REVIEW OF INVESTIGATIONS OF DELIVERY

15 May

REVIEW OF INVESTIGATIONS OF DELIVERY VIA THE FOLLICULAR PATHWAY
Mathematical Considerations

Scheuplein in 1967 (42) did  the  mathematical modeling for follicular  delivery of small  molecular weight electrolytes and  nonelectrolytes. He used  reported values of thickness, diffusion constant, area  of each  layer  of the  skin.  According to him, during the initial  part  of the experiment, the shunt pathway (follicular and  sweat glands) is dominant and  that introduces the lag period. At steady state, the transe- pidermal route  is dominant. The point  at which  the shunt pathway becomes equal to the bulk pathway is around 300 seconds, as per these  calculations. According to the paper, the concentration levels in the neighborhood of the ducts  and the follicles may  be very  much  higher than  in the bulk  of the epidermis in the early  stages  of diffusion, before  steady-state diffusion is achieved for small  molecules (ordinarily within the first one hour  after  application of an aqueous solution). The reason for this  behavior arises  from  the  limited area  of the  skin  surface  occupied by  these appendages, their  relatively large  diffusion constants, and  the nonlinear character of  diffusion prior   to  steady state.  Usually, measurements  of  skin  permeability done in vitro are invariably measurements of steady state because it is quite difficult to detect  concentrations within the first few minutes. However, this calculation was only  for  small  hydrophilic  molecules and  has  been  shown to  fail  many   times because it does  not take into consideration the nature of the vehicle.

This theory  was then extended in another report (43), where it was found that more  polar  steroid molecules in aqueous solutions had  a longer  lag time  than  the nonpolar ones.  The reason given  for this  was  that  the  diffusion constant of polar molecules is  smaller and  they  tend   to  prefer   the  follicular   route   as  compared with  the epidermal route,  and  hence  the long lag times  and  small  fluxes (Fig. 2).

In  still  another paper  (44),  aqueous  ibuprofen solution penetration  was monitored through  hydrated  human  skin.  The  parameters  of  the  experiment obtained were  fitted  into the equation as given  by the authors, accounting for the shunt and  bulk  pathway. It was  found that  the  shunt accounted for almost  25% of the  total  permeation at  steady state.  Also,  in  the  presence of shunt pathway, the lag time  was  eight  minutes, which  would have  otherwise been  92 minutes in the absence  of the appendages.

Studies Done to Investigate Physicochemical Properties of Drug  and Follicular Deposition

The importance of the  appendages was  shown by Kao et al. (45) in mouse skin. Three  different strains of mice were  used:  the normal-haired mice, the nonhaired mice, and the intermediate fuzzy-haired mice. The actives, benzopyrene and testos- terone, in acetone  under nonoccluded conditions were  then  applied to the excised dorsal skin  organ  culture of the  mice  and  the  permeation monitored hourly by scintillation counting for 16 hours. Some of the preparations were also investigated by fluorescence microscopy. It was concluded that the hair follicles could contribute significantly to skin  permeation, although there  were  differences in the  different strains of the mice. Testosterone permeated to a higher degree than  benzopyrene. It was  found that  for  testosterone, permeation was  rapid, peak  absorption was observed, and  peak  absorption was  higher and  faster  than   in  fuzzy-haired or hairless skin. Hence, it was concluded that testosterone that is absorbed extensively, the  transappendageal route,   played an  important role  in  the  beginning of  the

experiment, but  later  the  percutaneous absorption was  important. For benzopyr- ene,  that  was  less  well-absorbed and  peak  absorption was  not  seen  during the course  of the experiment, indicating that  the transappendageal route  is the domi- nant  route.  It is not  explained how  the  permeation took  place  for 16 hours after acetone  had  evaporated.

Skin    permeation   of   two    steroids,   hydrocortisone   (log P ¼ 1.61)   and testosterone  (log P ¼ 3.32),  in  95%  ethanol in  water was  evaluated in  vivo  on normal and  artificially damaged hairless mouse skin  in which  sebaceous glands disappeared during healing (41). The  test  compounds were  applied for  0.5, 2, and  6 hours. The results indicated that  the permeation into the dermis and  epider- mis was more through the normal skin than appendage-free skin. It was postulated that  sebaceous glands probably contributed to the  penetration of hydrocortisone and  testosterone. Testosterone, which  is a more  lipophilic molecule, was  found at a higher concentration than  hydrocortisone in the  SC. It was  also  observed that in  the  case  of normal skin,  the  epidermal and  dermal amounts of these  drugs increased with  application time,  but  this  increase was  smaller in the  case of scar skin.  It  was  hypothesized that  the  sebaceous glands acted  as  reservoir for  the steroids. In these  experiments, it was  not  mentioned whether they  were  carried out  under occluded conditions and  if the  vehicle  evaporated before  the  end  of the experiment.

In  another study done  by  Hueber et  al.  (41), percutaneous  absorption  of steroids on human skin  was  examined in vitro.  Percutaneous absorption studies on four steroids—progesterone (log P ¼ 3.87), testosterone (log P ¼ 3.32), estradiol (log P ¼ 2.49), and  hydrocortisone (log P ¼ 1.61)—were done  using  freshly  excised normal and  scar  (obtained from  abdominal and  mammary plasties) human skin. The steroids were dissolved in 95% ethanol in water and  applied. The experiments were  carried out  for eight  hours for progesterone and  testosterone and  up  to 24 hours for estradiol and hydrocortisone. It was found that permeation of the steroids was  significantly greater in  normal skin  as  compared to  scar  skin.  The  fluxes obtained for  progesterone and  testosterone were  significantly higher in  normal skin than in scar skin. The fluxes of estradiol and hydrocortisone became  significant after four hours. From the percentage absorbed f[(Normal skin  – scar skin)/normal skin]     100g,  it was  also  concluded from  the  study that  transfollicular absorption was  higher for progesterone and  testosterone, the  more  lipophilic steroids, than for  estradiol and  hydrocortisone. It  was  not  mentioned if the  experiment was

carried out under occluded or nonoccluded conditions.

Viprostol  is  a  synthetic  PGE2,  is  a  vasodilator,  and   its  deposition was monitored in  the  skin,  following application  in  different animals (mouse, rat, guinea pig, rabbit,  and  monkey) using  scintillation counting and  autoradiography (46). The active  was  incorporated in petrolatum base.  Autoradiography was  done only  for mice  and  monkeys. In mice,  the  radioactivity was  visible  in SC and  the hair  shafts  by 30 minutes. In two  hours, radioactivity was  also visible  throughout the  viable  epidermis and  only  the  hair  shaft  had  considerable radioactivity. Skin taken after 12 hours indicated the presence of radioactivity well down in the follicle and,  after 72 hours, radioactivity was visible only in the hair shaft and  hair follicle. The monkey skin also showed a similar pattern, but the time was longer.  By scintil- lation  counting, it was  concluded that  the drug was  present in the skin  for a rela- tively  long  period, following removal of the  drug from  the  application site.  The authors indicated that  there  was  formation of a significant drug depot in the skin and  that  viprostol penetrated through the follicular  route.

Rutherford and  Black (47) used  autoradiography to study the localization of germicides in guinea pig  skin.  The  deposition of two  germicides, zinc  pyride-2- thione   1 N  oxide   [zinc  pyrithione  (PTO)]  and   zirconium  pyride-2-thione  1 N oxide (zirconium PTO), from shampoos in guinea pig skin was investigated. Appli- cation  on the skin was rubbed for 10 minutes, after which  the animals were  killed and  the skin  excised.  Both germicides were  observed in the SC, hair  follicles,  and sebaceous glands. However, while  zirconium PTO was  detected in the upper epi- dermis, zinc  PTO was  not.  It was  concluded that  zinc  PTO’s solubility in sebum may  allow  it to become  localized in the hair  follicle, zirconium PTO is not soluble in  sebum, and  this  causes  it  to  penetrate the  dermis. This  suggested that  the physicochemical properties of the drug influence delivery into the PSU.

Studies Done to Investigate Vehicle Effects on  Follicular Deposition

Vehicles  Containing  Organic  Solvents

MacKee et al. (48) were among the first to investigate the nature of vehicle  influen- cing  the   deposition  of  iron,   bismuth,  sulfonamide  compounds,  and   dyes   in both  guinea pigs  and   human skin.  The  penetration was  monitored for  5,  30, and   60  minutes.  The   vehicles   investigated  were   (i)  ointment  bases,   which included lanolin,  (ii) organic  solvents, which  included PG, (iii) aqueous solutions, (iv)  mixtures of  water, surface   active  agents, and   solubilizers,  (v)  mixtures  of organic  solvents, surface  active  agents, solubilizers, and  coupling agents, (vi) mix- tures  of organic  solvents, surface  active  agents, and  solubilizers, (vii) mixtures of organic  solvents, water, surface  active  agents, and  solubilizers, and  (viii) mixtures of organic  solvents, surface  active  agents, and  water. None  of the  studies were above  one hour.  Histological examination of the skin biopsies was done  to investi- gate the distribution of the “active.” There was little or no penetration seen by the ointment bases,  or by PG, better  penetration with  the aqueous solutions with  the surface-active   agent    and    best   penetration   with    the   combination   vehicles. The authors first suggested the follicular  route  as being  important for delivery of actives.  In  this  particular study, the  time  in  which  it was  carried out  was  very short,  and  it  is  possible that  due  to  the  viscosity   of  the  oleaginous bases,  the effect was  not visible  until  one hour.

Rutherford and  Black (47) used  autoradiography to study the localization of germicides in  guinea pig  skin.  A trichlorocarbanilide (TCC)  compound in  soap vehicle  resulted in  predominantly transepidermal penetration through the  skin, whereas follicular  deposition occurred with  a nonsoap detergent. Application on the  skin  was  rubbed for 10 minutes, after  which  the  animals were  killed  and  the skin excised.  They concluded that  the vehicle  influenced the penetration route.

Montagna (49) studied penetration and local effect of vitamin A on the skin of the guinea pig. The effect of vehicles,  linoleic  acid, oleic acid, alcohol,  and  chloro- form  and  a paste  made of petrolatum, zinc  oxide,  and  talcum on  penetration  of the  0.5% vitamin A active  was  investigated.  Specimens of skin  were  in  contact with  these  agents for  10 minutes, one  hour,  and  two  hours and,  in  some  cases, four  and  eight  hours, after  which  skin  biopsies were  taken.  The penetration was tracked by the fluorescence of vitamin A. It was  found that  the penetration of the active  (dissolved in alcohol  and  chloroform) into  the  PSU was  seen  very  quickly (in 10 minutes). In oleic acid,  the  penetration to the  sebaceous ducts  took  almost two hours. With linoleic acid, it was almost  four to eight hours before fluorescence could  be detected in the sebaceous ducts.  With  the paste,  it was  even  slower.  The

author concluded that  the speed of vitamin A depended on the vehicle  used.  It is possible that  viscosity  effects played a role in delivery to the PSU.

Estradiol distribution and  penetration was  penetration of studies in rat  skin after topical  application, by high-resolution autoradiography (50). Estradiol in differ- ent  concentrations was  applied in  vehicles  dimethyl sulfoxide (DMSO),  ethylene glycol, and  sesame  oil in vivo. It was observed that  the rate of estradiol localization in the sebaceous glands was  dependent on the vehicle  and  dose.  At the end  of two hours, the  rate  of deposition of the drug into  the sebaceous glands was  more  with DMSO  as compared to ethylene glycol.  The concentration of estradiol is found to be the highest in the sebaceous glands at the end  of two hours, after  which  it starts to decrease. It was observed that  radioactivity was retained in the sebaceous glands for  24 hours or  longer  in  low  but  significant amounts  in  all  vehicles,  suggesting that  a drug depot effect may occur within the PSU.

The importance of the appendageal pathway was also observed in the percu- taneous absorption of 5% pyridostigmine bromide (hydrophilic) through various vehicles,  which  was  evaluated using  normal and  appendage-free scar  rat  skin  in vitro  during 72 hours (51). It was  found that  the drug absorbed was  higher from nerol  8% in  ethanol, followed by  azone  5% in  ethanol – PG  (90:10), followed by DMSO 10% in ethanol. PG 10% in ethanol inhibited pyridostigmine absorption as compared to the control,  which  was an ethanolic solution. In all cases, the absorp- tion  through the  appendage-free  skin  was  lower   than   the  absorption through control   skin.  The  percentage  of  appendageal pathway  was   calculated  by  the formula (1-Scar  skin  flux/normal  skin  flux)     100. It was  found that  in  the  first four hours, the appendageal absorption was most for the DMSO solution, followed by the proplylene glycol in ethanol, followed by ethanol, then  azone  solution, and then the nerol solution. At the end of 72 hours, proplylene glycol in ethanol had the highest percentage of appendageal transport, followed by DMSO, and  the  others were   not   that   significant.  The  authors  suggested  that   the   enhancers  (Nerol, azone)   affected   the   structure  of  the   epidermis,  whereas  the   other   solvents (DMSO, ethanol, and  PG) were  incorporated in the sebum and  dragged the drug into the sebaceous ducts. These experiments were done under occlusion. It was con- cluded that ethanol, DMSO, and PG in ethanol favored the transfollicular pathway, but the other  vehicles  did not. Since ethanol is primarily a lipid  solvent, it can solu- bilize sebum and allow the migration of the active in the sebaceous glands, explain- ing  why  it is primarily transfollicular. It was  concluded that  by  using  the  right vehicle  it was possible to favor  the transfollicular pathway and  target  the drug.

Radiography was  used  quantitatively by Fabin  et al. (39). Two drugs, tetra- hydrocannabinol (THC)  and  oleic acid,  were  evaluated for delivery into  hairless rat skin appendages with  different vehicles  in vivo. These vehicles  included poly- ethylene glycol  400 (PEG 400), transcutol, and  PG:ethanol (7:3). It was  found that after  two  hours, THC had  the highest penetration from  transcutol and  the lowest from PEG 400. After two hours, distribution of THC and  oleic acid from transcutol was  not  very  different. At  24  hours, the  transcutol  system had   delivered the maximum THC in the different skin  layers  and  PEG 400 delivered the lowest.  At

24 hours more, THC had been delivered in the different layers of the skin compared two  hours. It appears that  there  is a time-dependent effect in the distribution and localization of the  drug in the  follicle.  In the  same  experiments, when oleic acid was  added as  a vehicle  to  the  PG:ethanol system, the  penetration of THC  after two  hours was  much  higher with  compared to when oleic acid  was  not added. It was  concluded that  the  presence of oleic  acid  in the  delivery system applied to

the  skin  could   increase penetration  to  all  the  strata of  the  skin,  including the appendages.

The  percutaneous absorption of RA was  monitored in  haired and  hairless guinea pigs with  the expectation that  drug penetration would be lower  in hairless guinea pigs  because they  have  fewer  hair  follicles  (30). RA was  formulated in a

0.025% in an ethanolic gel formulation together with  and  without polyolprepoly- mer  (PP-2)  at  10%. In  vitro  permeation was  monitored on  haired and  hairless guinea  pigs   for   24   hours.  It   was   observed  from   penetration  profile   that hairless guinea pig  skin  was  more  permeable to RA than  haired skin  despite the lower  follicular  density. This was attributed to the different strains having different thickness of the SC and  different structure rather than  greater follicular  density. It was  suggested that  a  depot of  RA was  formed in  the  hair  follicle  of  the  hair guinea pig  due  to  the  PP-2.  The  addition of PP-2  to  the  formulation decreased the penetration of the drug. It is possible that by addition of a polymer, the viscosity of the formulation increased and  hence  there  was decrease in the penetration.

In summary, from these studies it can be concluded that vehicles  that interact with  sebum seem to be delivering the drug into the PSU. If sufficient  time is given for the experiment to run,  then  the more  viscous  materials may  help  in delivering the drug. Viscosity  of the vehicle  may  play  a role in the delivery.

Vehicles  Made  of Liposomes

Liposomes have  become  popular in the  delivery of drugs to the  PSU. In a study done  by  Li et  al.  (52), liposomes made of phosphatidylcholine (PC)-containing calcein   dye   were   investigated  for   delivery  to   the   PSU   using    mouse  skin histocultures by confocal  microscopy. It was  found that  liposomes-entrapped dye became  associated with  the hair  follicles in contrast to free dye without liposomes in 20 minutes. In a similar study (53) done  by the same authors, liposome, made of PC, mediated targeted delivery of melanin and  into  the  hair  follicles  of histocul- tured mouse skin  in 12 hours was  reported. This was  monitored by fluorescence microscopy. Similarly,   DNA  encapsulated  liposomes were  shown to  target   the hair follicle of histocultured mouse skin in 44 hours, as shown by autoradiography (54).  For  all  these   studies,  the  entrapped drug  was   separated  from   the  free drug using  gel filtration. It could  be concluded that  it takes  longer  for larger  mol- ecules  to be targeted to the PSU.

Liposomes have  been used  by Lieb et al. (18) for delivery into the PSU of the hamster ear  in vitro.  Carboxyfluorescein (CF) encapsulated in multilamellar ves- icles was prepared. The multilamellar vesicles  were  made of PC:Cholesterol (CH):Phosphatidylserine (PS) in the  ratio  of 1:0.5:0.1, respectively. Other  vehicles containing the  same  concentration of CF were  also  used,  which  included HEPES buffer  (pH ¼ 7.4), 5% PG in  HEPES,  10% ethanol in HEPES,  and  0.05% sodium lauryl sulfate   in  HEPES.  In  vitro   diffusion studies  for  24  hours,  quantitative fluorescence microscopy, and scraping technique was done  to investigate the depo- sition   into  the  follicles.  It  was  found that   the  most   intense fluorescence was observed with  the  liposomal formulations, whereas the  other  formulations were not much  different from  each other.

Liposomes made of PC:CH  and  PS at a mole  ratio  of 1.0:0.5:0.1 were  made containing 0.5% cimetidine (55) and  were  evaluated for deposition into  the  PSU of hamster ears.  Nonionic liposomes made of GDL:CH:POE-10  at a weight ratio of 57:15:28 containing cimetidine were  also  evaluated in vitro  and  in vivo.  These formulations  were   compared  with   (i)  aqueous  solution  of  pH ¼ 8.3,  (ii)  50%

alcohol  solution of pH ¼ 7.4, (iii) aqueous solution of pH ¼ 5.5, (iv) phospholipid liposome pH ¼ 5.5, (v) phospholipid  liposome pH ¼ 8.3, (vi) nonionic liposome of pH ¼ 5.5, and  (vii) nonionic liposome of pH ¼ 5.5. At pH ¼ 8.3, the drug is pre- dominately unionized. In vitro  deposition studies were  done  on excised  hamster ears when the formulation was applied for 24 hours, after which  the cells were dis- mantled and the distribution of the drug in the various strata of the skin was deter- mined. In the in vitro  studies, the aqueous solution showed significant deposition into the sebum-rich PSU of the hamster ear. In the in vivo  studies done  similarly, after 12 hours, maximum deposition in the PSU was observed by the phospholipid liposome at pH ¼ 5.5 as compared to the 50% alcohol  solution at pH ¼ 7.4 and  the nonionic liposome at pH ¼ 5.5. Therefore, when bioassay was done  and  a decrease in size of the sebaceous glands was monitored, it was found that only the hydroal- coholic solution and  the nonionic liposomes suppressed the growth of the glands. From these studies it was found that there were discrepancies in the in vivo and the in vitro  data,  and  caution needs  to be exercised about  the  activity  of the  drug in specific tissue.

Influence of  nonionic liposomal composition  on  topical  delivery of  alpha interferon (a-IFN) into PSUs was done  using  the hamster ear model  (56). The depo- sition  of hydrophilic protein, a-IFN into  the  PSU and  other  strata of the  hamster ear  12 hours after  topical  in  vivo  application from  three  nonionic formulations, a phospholipid formulation, and  an aqueous control  was  determined. The depo- sition  of cyclosporin (CsA), a hydrophobic peptide into the PSUs and  other  strata of the  ear  using  the  same  liposomal formulations and  a hydroalcoholic control, was  also  done.  The  nonionic liposome (Non-1)  was  made with  GDL,  POE-10, and  CH,  Non-2  was  made with  GDS (glyceryl  distearate) and  POE-10 and  CH, and   Non-3   was  made with   POE-10  and   CH.  The  liposomal formulation was made from  PC:CH  and  PS at a mole  ratio  of 1.0:0.5:0.1. The analysis was  done  the scintillation counting. The  deposition a-IFN of into  the  PSUs  was  in  the  order: Non-1 .. PC . Non-2 . Non-3 ¼ Aq. The deposition of CsA into  the  PSUs was in  the  order:   Non-1 .. Hydroalcoholic solution . PC . Non-2 ¼ Non-3.  It  was concluded that  in spite  of differences in the hydrophobicities of the peptide drugs, Non-1  liposome significantly enhanced the  deposition into  the  PSUs. Hence,  the vehicle  carrying the  drug made a major  impact on the  deposition  irrespective of whether it  was  hydrophilic or  hydrophobic. CsA  was,  however, deposited to  a greater extent  than  a-IFN. It was  explained that  since GDL melted at 308C, it was able  to cause  some  fluidization of the  liposomal bilayer  and  partial release  of its contents like POE-10 at body  temperature, whereas GDS melted at 548C and  hence did not cause this fluidization.

Expression plasmid DNA  for  the  human interleukin-1 receptor antagonist (IL-1ra)   protein  was   formulated  with   nonionic:cationic  (NC)   liposome and PC:cationic  (PC) liposomes and applied to auricular skin of hamsters (57). Confocal microscopy identified delivery of the plasmid DNA  proximal to the perifollicular cells. In the second part  of the study, the skin  was  treated for three  days  with  the NC  liposomes and  had  statistically significant levels  of transgenic IL-1ra present for five days  post-treatment. The  results indicated that  the  NC  liposomes could deliver expression plasmid DNA to perifollicular cells and mediate transient trans- fection  in vivo. The control  formulations were  made with  empty niosomes, that  is, no DNA,  and  were  done  with  infinite  dosing. The control  used  should have  been empty niosomes with  the  plasmid DNA  outside it and  would have  indicated  if drug or niosome had  the property of reaching the follicle.

The  sebaceous gland  deposition of isotretinoin after  topical  application on human facial  skin  in  vitro  was  measured 16 hours after  application by  taking skin biopsies and  separation of the skin compartments (58). Ethanolic gel was  the control  and  was  compared with  a liposomal formulation, Natipide II (made  of PC) formulation, and  a mixed  micelle  formulation. All the experiments were  per- formed  with   skin   from   the   periauricular  skin   of  women  undergoing  plastic surgery. Autoradiography and  fluorescence microscopy elucidated the penetration pathway of the active. The results showed that neither the liposomes nor the mixed micellar  system revealed an improved sebaceous gland  deposition of isotretenoin as compared with  the ethanolic gel.

The significance of the sebaceous gland  pathway in the cutaneous permeation of an antiandrogen, RU58841 in liposomes, was studied with  normal and  scar skin in hairless rat  (59). RU 58841 was  made into  a liposomal formulation containing lipoid  E 100-35 and  a-tocopherol in  a phosphate buffer  of pH ¼ 7, whereas the control   solution  was   made of  ethanol  PG:water (40:10:50 w/w).  The  in  vitro cutaneous permeation studies were  carried out  for 24 hours. The cumulative per- centage of RU 58841 absorbed was  three-fold higher through normal skin after 24 hours, whereas that  of the  liposomes was  four-fold higher through normal skin as compared to scar  skin.  However, the permeation of the ethanolic solution was much   higher through  normal skin  as  compared to  the  liposomal formulation through the  skin.  In  in  vivo  studies  carried out  for  24  hours,  it  was   found that  the  epidermis and  dermis of normal skin  contained more  amounts of the active than the scar tissue. An autoradiography showed that with the ethanolic sol- ution,  the drug was mainly localized in the SC/epidermis and with the liposomes it was  localized in  the  sebaceous glands.  The  overall   permeation was,  however, higher through the normal skin for the ethanolic solutions as compared to the lipo- somal solutions. When the correction for targeting was applied, liposomes targeted better  than  the ethanolic solution.

Mechanism of Action of Liposomes

Weiner  et al. (60) have hypothesized the use of liposomes in the following way. For hydrophobic drugs, a major  fraction  of the added drug would be encapsulated or intercalated within the  bilayers of the  liposomes. The  transfer of drug from  the lipid bilayers into the skin could occur as long as the bilayers were in liquid crystal- line state.  If the liquid crystalline phase  is altered to the gel state,  transport of the drug would cease  or  be  negligibly low.  Dehydration of  liposomal suspensions has  been  shown to reduce transitions from  the  liquid crystalline phase  to the  gel state.  If, however, dehydration was  complete and  the  bilayers were  transformed from  the liquid crystalline state  to a gel state,  then  the transfer of the drug would cease.  If dehydration to an equilibrium stage  occurs,  wherein a constant amount of water is always retained, then the transport of the drug is steady and continuous. A second consequence of dehydration involves the formation of a strong adhesive patch  of the  liposomal bilayers on the  skin.  The formation of such  patches maxi- mizes  the intimacy of contact  between the drug-laden bilayers and  the skin.

For hydrophilic drugs, too, a similar mode  of action  is explained. Liposomal systems that  undergo total  dehydration, drug transport ceases,  as the  drug is no longer  in a dissolved state.  For liposomal systems that  retain  a constant amount of water within the bilayers following dehydration to an equilibrium, drug trans- port  would continue over extended periods. A major  consequence of dehydration for   hydrophilic  drugs   involves  the   enhancement   or   enrichment   of   drug

concentration in the aqueous phase of the bilayers, leading to an enhancement in flux of drug into and across skin. When a follicular  pathway is present, upon dehy- dration, the liposomal bilayers can partition and  can pack  into the follicular  ducts containing the  lipids.   The  filling  of  the  follicular   openings with  the  liposomal bilayers not  only  results in entrapped drugs being  carried into  the  follicles,  but also allows  partitioning of free drugs into the bilayer  matrix  within the follicles.

Vehicles  Made  of Lipid Melts

In these studies, the melted form of the lipid components, which  were used to make the niosomes (namely, GDL:CH:POE-10),  were evaluated as a vehicle for the trans- port  of CsA and  other  agents into and  through hairless mouse skin (58,60,61). The experiments were  carried out  under nonoccluded conditions. At  predetermined times  of two, four, eight  and  24 hours, the diffusion setup was dismantled and  the drug in various strata  of the skin determined using  stripping and  digesting of the skin  using  scintillation counting. The  profiles  of the  extent  and  uptake of CsA from the lipid  melt formulations are similar to those  of the liposomal formulations of the same  compositions. In these  same  experiments, the ratio  of GDL to POE-10 was  varied. In all these  formulation, CH  was  kept  at a constant concentration of

15% and  GDL to POE-10 ratios  examined were  0:85, 15:70, 45:40, and  57:28. It was found that the 45:40 lipid  melt was more  effective in delivery of the active into and through the mouse skin. The authors explained that that particular combination of lipid  melt  had  the lowest  melting point  (   238C) and  that  was  responsible for the better   uptake.  Nevertheless, the  remaining melts   (except   0%  GDL)  were   also liquids at body  temperature and  the  melting point  does  not  seem  to explain the point   enough.  The   microautoradiographs  and   light   microscope  images   also confirm  these results. It was thus concluded that the similarity of the kinetic profiles for CsA transport into and  across  living  skin strata of hairless mice from  the lipo- somal  and  lipid  melt formulations along  with  the microautoradiographic evidence for localization within the PSUs implied that transport of CsA into the highly hydro- philic viable skin strata occurs mainly via the follicular  route.

Similar  studies were  also  done  on  the  delivery of hydrocortisone (hydro- phobic)  and  mannitol (hydrophilic) through the same  lipid-based formulations in and  through hairless mouse skin in vitro (62). The results after 24 hours indicated that  the extent  of hydrocortisone uptake rose with  increasing the GDL to POE-10 ratio,  whereas for mannitol, the uptake was the opposite decreasing with  increase in  this  ratio  in  liposomes. Lipid  melt  formulations for  mannitol were  not  made because it is a hydrophilic drug and  niosomes of the  above  compositions along with  water were  made. From  the  autoradiographic studies, it was  reported that the  nonionic lipid-based formulations were  predominantly transfollicular. It was also suggested that  a hydrophilic molecule like mannitol could  not  partition into the  lipid  environment of the  sebum-filled follicles  or into  and  across  the  SC and was  transported  mainly via  the  transepidermal route.   This  suggested that  the two macromolecules were transported through the skin via two different pathways. It was  thus  was  possible to tailor  formulations for specific  and  targeted delivery across  a certain  route.

Studies Done  on Microbeads: Size  Effects  on Follicular Deposition

Fluorescent polystyrene (16) microbeads of different sizes  were  made in aqueous medium and  Miglyolw  (hydrophobic vehicle)  as  suspensions and  were  applied on  live  female  hairless rat’s  back  and  freshly  excised  human skin.  Suspensions were  applied with  massaging for  five  minutes. The  experiment was  carried on

for 15 minutes, after which  skin biopsy  was done  and observed under a fluorescent microscope. It was found that the best follicular  penetration was observed with  the most   lipophilic vehicle   Miglyolw. The  9-  to  10-mm  beads   concentrated at  the opening of the follicles, but  did  not penetrate; however, the 7-mm beads  were  fre- quently observed deep  down in the follicular  canal,  but  rarely  penetrated the SC. The  smallest beads  ,3 mm penetrated the  follicles  well,  but  were  also  observed in the superficial layers  of the SC.

In the  same  paper, poly  b-alanine beads  labeled with  dansyl chloride were formulated in an aqueous gel, hydroalcoholic gel, and silicone oil. The suspensions were  applied to  the  rat’s  back  and  human skin.  Here  too,  the  best  results were observed with the most lipophilic vehicle (silicone oil). The 5 mm showed selectivity for the follicular  canal.

The site-specific delivery of adalapene to the hair follicles was attempted both in vivo  and  in vitro  (63). The drug was  loaded in 50:50 poly  (DL-lactic – coglycolic acid).  Different sizes  (1, 5, and  20 mm) of  the  microspheres were  evaluated in vitro  by investigating cutaneous penetration of the  microspheres through human skin  and  the female  hairless rat skin. The permeation was  allowed to proceed for

35 and  300 minutes, after  which  the  cells were  dismantled and  the  skin  samples were  frozen  and  then  evaluated by  fluorescence microscopy. It was  found that the  1-mm microspheres were  randomly distributed into  the  SC and  hair  follicles. The 20-mm microparticles did  not penetrate the skin and  remained on the surface where the  5 mm were  found in  the  hair  follicles.  In  addition, a time-dependent effect  was  seen  in  this  evaluation where greater penetration was  observed after

300 minutes than  after 35 minutes.

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