Protein delivery with infusion pumps.

When a therapeutic effect is optimized by precise control of specific temporal patterns of plasma levels, infusion offers distinct advantages over oral administration, bolus injection, or depot delivery of polypeptides. The limitations of oral delivery are well known, and although research is under way into development of carrier systems that prevent degradation of labile agents, it is unlikely that the variances in absorption will meet the need for precise control. Depot delivery from subcutaneous or intramuscular implants presents a difficult situation when local tissue reactions to the agent sometimes occur. Removal of a depot system in the event of adverse reactions presents additional difficulties. Bolus injections are unable to sustain constant plasma levels unless the drug half-life is long or the injections are frequently administered. Insulin injections, for example, would be required every 30-60 minutes to approximate the plasma levels provided by a continuous infusion; such frequent injections would not be practical on a 24-hour basis. For the developer of new polypeptides, parenteral administration offers the most direct route to the marketplace. The step from periodic injections to tightly controlled infusion is a logical progression as compared with modification of the molecules or vehicles to obtain equivalent profiles. In Table II several different types of devices that can be used for infusion of proteins are compared. Microelectronics have played a major role in the miniaturization of infusion devices and undoubtedly will continue to do so. Micromachining, a spin-off technology of integrated circuit manufacture, will also find application in small infusion devices. In the future, we will have cost-effective disposable devices (Saaman et al., 1994) built on this technology that are programmable and thus can be adapted to meet each individual therapeutic need (Horres, 1994). We can also expect to see more closed-loop drug delivery systems where biosensors and infusion devices are combined to optimize a particular therapy. Recent positive results obtained in diabetics by a decade on tight glucose control may forecast a resurgence of popularity of insulin pumps. At the other end of the spectrum, low-cost, small, and simple-to-use osmotically powered systems are close to being marketed; these systems will make infusion almost as convenient as transdermal patches. We will also see major advances in how drugs and devices are interfaced. Prefilled and ready-to-use drug cartridges have proven to be efficient in surgical and emergency medicine and can greatly improve most infusion applications. It is anticipated that coded, prefilled cartridges or pouches will be automatically, recognized by preprogrammed pumps to reduce operator labor and entry error.

Stratum corneum lipids of human epidermal keratinocyte air-liquid cultures: implications for barrier function.

PURPOSE: The purpose of this study is to investigate the permeability barrier, i.e., the stratum corneum (SC) lipids, of human epidermal keratinocyte air-liquid cultures and compare them with those of human SC. METHOD: The SC lipids composition was analyzed by TLC technique, the organization by electron microscopic procedure, and the phase transition temperature by infrared spectroscopic method. RESULTS: Electron microscopy demonstrated that The SC lipids of cultures were largely retained inside the comeocytes, and that the intercellular lipids lack both the basic unit repetition (i.e., broad: narrow: broad: broad: narrow: broad of electron lucent bands) and the covalently-bound lipid envelope normally found in human SC. These characteristics are similar to those found in SC from patients with atopic dermatitis or psoriasis, or from animals with essential fatty acid deficiency, suggesting that the cultures may be hyperproliferative. In addition, the high free sterol content and the altered fatty acid/ceramide composition of these cultures argue that the compromised barrier function is linked to hyperproliferation and lipid synthesis, or vice versa. Infrared spectroscopic analyses confirm that there are major conformational differences between the lipids of human and cultured SC. CONCLUSIONS: The profound differences in SC lipid composition, organization and conformational properties attest that permeability alone is not a sufficiently sensitive marker to define barrier equivalence between cultures and human skin.

Characterization of low-temperature (i.e., < 65 degrees C) lipid transitions in human stratum corneum.

This study aims to characterize human stratum corneum (SC), focusing on those lipid transitions that occur at or below physiologically relevant temperatures. In the past, a lipid transition near 35 degrees C had been thought to be variable and a consequence of superficial sebaceous lipid contamination. However, analysis here indicates that it is widely present, and cannot be attributed to sebum production. We demonstrate that this transition represents a solid-to-fluid phase change for a discrete subset of SC lipids. The reversibility of this transition upon reheating, and its absence in extracted lipid samples imply that these lipids are not uniformly present throughout the SC, but would appear to be differentially distributed in response to terminal differentiation. Further, such an arrangement could involve a close association with other nonlipid (e.g., protein) components. Evidence for a new transition at approximately 55 degrees C is presented that suggests the loss of crystalline orthorhombic lattice structure. The existence of orthorhombic structure at physiologic temperature is reasoned to involve ceramides and/or free fatty acids. Localization of these lipids at the level of the corneocyte envelope supports a comprehensive picture of water transport across the SC, whereby diffusion occurs primarily via the intercellular lipids. This view, coupled with the hydration-induced changes in lipid disorder observed here provides additional insight into the mechanism by which skin occlusion increases permeability. Summarily, these results i) emphasize the inherent danger of over-interpreting experiments with isolated SC lipids, ii) emphasize the potential advantage(s) of employing several biophysical techniques to study SC structure, and iii) indicate that a full characterization of lipid phase behavior is requisite to our eventual understanding of SC structure and permeability function, particularly those phase transitions that occur near or at normal skin temperature.

Polymorphism in stratum corneum lipids.

Fourier transform infrared spectroscopy (FTIR) was employed to investigate the thermotropic phase behavior of stratum corneum lipid multilamellae. Stratum corneum (SC), the uppermost layer of mammalian skin, is unusual in many respects. It has been demonstrated that the lipids of the stratum corneum provide the primary electrical and transport resistance in the skin. These lipids are unusual in their composition, structure and localization; they contain only cholesterol, fatty acids and ceramides and they form broad, multi-lamellar sheets which are located extracellularly. The FTIR results from both the symmetric CH2 stretching and the CH2 scissoring vibrations suggest that the SC lipids exhibit polymorphic phase behavior below the main phase transition temperature. The multiple phases are most likely crystalline mixtures of different alkyl chain packings, along with solid-liquid phases. Similarities between the FTIR results reported here for SC lipids and those obtained for cholesterol-containing gel phase phospholipids suggest that the non-uniform distribution of cholesterol occurs in each system.

Topical penetration of piroxicam is dependent on the distribution of the local cutaneous vasculature.

The mechanism of the topical delivery of piroxicam, a nonsteroidal antiinflammatory drug, has been controversial as to whether systemic absorption is required for topical efficacy. This study, using in vivo pigs treated with topical 3H-piroxicam gel, was designed to assess the role of systemic absorption on its delivery to deep tissues. Further, the role of the structure of the cutaneous vasculature (e.g., direct cutaneous or musculocutaneous) was studied. Finally, piroxicam delivery was measured using in vitro diffusion cells with pig skin obtained from the same sites to determine inherent permeability independent of vascular anatomy. These studies showed that penetration of the radiolabel occurred in subcutaneous and muscle tissue only under the dosed sites and not at the remote sites, ruling out systemic absorption as a prerequisite for local delivery. Tissue penetration in vivo was enhanced at the musculocutaneous compared to the direct cutaneous sites. In contrast, in vitro flux was identical in skin harvested from the two vascular sites, suggesting that the vasculature plays a pivotal role in deep tissue penetration of piroxicam. In conclusion, local delivery of topical drugs occurs independent of systemic absorption and the nature of the cutaneous vasculature at different sites must be taken into consideration for optimal delivery.

Local enhanced topical delivery (LETD) of drugs: does it truly exist?

There is considerable uncertainty over whether and to what extent topically applied drugs can be delivered directly to anatomical sites beneath the skin, without prior entry into the systemic blood circulation. The in vivo studies reported in this work were designed to assess whether local enhanced topical delivery (LETD) can be achieved with piroxicam, a nonsteroidal antiinflammatory drug. Equivalent doses of tritium-labeled drug were administered by the i.v. or topical routes to male rats. The topical plasma profile reveals a maximum concentration (Cpmax) at 12 hr, compared to a typical, multiexponential decline in plasma concentration after i.v. dosing. All four muscles from the topically dosed shoulder exhibit two distinct peaks, the first at 4 hr and a later one at 12 hr (which coincides with the topical Cpmax). The contralateral muscles from the nondosed shoulder, in contrast, produce only a single peak at 12 hr after topical dosing. After the i.v. administration of piroxicam, the concentration-time profiles for each muscle closely parallel that seen for the i.v. plasma. Tissue-to-plasma ratios (T/P) show that the topical nondosed and the i.v. muscles are nearly constant over the entire time course of this study, indicating a pseudo-equilibrium between the plasma and those muscles. However, the early T/P ratios for the topically dosed muscles are markedly elevated and gradually decline to a constant value only after 12 hr, indicating that a similar pseudo-equilibrium is not established in this case. Thus, these results strongly imply that the topical administration of a drug can lead to LETD for tissues subjacent to the skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical methods for studying stratum corneum lipids.

The skin's barrier to water loss and drug transport is closely related to the biophysical properties of the stratum corneum lipids. A number of in vitro and in vivo methods have been used to evaluate the physical properties of stratum corneum lipids. Two particularly useful techniques, infrared spectroscopy and differential scanning calorimetry, have provided a wealth of information about stratum corneum structure and function. The results of these studies demonstrate that the alteration of stratum corneum lipid packing results in increased skin permeability. Furthermore, the magnitude of increased permeability can be predicted from these physical properties. Therefore, these results provide both insight into the mechanism of transport through the stratum corneum, as well as a useful paradigm for selection of agents that reversibly alter the permeability of the skin.

Secondary structural analysis of two recombinant murine proteins, interleukins 1 alpha and 1 beta: is infrared spectroscopy sufficient to assign structure?

The secondary structure for two murine recombinant proteins, interleukins 1 alpha and 1 beta (rmIL-1 alpha and -1 beta), has been analyzed by Fourier transform infrared (IR) spectroscopy and then compared to results obtained by X-ray diffraction, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. The IR results obtained here for rmIL-1 alpha and -1 beta suggested that their secondary structures consisted predominantly of beta-sheets or strands. However, the analysis also revealed a significant absorption band near 1656 cm-1, which is typically assigned to alpha-helical or random structures. When these same murine polypeptides were analyzed by CD, no evidence of alpha-helical structures was observed. Further, published X-ray diffraction and NMR studies characterizing the human forms of IL-1 alpha and -1 beta indicate the absence of alpha-helices and that the human proteins are composed mainly of beta-strands (i.e., greater than 55%), with approximately 24% of the amino acids involved in large loops connecting the strands. The murine IL-1 proteins, when compared to their respective human counterparts, each show greater than 80% sequence homology. Given this fact, the CD analyses, and the result that this IR band amounted to 21% of the overall integrated area, the absorption peak at 1656 cm-1 was attributed to the presence of large loops rather than to alpha-helical or random structures. Such a structural assignment appears reasonable and is totally consistent with the established existence of large loops in the human forms as well as in other proteins found to fold similarly (viz., human bFGF).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of stratum corneum morphology on water permeability.

The stratum corneum (SC) provides the barrier to water loss for the skin of mammals. A significant body of evidence now exists suggesting that extracellular SC lipids are primarily responsible for this barrier. We have measured the permeability (P) and lag-time (Tlag) for water vapor transport through the SC and found that P is about 1000 times less than the values obtained for most other lipid membranes. In addition, we have measured the water partitioning into the lipid microdomain of the SC using a differential scanning calorimetry technique. These combined data provide an estimate of the diffusion coefficient (D) and diffusion pathlength (delta). The results show that the intrinsic diffusion of water is comparable to values obtained with other lipid membranes. The value obtained for delta, however, is fiftyfold greater than the sample thickness. These results are interpreted in terms of the unique morphology of the SC, where lipids form an extracellular continuum that is highly tortuous. Thus, the exceedingly low permeability of the SC may be due, in large part, to its unique morphology.

Evidence that oleic acid exists in a separate phase within stratum corneum lipids.

Oleic acid is known to be a penetration enhancer for polar to moderately polar molecules. A mechanism related to lipid phase separation has been previously proposed by this laboratory to explain the increases in skin transport. In the studies presented here, Fourier transform infrared spectroscopy (FT-IR) was utilized to investigate whether or not oleic acid exists in a separate phase within stratum corneum (SC) lipids. Per-deuterated oleic acid was employed allowing the conformational phase behavior of the exogenously added fatty acid and the endogenous SC lipids to be monitored independently of each other. The results indicated that oleic acid exerts a significant effect on the SC lipids, lowering the lipid transition temperature (Tm) in addition to increasing the conformational freedom or flexibility of the endogenous lipid alkyl chains above their Tm. At temperatures lower than Tm, however, oleic acid did not significantly change the chain disorder of the SC lipids. Similar results were obtained with lipids isolated from the SC by chloroform:methanol extraction. Oleic acid, itself, was almost fully disordered at temperatures both above and below the endogenous lipid Tm in the intact SC and extracted lipid samples. This finding suggested that oleic acid does exist as a liquid within the SC lipids. The coexistence of fluid oleic acid and ordered SC lipids, at physiological temperatures, is consistent with the previously proposed phase-separation transport mechanism for enhanced diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Oleic acid: its effects on stratum corneum in relation to (trans)dermal drug delivery.

Calorimetric studies with porcine stratum corneum (SC) have shown that the lipid phase transitions associated with the intercellular bilayers are markedly affected by treatment with oleic acid. Specifically, the transition temperatures (Tm) and cooperativity are reduced, whereas no effect was observed on the endotherm associated with keratin denaturation, suggesting that oleic acid primarily affects the SC lipids. The decrease in the lipid-associated Tm's was further correlated with the amount of oleic acid taken up by the SC. Parallel experiments with silastic implied that the uptake is dependent on the thermodynamic activity of oleic acid in the vehicle itself. The in vitro transport of Piroxicam across human and hairless mouse skin (HMS) was significantly enhanced by oleic acid, as a function of the extent of oleic acid uptake, with an attendant change in Tm. These results emphasize the role of SC lipids in percutaneous absorption. Transport also depended on the donor concentration of ionized drug suggesting that the enhanced transport mechanism cannot be accounted for solely on the principles of the classical pH-partition hypothesis. Accordingly, a model of skin permeability enhancement involving solid-fluid phase separation within the SC lipids is proposed for oleic acid, consistent with the existing phospholipid literature. In conjunction with the use of oleic acid as an enhancer, very soluble hydrophilic salts were recognized as key factors in attaining maximum delivery. Oleic acid uptake, lipid delta Tm, and enhanced drug flux were all found to correlate, exhibiting a bell-shaped curve as a function of the ethanol vehicle concentration. Therefore, uptake and/or DSC experiments are useful for formulating enhanced topical delivery systems.

Lipid biophysics of water loss through the skin.

The regulation of water loss through the skin is a poorly understood but crucial process in maintaining terrestrial life-forms. In mammalian skin, the outermost layer, called the stratum corneum (SC), is rate-limiting to water loss. We have evaluated temperature-dependent changes in water vapor permeability and infrared spectra of porcine SC. In particular, we have analyzed the infrared absorption peaks due to the extracellular lipids of the SC. These results show a remarkable correlation between water permeability and the frequency of the C-H stretching vibrations over a broad range of temperature. Since the spectral changes reflect an increased number of alkyl gauche conformers, these results suggest that water permeability is dependent upon the hydrocarbon-chain disorder of SC lipids.

The kinetics of timolol in the rabbit lens: implications for ocular drug delivery.

This study examines the uptake and distribution of timolol in the rabbit lens following topical instillation using a heuristic approach. The implications of anisotropic drug diffusion through the lens are presented here and discussed in the context of actual in vivo data. The dynamics of timolol in the lens involve an initial, rapid uptake of the drug by the capsule and epithelium followed by slower, anisotropic diffusion through the cortex body. Kinetically, the capsule and epithelium can be treated as a separate compartment which is distinct from the cortex and which serves to provide a concentration gradient for subsequent diffusion of timolol into the dense interior structures of the lens. Model simulations support the hypothesis that the preferred route of penetration of timolol into the vitreous humor via the lens is the diffusion of drug around the capsule/epithelium and peripheral cortical layers. It is also shown that due to the high and increasing diffusional resistance toward the center of the lens, as well as the diminishing drug concentrations in the capsule and epithelium, steady-state levels in the lens may be extremely difficult to achieve in some therapeutic situations. This phenomenon could have a significant impact on the success or failure of a drug treatment involving the lens and ocular tissues.