Variation among Plasmodium falciparum strains in their reliance on mitochondrial electron transport chain function

Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.     

Synthesis and in vitro activities of ferrocenic aminohydroxynaphthoquinones against Toxoplasma gondii and Plasmodium falciparum

Fourteen ferrocenyl aminohydroxynaphthoquinones, analogues of atovaquone, were synthesized from the hydroxynaphthoquinone core. These novel atovaquone derivatives were tested for their in vitro activity against two apicomplexan parasites of medical importance, Toxoplasma gondii and Plasmodium falciparum, including resistant strains to atovaquone (T. gondii) and chloroquine (P. falciparum). Three of these ferrocenic atovaquone derivatives composed of the hydroxynaphthoquinone core plus an amino-ferrocenic group and an aliphatic chain with 6-8 carbon atoms were found to be significantly active against T. gondii. Moreover, these novel compounds were also effective against the atovaquone-resistant strain of T. gondii (Ato(R)).     

Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To that end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone-mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance-conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side-chains may determine atovaquone-binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.     

Therapeutic evaluation of free and liposome-encapsulated atovaquone in the treatment of murine leishmaniasis

The use of drug delivery systems may reduce the toxicity and improve the activity of anti-leishmanial compounds. The activity of atovaquone (ATV)-loaded liposomes was compared by determination of median effective doses (ED(25) and ED(50)), with that of free ATV in a murine model of visceral leishmaniasis induced by Leishmania infantum. On day 0, mice were infected intravenously with 4.10(7) promastigotes and treated via the tail vein on days 15, 17 and 19 by free drug in a DMSO/cremophor/water solution (0.2 to 1.6 mg/kg body weight) or by liposomal drug (0.04 to 0.32 mg/kg body weight). Mice were killed and livers and spleens were removed and weighed on day 21 p.i. and liver parasite burdens evaluated using the Stauber method. Effective doses were determined using the Hill representation relating the percentage of parasite suppression to the dose. Liposomal ATV was significantly more effective than the free drug in reducing liver parasites (61.6% of parasite suppression at a dose of 0.32 mg/kg vs 34.9% at a dose of 1.6mg/kg). Liposomal ATV was 23 times more active than the free drug (ED(25) value=0. 02+/-0.01 mg/kg vs 0.46+/-0.15 mg/kg for free drug). It was not possible to obtain the ED(50) for free ATV because the dose-response curve reached a plateau around 33% of parasite suppression. Conversely, the ED(50) for liposomal ATV was 0.17+/-0.05 mg/kg. 100% efficacy of bound ATV could be obtained with a concentration of 1. 77+/-0.35 mg/kg. A significant decrease in spleen weights was also observed reflecting a leishmanicidal activity of ATV. These results suggest that liposome loaded ATV is more efficacious than the free drug against Leishmania infantum in this murine model.     

Uncovering the molecular mode of action of the antimalarial drug atovaquone using a bacterial system

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c1 of the cytochrome bc1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc1 complex.     

Plasmodium falciparum: the effects of atovaquone resistance on respiration

Atovaquone is an antimalarial agent that specifically inhibits the cytochrome bc(1) complex of the cytochrome pathway. High-level atovaquone resistance is associated with a point mutation in the cytochrome b gene. A pair of isogenic clinical isolates of Plasmodium falciparum derived from before and after the acquisition of atovaquone resistance was used to determine whether the change in the cytochrome b gene resulted in changes in respiration in response to atovaquone. Since P. falciparum appears to utilize a branched respiratory system comprising both the cytochrome and an alternative respiratory pathway, the proportion of each pathway utilized by the sensitive and resistant parasites was investigated. Atovaquone inhibited total parasite oxygen consumption by up to 66% in the sensitive isolate but only up to 28% in the resistant isolate. Both the atovaquone-sensitive and the atovaquone-resistant parasites were comparably sensitive to the alternative pathway inhibitor, salicylhydroxamic acid. Atovaquone appeared to partially inhibit the rate of oxygen consumed through the alternative pathway in only the atovaquone-sensitive isolate. Cross resistance was noted between atovaquone and a new antimalarial agent WR243251. However, the level of WR243251 resistance was very modest compared to the level of atovaquone resistance. WR243251 was shown to rapidly reduce the rate of parasite oxygen consumption by almost 80% in the atovaquone-sensitive isolate and by 57% in the atovaquone-resistant isolate. Drug interaction studies suggest that atovaquone and WR243251 may inhibit growth additively or with mild synergy. Together, these results suggest that while WR243251 may inhibit respiration, its target of action probably differs from that of atovaquone.     

Cytochrome b mutations that modify the ubiquinol-binding pocket of the cytochrome bc1 complex and confer anti-malarial drug resistance in Saccharomyces cerevisiae

Atovaquone is a new anti-malarial agent that specifically targets the cytochrome bc1 complex and inhibits parasite respiration. A growing number of failures of this drug in the treatment of malaria have been genetically linked to point mutations in the mitochondrial cytochrome b gene. To better understand the molecular basis of atovaquone resistance in malaria, we introduced five of these mutations, including the most prevalent variant found in Plasmodium falciparum (Y268S), into the cytochrome b gene of the budding yeast Saccharomyces cerevisiae and thus obtained cytochrome bc1 complexes resistant to inhibition by atovaquone. By modeling the variations in cytochrome b structure and atovaquone binding with the mutated bc1 complexes, we obtained the first quantitative explanation for the molecular basis of atovaquone resistance in malaria parasites.     

Efficacy of atovaquone and sulfadiazine in the treatment of mice infected with Toxoplasma gondii strains isolated in Brazil

The efficacy of atovaquone and sulfadiazine was examined alone or in combination for the treatment of mice infected with six Brazilian Toxoplasma gondii strains previously genotyped using the PCR-RFLP assays of the SAG2 gene, in addition to RH strain. Swiss mice were infected intraperitoneally with 10(2) tachyzoites from each strain of T. gondii and treated with 6.25, 12.5, 25 and 50 mg/Kg/day of atovaquone or 40, 80, 160 and 320 mg/Kg/day of sulfadiazine. In a second experiment, mice were treated with the association of previously determined doses of each drug. Treatment started 48 hours post-infection, and lasted 10 days. The susceptibility of T. gondii to atovaquone and to sulfadiazine was different according to the parasite strain. It was observed strains that are susceptible to atovaquone, and strains that are resistant to it. Type I strains were more susceptible to the activity of sulfadiazine and more resistant to atovaquone. Yet type III strains were susceptible to atovaquone and to sulfadiazine. Association of atovaquone and sulfadiazine presented a synergic effect in the treatment of mice infected with RH type I strain and an additive effect in the treatment of mice infected with one type I strain and with two type III strains.     

In vivo antimalarial activity of novel 2-hydroxy-3-anilino-1,4-naphthoquinones obtained by epoxide ring-opening reaction

1,4-Naphthoquinone derivatives are known to have relevant activities against several parasites. Among the treatment options for malaria, atovaquone, a 1,4-naphthoquinone derivative, is widely applied in the treatment and prophylaxis of such disease. Based on the structure simplification of atovaquone, we designed and synthesized four novel naphthoquinoidal derivatives. The compounds were obtained by the underexplored epoxide-opening reaction of 1,4-naphthoquinone using aniline derivatives as nucleophiles. The antiplasmodial activity of the synthesized compounds was performed in vivo using Peter’s 4 days suppression test. Significant parasitemia reduction and increased survival were observed for some of the compounds.     

Antimalarial quinolones: synthesis, potency, and mechanistic studies

In the present article we examine the antiplasmodial activities of novel quinolone derivatives bearing extended alkyl or alkoxy side chains terminated by a trifluoromethyl group. In the series under investigation, the IC50 values ranged from 1.2 to approximately 30 nM against chloroquine-sensitive and multidrug-resistant Plasmodium falciparum strains. Modest to significant cross-resistance was noted in evaluation of these haloalkyl- and haloalkoxyquinolones for activity against the atovaquone-resistant clinical isolate Tm90-C2B, indicating that a primary target for some of these compounds is the parasite cytochrome bc1 complex. Additional evidence to support this biochemical mechanism includes the use of oxygen biosensor plate technology to show that the quinolone derivatives block oxygen consumption by parasitized red blood cells in a fashion similar to atovaquone in side-by-side experiments. Atovaquone is extremely potent and is the only drug in clinical use that targets the Plasmodium bc1 complex, but rapid emergence of resistance to it in both mono- and combination therapy is evident and therefore additional drugs are needed to target the cytochrome bc1 complex which are active against atovaquone-resistant parasites. Our study of a number of halogenated alkyl and alkoxy 4(1H)-quinolones highlights the potential for development of "endochin-like quinolones" (ELQ), bearing an extended trifluoroalkyl moiety at the 3-position, that exhibit selective antiplasmodial effects in the low nanomolar range and inhibitory activity against chloroquine and atovaquone-resistant parasites. Further studies of halogenated alkyl- and alkoxy-quinolones may lead to the development of safe and effective therapeutics for use in treatment or prevention of malaria and other parasitic diseases.     

Atovaquone enhances doxorubicin’s efficacy via inhibiting mitochondrial respiration and STAT3 in aggressive thyroid cancer

The clinical management of anaplastic thyroid carcinoma and follicular thyroid carcinoma is challenging and requires an alternative therapeutic strategy. Although atovaquone is an FDA-approved anti-malarial drug, studies has recently demonstrated its anti-cancer activities. In line with these efforts, our study shows that atovaquone is an attractive candidate for thyroid cancer treatment. We show that atovaquone significantly inhibits growth, migration and survival in a concentration-dependent manner in 8505C and FTC113 cells. Mechanistically, atovaquone inhibits mitochondrial complex III activity, leading to mitochondrial respiration inhibition and reduction of ATP production in thyroid cancer cells. The inhibitory effects of atovaquone is reversed in mitochondrial respiration-deficient 8505C ρ0 cells, confirming mitochondrial respiration as the mechanism of atovaquone’s action in thyroid cancer. In addition, atovaquone suppresses phosphorylation of STAT3 in thyroid cancer wildype but not ρ0 cells, demonstrating that STAT3 phosphorylation inhibition by atovaquone is a consequence of mitochondrial respiration inhibition. Notably, we further demonstrate that atovaquone significantly augments doxorubicin’s inhibitory effects via suppressing mitochondrial respiration and STAT3. Our findings suggest that atovaquone can be repurposed for thyroid cancer treatment. Our work also highlights that targeting mitochondrial respiration may represent potential therapeutic strategy in thyroid cancer.     

Ubiquinone synthesis in mitochondrial and microsomal subcellular fractions of Pneumocystis spp.: differential sensitivities to atovaquone

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ7, CoQ8, CoQ9, and CoQ10, as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p-hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell.     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

Characterization of a Plasmodium falciparum Orthologue of the Yeast Ubiquinone-Binding Protein,Coq10p

Coenzyme Q (CoQ, ubiquinone) is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10), which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.     

Iron chelators as anti-infectives; malaria as a paradigm

Malaria is the major life threatening parasitic disease and the cause of a global public health problem. The failure of vector eradication programs and the appearance and spread of drug resistant parasites have posed the urgent challenge of developing effective, safe and affordable anti-malarial drugs. The design of such drugs is largely based on the targeting of agents to the parasite-based machinery for host digestion and to the products of hemoglobin catabolism. Iron chelators, by depriving intracellular parasites from essential iron, lead to selective suppression of parasite growth. However, by acting on parasite-impaired macrophages, chelators can also expedite resumption of phagocytosis and elimination of parasites. In order to be clinically effective, chelators need to be maintained in the blood for extensive time periods. Therapeutic doses can be attained with appropriate drug combinations and formulations or delivery devices and these must be presented in a form well tolerated by the host. The early documentation that chelation therapy has activity against human malaria has paved the road for the design of novel and more efficient remedies based on short-term iron deprivation.     

Targeting mitochondria by anthelmintic drug atovaquone sensitizes renal cell carcinoma to chemotherapy and immunotherapy

Targeting mitochondria respiration is an effective therapeutic strategy in renal cell carcinoma (RCC). Atovaquone is a FDA‐approved antibiotic but is also known as a mitochondrial inhibitor. We found that atovaquone inhibited proliferation and induced apoptosis of RCC cells. Mechanistically, atovaquone inhibits mitochondrial respiration in a concentration‐dependent and time‐dependent manner, via targeting mitochondrial respiratory complex III. Although increased glycolysis was observed in atovaquone‐treated cells, atovaquone decreased ATP levels. As a consequence of mitochondrial respiration inhibition, reactive oxygen species levels were increased by atovaquone. The complete rescue of atovaquone's effects by an antioxidant suggests the important role of oxidative stress in the action of atovaquone in RCC. Importantly, atovaquone enhanced the in vitro and in vivo efficacy of 5‐fluorouracil (5‐FU) and interferon‐α (IFN‐α). Our preclinical findings suggest that atovaquone is a useful addition for RCC treatment. Our work also further demonstrates that RCC is more dependent on mitochondrial respiration than glycolysis.     

Using an antimalarial in mosquitoes overcomes Anopheles and Plasmodium resistance to malaria control strategies

The spread of insecticide resistance in Anopheles mosquitoes and drug resistance in Plasmodium parasites is contributing to a global resurgence of malaria, making the generation of control tools that can overcome these roadblocks an urgent public health priority. We recently showed that the transmission of Plasmodium falciparum parasites can be efficiently blocked when exposing Anopheles gambiae females to antimalarials deposited on a treated surface, with no negative consequences on major components of mosquito fitness. Here, we demonstrate this approach can overcome the hurdles of insecticide resistance in mosquitoes and drug resistant in parasites. We show that the transmission-blocking efficacy of mosquito-targeted antimalarials is maintained when field-derived, insecticide resistant Anopheles are exposed to the potent cytochrome b inhibitor atovaquone, demonstrating that this drug escapes insecticide resistance mechanisms that could potentially interfere with its function. Moreover, this approach prevents transmission of field-derived, artemisinin resistant P. falciparum parasites (Kelch13 C580Y mutant), proving that this strategy could be used to prevent the spread of parasite mutations that induce resistance to front-line antimalarials. Atovaquone is also highly effective at limiting parasite development when ingested by mosquitoes in sugar solutions, including in ongoing infections. These data support the use of mosquito-targeted antimalarials as a promising tool to complement and extend the efficacy of current malaria control interventions.     

Effect of Farnesyltransferase Inhibitor R115777 on Mitochondria of Plasmodium falciparum

The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (ΔΨm) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.     

Factors Enhancing the Host-Cell Penetration of Toxoplasma gondii

The penetration into HeLa cells of Toxoplasma gondii was studied with a cell culture technique. The influence on the rate of penetration and the number of penetrating Toxoplasma parasites was tested by use of preparations of disintegrated parasites mixed with test parasites. These preparations were found to contain factors enhancing the penetrating rate of the parasites. This effect was demonstrable by use of untreated parasites as well as parasites lacking active motility owing to a previous exposure to Formalin. The preparations of disintegrated parasites contained, in addition, components inhibitory to the penetration-enhancing factors. These inhibitory components were able to reduce the penetrating capacity of normal Toxoplasma parasites, suggesting that the studied enhancing factors may play a role in the natural process of penetration. The efficacy of various techniques for disintegration of Toxoplasma parasites was investigated for release of penetration-enhancing factors from Toxoplasma parasites. The methods used resemble those used for liberation of lysosomal enzymes. Reduced osmotic pressure was obviously not adequate for release of enhancing factors, whereas the freezing and thawing procedure, sonic treatment, and irradiation produced high yields. It was difficult to evaluate the effect of incubation at acid pH on release of enhancing activity, because the penetration-promoting factors seemed unstable on both the acid and the alkaline sides of pH 7.6.