  involves placing a  drop  of adhesive on a clean  region  of the  skin  and  then  gently  pressing a slightly moist glass  slide  on top  of the  area.  After  30 to 40 seconds, the  slide  is removed along with  the superficial horny layer  of SC and  follicular  casts.  Lavker  et al. (32) have investigated the earlier  events  of comedo formation in relation to the bacterial con- tents   using   this  technique.  Mills  and   Kligman (33)  used   this  model   to  assay comedolytic  substances.  Microcomedones  were   induced on  the  back  of  adult males  by  a two-week occlusive  exposure to  10% crude coal  tar.  The  test  agents were  then  applied for two  weeks  and  the reduction in the density of microcome- dones is  determined by  the  noninvasive cyanoacrylate technique. This  method was  used  to screen  anti-acne formulations. Mills et al. (34) analyzed SA levels  in sebaceous follicles  after  topically applying it in  a 2% hydroalcoholic vehicle  or gel base.

Azelaic acid (20%) (AzA) cream  was used  on human volunteers for the treat- ment of acne (35). Surface AzA was removed by washing with acetone and then col- lected  the  follicular casts  using  follicular biopsy  method and  analyzed by HPLC method. Initial  samples were  taken  in five minutes and  thereafter over  a period of five hours, five times  with  no fixed intervals.

Invasive  Technique

Often,  a  full  thickness biopsy   containing several   sebaceous follicles  is  needed. Biopsies  are  inevitable if  sebaceous glands are  to  be  investigated. It  has  been used  for human tissues  where the tissue  is used  within 24 hours of excision  (36). After  the  permeation experiments in  vitro,  the  SC is removed by  stripping and then  the  biopsies are  taken  from  the  formulation-treated skin.  The  biopsies are immersed in CaCl2 for a few hours and then the epidermis and dermis is separated. Under microscopic observation, the epidermis with  the attached PSU is removed from  the  dermis. These  sebaceous glands are  cut  off from  the  under side  of the epidermis and  then  analyzed by a suitable analytical technique.

Mechanical Separation of the  Follicles

This has been documented recently by Lieb (37). The procedure involved using  the Syrian hamster ear as a model. The scheme  involved mounting the ears, ventral side up on the diffusion cells, which  maintained the temperature at 328C. An aliquot of the test preparation was  then  applied to the skin  surface.  At predetermined time intervals, the  skin  specimens were  separated by  gentle  peeling or  scraping into three  layers:  epidermis, ventral dermis, and  dorsal dermis. The sebaceous glands in the  ventral dermis were  removed by  gentle  dragging of a dull  scalpel  across the  bottom surface  of the  layer.  The pilosebaceous material appeared as a milky material. The scraping process  was  considered complete when the skin areas  pre- viously containing glands appear empty under the microscope. The scraped sebac- eous  glands and  skin layers  were  assayed separately by scintillation counting.

Follicle-Free Models

This has been explained earlier  in the rat model. Illel and  Schafer (38) reported the first systematic, quantitative in vivo studies of follicular  delivery using this method. By employing the Franz diffusion cell method, it is possible to compare penetration of drug across  both  follicle-free  and  normal skin (30).

Skin  Autoradiography by Computerized Image Analysis

In  this  technique, the  spatial distribution of  a  radiolabeled substance within a biological  specimen is detected by exposure of the specimen to radiation-sensitive film. The main  drawback of qualitative autoradiography is that  it merely enables visualization but  not  quantification of the penetrant in the skin.  Furthermore, the signals   appearing on  the  images  generally represent the  tissue-bound residues rather  than   the   freely   diffusable  substance.  The   technique,  therefore,  only provides an indication of drug transport through the integument.

Some   workers  have   used   quantitative  autoradiography  (39).  With   this method, it is possible to visualize and  measure by means of a software program the  concentrations in  the  hair  follicle,  glands, and  various skin  layers  without

resorting to  mechanical  horizontal  sectioning. The  general procedure  involves freezing the test skin sample at 21308C, and then vertically sectioning them by cryo- stat  in order  to yield  6-mm thick  slices.  These  sections  are  then  fixated  on  glass slides,  placed in film cassettes, and  covered by tritium-sensitive autoradiography film.  Following a four-  to  seven-week exposure period, the  autoradiograms are developed. An  imaging  system  consisting of  a  video   camera,   high-resolution light  microscope, IBM-compatible computer, and  appropriate software transforms the optical  density readings of the experimental autoradiograms into drug concen- trations values  and  projects  the resultant digitized images  on a TV screen,  where they are displayed in pseudocolor mode.  This provides a more visually meaningful picture of drug localization. In addition to autoradiography, the original skin sec- tions  are stained with  hematoxylin and  eosin dye. In the final step, the instrumen- tation  is used  to  superimpose the  autoradiography image  with  the  histological image. The computerized system is then used  to quantify drug deposition densities into  drug concentration. These  values require reference to calibration graph data, which  are derived from  a separate set of experiments. In these  calibration experi- ments,  good  reproducibility was  shown between the  optical  density readings of standard sections  containing the same  drug concentration (40).

Confocal Laser Microscopy

This  method depends upon the  analysis of re-emitted light,  with  the  measuring depth dependent upon the  beam   wavelength.  However,  in  fluorescence spec- troscopy, the  remitted light  is  detected after  passing a  second monochromator, that  is, only  a specific  emission wavelength is detected. The  procedure involves application of a self-fluorescent marker in a formulation on the skin. After a prede- termined time, the diffusion cells are dismantled and the area of the skin exposed to treatment is punched out. The peeling and scraping technique previously described is used  to mechanically isolate  the sebaceous glands. The scraped glands are then suspended in  a buffer  and  sonicated in  order  to  release  the  fluorescent marker. This solution could  then  be assayed for the marker with  a fluorescent microscope. This technique has been used  quantitatively by Weiner  et al. (18).

Horizontal Sectioning of Strata

This   technique   involves   the   application   of   the   test   compound   (typically radiolabeled) to a specific  area  of skin.  After  a defined exposure time,  the  excess drug is removed. The  horny layer  is stripped off by the  consecutive application of an adhesive tape.  The underlying tissue  can then  be excised,  and  the epidermis and dermis are sectioned parallel to the skin surface  with a freezing microtome. The quantity of drug in each horny layer strip or tissue slice can be determined by using a suitable analytical technique. From  the derived data,  it is possible to generate a profile  representing drug concentration versus depth for the  investigated tissue. A disadvantage of this technique is the fact that  unless  sufficient  data  is available from  the literature about  the thickness of each skin  layer  at the required regional site, preliminary experiments are needed to assess  this.  Another major  limitation of this technique is that  it does  not yield  any  data  about  penetrant concentrations in  the  PSUs.  It  usually has  to  be  used   in  conjunction  with   other   techniques described earlier  (41).

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